501-Stem Cell and Tissue Engineering-Song Li Song Li Nicolas L'Heureux Jennifer Elisseeff-9814317

STEM CELL AND

TISSUE ENGINEERING

World Scientific

edited by

Song LiUniversity of California, Berkeley, USA

Nicolas L’HeureuxCytograft Tissue Engineering, USA

Jennifer ElisseeffJohns Hopkins University, USA

7829tp.indd 2 7/27/10 11:42 AM

N E W J E R S E Y • L O N D O N • S I N G A P O R E • B E I J I N G • S H A N G H A I • H O N G K O N G • TA I P E I • C H E N N A I

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STEM CELL AND TISSUE ENGINEERING

Contents

Contributors xiii

Preface xxiii

1 Tissue Engineering: From Basic Biology to Cell-Based 1ApplicationsRobert M. Nerem

1. Introduction 12. Cell Source 33. Stem Cells 54. From Benchtop Science to Cell-Based Applications 75. Concluding Comments 8Acknowledgments 8References 9

2 Recent Advances and Future Perspectives 13on Somatic Cell ReprogrammingKun-Yong Kim and In-Hyun Park

1. Introduction 132. Nuclear Reprogramming 143. Reprogramming by Defined Factors 16

v

4. Recent Advances in Reprogramming Methods 175. Future Perspectives on Reprogramming and iPS Cells 19Acknowledgments 23References 23

3 Hematopoietic Stem Cells 31Jennifer J. Trowbridge

1. Introduction 312. Hematopoietic Stem Cell Sources 323. Applications 364. Challenges for Tissue Engineering 37Acknowledgments 41References 42

4 Mesenchymal Stem Cells for Tissue Regeneration 49Ngan F. Huang and Song Li

1. Introduction 492. MSC Sources and Phenotype 503. Differentiation of MSCs in vitro 524. Tissue Engineering and Regeneration Using Bone 54

Marrow MSCs and ASCs5. Future Directions 61Acknowledgments 62References 62

5 Delivery Vehicles for Deploying Mesenchymal Stem Cells 71in Tissue RepairMichael S. Friedman and J. Kent Leach

1. Introduction 712. Delivery of MSCs for Repairing Cardiovascular Tissues 723. Delivery Vehicles for Deploying Stem Cells in 78

Skin Regeneration4. Biomaterials for Implanting MSCs for Regenerating 81

Osteochondral Tissues5. Conclusions 88References 88

vi Contents

6 Stem Cells for Cardiac Tissue Engineering 95Jennifer L. Young, Karen L. Christman and Adam J. Engler

1. Cell Therapies for Myocardial Infarction and Heart Failure 952. Cellular Cardiomyoplasty Revisited: The Influence of 98

in vitro Mechanics3. Tissue Engineering Approach: Utilizing Biomaterial 102

ScaffoldsReferences 107

7 Cardiovascular System: Stem Cells in Tissue-Engineered 115Blood VesselsRajendra Sawh-Martinez, Edward McGillicuddy,Gustavo Villalona, Toshiharu Shin’okaand Christopher K. Breuer

1. Introduction 1152. Critical Elements of an Artificial Blood Vessel 1173. Approaches to Creating TEBVs 1194. Conclusion 127Acknowledgments 128References 128

8 Stem Cells for Vascular Regeneration: An 135Engineering ApproachLaura E. Dickinson and Sharon Gerecht

1. Introduction 1352. Cell Sources 1363. Engineering Vascular Differentiation 1414. Three-Dimensional Space 142Acknowledgments 152References 152

9 Stem Cells and Wound Repair 159Sae Hee Ko, Allison Nauta, Geoffrey C. Gurtnerand Michael T. Longaker

1. Clinical Burden of Wound Healing 159

Contents vii

2. Physiology of Wound Healing 1613. Stem Cells and Wound Repair 1654. Conclusion 173References 174

10 Engineering Cartilage: From Materials to Small 181MoleculesJeannine M. Coburn and Jennifer H. Elisseeff

1. Introduction 1812. Structure of Articular Cartilage of the Knee 1813. Osteoarthritis of the Knee 1834. Surgical Strategies for Repairing Focal Cartilage Defects 1855. Scaffolds for Assisting Operative Techniques 1876. Mesenchymal Stem Cells for Cartilage Tissue 192

Engineering7. Hydrogels for Directed Differentiation of Mesenchymal 193

Stem Cells8. Fiber-Hydrogel Composites 1979. Small Molecules for Directing Chondrogenesis 199

10. Conclusion 201Acknowledgments 202References 202

11 Adult Stem Cells for Articular Cartilage Tissue 211EngineeringSushmita Saha, Jennifer Kirkham, David Wood,Stephen Curran and Xuebin B. Yang

1. Introduction 2112. Human Bone Marrow Mesenchymal Stem Cells 213

(hBMMSCs)3. Adipose Derived Mesenchymal Stem Cells (ASCs) 2164. Periosteum Derived Stem/Progenitor Cells 217

(PDSCs/PDPCs)5. Synovium Derived Mesenchymal Stem Cells (SMSCs) 2186. Human Dental Pulp Stem Cells (HDPSCs) 2197. Umbilical Cord/Cord Blood Derived Stem Cells 220

viii Contents

8. Other Potential Cell Sources with a Chondrogenic 220Potential

9. Conclusion and Future Directions 222Acknowledgments 222References 222

12 Stem Cells for Disc Repair 231Aliza A. Allon, Zorica Buser, Sigurd Bervenand Jeffrey C. Lotz

1. Introduction 2312. The Demanding Intervertebral Disc Environment 2333. Evaluating a Stem Cell-Based Therapy 2344. Non-Stem Cell-Based Regeneration Strategies 2365. Stem Cells for Disc Repair 2376. Conclusion 244References 244

13 Skeletal Tissue Engineering: Progress and Prospects 251Nicholas J. Panetta, Deepak M. Gupta andMichael T. Longaker

1. Introduction 2512. Lessons Learned from Endogenous Skeletal Tissue 255

Development, Healing and Regeneration3. Progenitor Cell-Based Skeletal Tissue Engineering 2574. Pro-osteogenic Molecular Biology 2615. Advances in Skeletal Tissue Engineering Scaffolds 2666. Summary and Future Directions 268References 269

14 Clinical Applications of a Stem Cell Based Therapy 277for Oral Bone ReconstructionBradley McAllister and Kamran Haghighat

1. Introduction 2772. Procurement Methodology for Stem Cell Containing 279

Allograft

Contents ix

3. Ridge Augmentation 2824. Sinus Augmentation 2845. Discussion 289Acknowledgments 292References 292

15 Therapeutic Strategies for Repairing the Injured Spinal 297Cord Using Stem CellsMichael S. Beattie and Jacqueline C. Bresnahan

1. Introduction 2972. Secondary Injury and Endogenous Repair After SCI 2993. Therapeutic Targets for Transplanted Stem and 300

Progenitor Cells4. Animal Models of Spinal Cord Injury 3015. Types of Stem and Progenitor Cells Used for 305

Transplantation in SCI6. Evidence for Effects on Regeneration and Sprouting 3067. Evidence for Effects on Neuroprotection 3078. Evidence for Replacement of Neurons 3079. Evidence for Oligodendrocyte Replacement and 308

Remyelination10. Keys to Future Progress 30911. Are Stem and Progenitor Cell Therapies Ready for 311

Clinical Trials?Acknowledgments 312References 312

16 Potential of Tissue Engineering and Neural Stem 321Cells in the Understanding and Treatment ofNeurodegenerative DiseasesCaroline Auclair-Daigle and François Berthod

1. Introduction 3212. Neurodegenerative Diseases and Their Current Treatments 3223. Tissue Engineering as a Tool to Better Understand 324

Neurodegenerative Diseases4. Neural Stem Cells to Treat Neurodegenerative Diseases 329

x Contents

5. Conclusion 339Acknowledgments 339References 340

17 High-Throughput Systems for Stem Cell Engineering 347David A. Brafman, Karl Willert and Shu Chien

1. Introduction 3472. Sources of Stem Cells Suitable for High-Throughput 348

Screening Approaches3. The Stem Cell Niche: A Cellular Microenvironment 349

That Controls Stem Cell Behavior4. High-Throughput Intrinsic Systems for Stem Cell 363

Investigations5. Conclusions and Future Trends 365References 367

18 Microscale Technologies for Tissue Engineering and 375Stem Cell DifferentiationJason W. Nichol, Hojae Bae, Nezamoddin N. Kachouie,Behnam Zamanian, Mahdokht Masaeli andAli Khademhosseini

1. Introduction 3752. Control of Cellular and Tissue Microarchitecture 3773. Microscale Technologies to Investigate and Control 380

Stem Cell Behavior4. Assembly Techniques for Creating Engineered Tissues 385

from Microscale Building Blocks5. Conclusions and Future Directions 391References 391

19 Quality Control of Autologous Cell- and Tissue-Based 397TherapiesNathalie Dusserre, Todd McAllister and Nicolas L’Heureux

1. Introduction 3972. Regulations Pertaining to Quality Control of Cell- 398

and Tissue-Based Products

Contents xi

3. cGMP, cGTP and Quality System 4014. Core Requirements of a Quality Program 4025. Tailoring Quality Control to the Manufacturing Process 4076. Conclusion 417Acknowledgments 418References 418

20 Regulatory Challenges for Cell-Based Therapeutics 423Todd McAllister, Corey Iyican, Nathalie Dusserreand Nicolas L’Heureux

1. Introduction 4232. Regulatory Challenges at Each Phase of Clinical 425

Development3. Regional Considerations for Clinical Trials 4304. Use of a Clinical Research Organization 4355. Universal Regulatory Considerations for Cell-Based 437

TherapeuticsReferences 439

Index 441

xii Contents

Contributors

Aliza A. AllonDepartment of Orthopedic Surgery University of California San FranciscoSan Francisco, CA 94110

Caroline Auclair-DaigleLaboratoire d’Organogénèse Expérimentale (LOEX)Centre de recherche FRSQ du CHA universitaire de QuébecHôpital du Saint-Sacrement, and Département de chirurgieFaculté de médecine, Université Laval Québec, Canada

Hojae BaeHarvard-MIT Division of Health Sciences and TechnologyCenter for Biomedical Engineering, Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolMassachusetts Institute of TechnologyCambridge, MA 02139

xiii

Michael S. Beattie Brain and Spinal Injury CenterDepartment of Neurological SurgeryUniversity of California San FranciscoSan Francisco, CA 94110

François BerthodLaboratoire d’Organogénèse Expérimentale (LOEX)Centre de recherche FRSQ du CHA universitaire de QuébecHôpital du Saint-Sacrement, and Département de chirurgieFaculté de médecine, Université LavalQuébec, Canada

Sigurd BervenDepartment of Orthopedic Surgery University of California San FranciscoSan Francisco, CA 94110

David A. BrafmanDepartment of BioengineeringInstitute of Engineering in MedicineUniversity of California San DiegoSan Diego, CA 92093

Jacqueline C. BresnahanBrain and Spinal Injury CenteDepartment of Neurological SurgeryUniversity of California San FranciscoSan Francisco, CA 94110

Christopher K. BreuerInterdepartmental Program in Vascular Biology and Therapeutics Yale University School of MedicineNew Haven, CT 06520

xiv Contributors

Zorica BuserDepartment of Orthopedic Surgery University of California San FranciscoSan Francisco, CA 94122

Shu ChienDepartments of Bioengineering, Cellular and Molecular Medicine,

and Medicine; Institute of Engineering in Medicine University of California San DiegoSan Diego, CA 92093

Karen L. Christman Department of BioengineeringUniversity of California San DiegoLa Jolla, CA 92093

Jeannine M. Coburn Department of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimore, MD 21218

Stephen CurranSmith & Nephew Research Centre, York Science ParkYork YO10 5DF, United Kingdom

Laura E. DickinsonDepartment of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimore, MD 21218

Nathalie DusserreCytograft Tissue EngineeringNovato, CA 94949

Jennifer H. ElisseeffDepartment of Biomedical EngineeringJohn Hopkins UniversityBaltimore, MD 21218

Contributors xv

Adam J. EnglerDepartment of BioengineeringUniversity of California San DiegoLa Jolla, CA 92093

Michael S. FriedmanThermoGenesis CorporationRancho Cordova, CA 95742-6303

Sharon GerechtDepartment of Chemical and Biomolecular EngineeringJohn Hopkins UniversityBaltimore, MD 21218

Deepak M. GuptaDepartment of SurgeryStanford University School of MedicineStanford, CA 94305-5406

Geoffrey C. GurtnerHagey Laboratory for Pediatric and Regenerative MedicineDivision of Plastic and Reconstructive SurgeryDepartment of SurgeryInstitute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanford, CA 94305

Kamran Haghighat Private PracticePortland, OR 97205

Ngan F. HuangDivision of Cardiovascular MedicineStanford University Stanford, CA 94305-5406

xvi Contributors

Corey IyicanCytograft Tissue EngineeringNovato, CA 94949

Nezamoddin N. Kachouie Harvard-MIT Division of Health Sciences and TechnologyCenter for Biomedical Engineering, Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolMassachusetts Institute of TechnologyCambridge, MA 02139

Ali KhademhosseiniHarvard-MIT Division of Health Sciences and TechnologyCenter for Biomedical Engineering, Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolMassachusetts Institute of TechnologyCambridge, MA 02139

Kun-Yong KimDepartment of Genetics, Yale Stem Cell CenterYale University School of MedicineNew Haven, CT 06520

Jennifer KirkhamBiomaterials and Tissue Engineering Group, Leeds Dental InstituteUniversity of LeedsLeeds LS2 9LU, United Kingdom

Sae Hee KoHagey Laboratory for Pediatric and Regenerative MedicineDivision of Plastic and Reconstructive SurgeryDepartment of SurgeryInstitute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanford, CA 94305

Contributors xvii

J. Kent LeachDepartment of Biomedical EngineeringUniversity of California DavisDavis, CA 95616

Nicolas L’Heureux Cytograft Tissue EngineeringNovato, CA 94949

Song LiDepartment of Bioengineering University of California BerkeleyBerkeley, CA 94720-1762

Michael T. LongakerHagey Laboratory for Pediatric and Regenerative MedicineDivision of Plastic and Reconstructive SurgeryDepartment of SurgeryInstitute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanford, CA 94305

Jeffrey C. LotzDepartment of Orthopedic Surgery University of California San FranciscoSan Francisco, CA 94110

Mahdokht Masaeli Department of Electrical and Computer EngineeringNortheastern UniversityBoston, MA 02115

Bradley McAllisterDepartment of PeriodontologyOregon Health Sciences UniversityPortland, OR 97239

xviii Contributors

Todd McAllisterCytograft Tissue EngineeringNovato, CA 94949

Edward McGillicuddyInterdepartmental Program in Vascular Biology and Therapeutics Yale University School of MedicineNew Haven, CT 06520

Allison Nauta Hagey Laboratory for Pediatric and Regenerative MedicineDivision of Plastic and Reconstructive SurgeryDepartment of SurgeryInstitute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanford, CA 94305Georgetown University Hospital, Washington DC 20007

Robert M. NeremThe Georgia Tech/Emory Center (GTEC) for Regenerative MedicineEmory UniversityAtlanta, GA 30322

Jason W. Nichol Harvard-MIT Division of Health Sciences and TechnologyCenter for Biomedical Engineering, Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolMassachusetts Institute of TechnologyCambridge, MA 02139

Nicholas J. PanettaDepartment of SurgeryStanford University School of MedicineStanford, CA 94305-5406

Contributors xix

In-Hyun ParkDepartment of Genetics, Yale Stem Cell CenterYale University School of MedicineNew Haven, CT 06520

Sushmita SahaBiomaterials and Tissue Engineering Group, Leeds Dental InstituteUniversity of LeedsLeeds LS2 9LU, United Kingdom

Rajendra Sawh-Martinez Interdepartmental Program in Vascular Biology and Therapeutics Yale University School of MedicineNew Haven, CT 06520

Toshiharu Shin’okaInterdepartmental Program in Vascular Biology and Therapeutics Yale University School of MedicineNew Haven, CT 06520

Jennifer J. TrowbridgeDepartment of Pediatric Oncology, Dana-Farber Cancer Institute Division of Hematology/Oncology, Children’s Hospital BostonHarvard Stem Cell Institute, Harvard Medical SchoolBoston, MA 02215

Gustavo Villalona Interdepartmental Program in Vascular Biology and Therapeutics Yale University School of MedicineNew Haven, CT 06520

Karl WillertDepartment of Cellular and Molecular MedicineInstitute of Engineering in MedicineUniversity of California San DiegoSan Diego, CA 92093

xx Contributors

David Wood Biomaterials and Tissue Engineering Group, Leeds Dental InstituteUniversity of LeedsLeeds LS2 9LU, United Kingdom

Xuebin B. YangBiomaterials and Tissue Engineering Group, Leeds Dental InstituteUniversity of LeedsLeeds LS2 9LU, United Kingdom

Jennifer L. Young Department of BioengineeringUniversity of California San Diego La Jolla, CA 92093

Behnam ZamanianHarvard-MIT Division of Health Sciences and TechnologyCenter for Biomedical Engineering, Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolMassachusetts Institute of TechnologyCambridge, MA 02139

Contributors xxi

Preface

Cells are the building blocks of tissues and organs. Therefore, cell sourceis a critical issue for tissue engineering. An ideal cell source should be suf-ficient in quantity, compatible with the immune system of the recipientand free of pathogens or contamination. Depending on the specific tissueengineering application, the cell source can be autologous, allogeneic orxenogenic. Traditionally, fully differentiated cell types are used to engi-neer tissues. However, for many cell types, differentiated cells from adulttissues often have little or no proliferation potential.

In the past few years, the advancement of stem cell biology has openeda new avenue for tissue engineering. Stem cells can be isolated from adulttissues, fetal tissues or embryos, are highly expandable, and can bedirected to differentiate into specific cell types. Furthermore, recentbreakthroughs in cell reprogramming make it possible to take tissue biop-sies from patients and reprogram the cells into pluripotent stem cells orspecific cell types such as neurons or cardiomyocytes. This progress hasallowed tissue engineers to have access to unlimited, immune-acceptablecell sources. To fully harness the therapeutic potential of stem cells, weneed to understand how stem cells respond to microenvironmental factorsincluding both biochemical and biophysical cues. This is not onlyrequired for controlling cell fate in vitro, but it is also important for thedesign of scaffolds and tissue constructs that can maximize the recruitment

xxiii

of adult stem cells following implantation. In addition, to cultivate cellsfor clinical applications, quality control and FDA requirements must befulfilled.

Tissue engineering using stem cells is an emerging and fast-growingfield. There is a pressing need for a book that provides a comprehensiveintroduction to the field and summarizes its recent progress. We haveinvited experts in their respective fields to provide insightful reviews ofspecific topics on stem cells and tissue engineering. We hope that thisbook is timely and useful for researchers and students. Chapters 1 to 4 ofthis book introduce tissue engineering (Chapter 1) and discuss differenttypes of stem cells (Chapters 2 to 4). Chapters 5 to 8 discuss the use ofstem cells and biomaterials for the regeneration of cardiac tissue, bloodvessels and the vascular network. Chapter 9 reviews the role of stem cellsin general wound repair. Chapters 10 to 14 focus on skeletal tissue engi-neering, including cartilage, intervertebral disc and bone. Chapters 15 and16 review the use of stem cells to treat spinal cord injury and neurode-generative diseases. Chapters 17 and 18 illustrate state-of-art technologiesused in stem cell engineering, including high-throughput systems andmicrotechnologies. Chapters 19 and 20 discuss quality control and regu-latory issues. Although this book does not have the capacity to cover theuse of stem cells for all tissues and organs, we hope that the general con-cepts and approaches illustrated in this book are helpful for researcherswho are interested in other tissues and organs that are not discussed here.

We thank all the contributors for their hard work and valuable contri-butions. We also thank Joy Quek and the other staff of World ScientificInc. for their tremendous effort in editing and organizing this book.

Song Li, Ph.D.University of California, Berkeley

Nicolas L’Heureux, Ph.D.Cytograft, Inc.

Jennifer Elisseeff, Ph.D.Johns Hopkins University

xxiv Preface

1

Tissue Engineering: From BasicBiology to Cell-Based Applications

Robert M. Nerem

1. Introduction

With the advent of the 21st century, the use of tissue engineering-basedtherapies to treat a variety of diseases and/or injuries has moved frombeing a dream of what might be possible in the future to the realm of theachievable. Even so, there is still much that needs to be done, much stillto be learned; however, it is clear that major advances have been made inthe last two decades.

The term tissue engineering is used here to describe a wide variety ofapproaches. This includes the replacement, the repair, and/or the regener-ation of tissues and organs. Different terms have been used to describe thisharnessing of the intrinsic biological abilities of the human body and ofliving cells. Although research in this general area goes back nearly half acentury and the possibilities of such approaches was described even ear-lier, it was in 1987 that the term tissue engineering was introduced.1 Thenin the 1990s the term regenerative medicine came into use. For some theseterms are interchangeable. For others tissue engineering is used for

1

approaches that are aimed at fabricating substitute tissues outside of thebody that then can be implanted into the body as replacements. For somethe term regenerative medicine means stem cell technology. For thisauthor, however, tissue engineering has a much broader meaning. Thus, tominimize any confusion associated with the choice of terms, it is the termtissue engineering that will be used in this introductory chapter and it willbe used in the broadest sense to include replacement, repair, and regener-ation, i.e. the wide variety of approaches that harness the intrinsicbiological ability of the body and the use of living cells, whether of exoge-nous or endogenous origin.

It should be noted that over the past two decades the industry associ-ated with this field has had its “ups and downs.” There were productsbeing developed in the 1990s, and these were largely skin substitutes. Theleading companies were Advanced Tissue Sciences (ATS) andOrganogenesis (OI). As we entered the 21st century, however, we encoun-tered what might be called “the sobering years.” Both ATS and OI enteredbankruptcy. Today ATS no longer exists, but OI has reinvented itself andis a profitable company. In fact, in the last five years there has been aresurgence of the industry. In 2007, the last year for which there is dataavailable, total industrial activity was US$2.4. billion with more than halfof this being the sale of commercial products.2 For development stagefunding the largest component is that of stem cells.

Whatever the approach being used in tissue engineering, a criticalissue is the source of the cells to be employed. This thus will beaddressed in the next section. One possibility of course is to use stemcells, and since the early reports of human stem cells a decade ago3–7

there has been a surge of activity. As stem cells and tissue engineeringare the focus of this book, a brief introduction to stem cells isprovided. To employ stem cells in a cell-based therapy will require,however, the translation of the basic benchtop science to the variety ofapplications that are possible. This is an area that has been largelyoverlooked, certainly not addressed to the extent necessary, and in thenext to last section of this introductory chapter the issues that need tobe addressed as one moves from the basic stem cell biology researchto applications will be briefly discussed. The chapter then ends withsome concluding comments.

2 R. M. Nerem

2. Cell Source

Whatever the tissue engineering approach, whether it be one of replace-ment or that of repair and regeneration, a critical issue is that of cellsource, i.e. from where will the cells come that are to be employed in thetreatment or therapy. In addressing this issue, there are several questionsthat need to be asked. These are as follows.

• Will the source of cells be endogenous or exogenous?• Will one use undifferentiated stem cells, progenitor cells, or fully

differentiated somatic cells?• Will one employ an autologous cell strategy or an allogeneic or even

xenogeneic strategy?• Are there differences associated with the age of the donor or with the

disease state?• Are there sex differences that must be taken into account?

Let us consider these one by one.

To start with, is the strategy one of recruiting cells from within thepatient or one using an exogenous source? If the former, then the approachis an autologous one, and the challenge is how to recruit the cells. If thelatter, then one must proceed to a series of additional questions.

This book focuses on stem cells; however, it also includes the use ofprogenitor cells that in fact can be derived from stem cells. Furthermore,and as will be discussed in the next section, there are different types ofstem cells, e.g. embryonic versus adult, and these may be different in theirability to be differentiated into the particular type of cell to be used in thetherapy. Even starting with a stem cell, however, does one use the stemcell directly in the therapy, does one use a progenitor cell derived from thestem cell, or does one use a fully differentiated cell?

One question for any clinical therapeutic strategy is that of autolo-gous vs. allogeneic vs. xenogeneic cells. Although the use of autologouscells is attractive from the viewpoint of immunogenicity, the use ofautologous cells does not in general provide for off-the-shelf availabil-ity to the clinician. Why is off-the-shelf availability important? Forsurgeries that must be carried out on short notice, e.g. following a heart

Tissue Engineering and Stem Cells 3

attack, off-the-shelf availability of the cells to be employed in thetherapy is essential; however, even when the time of surgery is elective,one can argue that only with off-the-shelf availability will the widerpatient population that is in need be served. There is of course oneexception to the above generalization, and this is if the cells to beemployed are to be recruited from within the body of the patient. In thiscase what is needed by the clinician off-the-shelf is in fact not the cellsthemselves, but perhaps only an acellular implant to be used in recruit-ing the cells and/or to serve as a target for the cells. In contrast,allogeneic cells or even xenogeneic cells do provide for off-the-shelfavailability. Here the challenge is that of immunogenicity. The problemof achieving immune acceptance with xenogeneic cells is particularlysevere; however, even for allogeneic cells for at least some cell types,e.g. vascular endothelial cells, a strategy for creating immune accep-tance would have to be used.

Finally, there are the questions of differences due to age, due to thedisease state of the patient, or due to the sex of the donor. Although largelyunexplored, these can be significant issues, and it is important that futureresearch addresses these questions. For example, there is a report thatpatient characteristics affects the number of human cardiac progenitorcells that will be available.8 There also is evidence that the disease statecan have influence. In this case an example is that in patients with coro-nary artery disease there was observed a functional impairment of thehematopoietic progenitor cells.9

There also are sex differences in the basic characteristics of cellswhether they be stem cells, progenitor cells, or fully differentiated cells.This is a very important area, one which gives rise to a variety of ques-tions. For example, if the cells are to be cultured, are different culturingprotocols required for female cells as compared to male cells? For theclinical therapy itself, will the outcome be different depending on sex ofthe donor versus the sex of the patient? In this latter case, there are reportsin the literature documenting differences.10,11

There thus are a variety of questions to be answered, and although thereis developing a rich literature, further research is required. In the nextsection, however, we move on to a very brief discussion of stem cells.

4 R. M. Nerem

3. Stem Cells

As noted earlier, since the early reports in the late 1990s there has been asurge of activity and this increases at an ever accelerating level. Furtherdetails will be found in the literature12,13 and in subsequent chapters. Thusthis section of this introductory chapter will only attempt to provide abroad and very brief overview of the different types of stem cells andother pluripotent cells available for use in tissue engineering. From anoverall point of view, however, one may consider three general types ofstem cells as follows.

3.1 Embryonic stem cells (ESCs)

These can be isolated from the inner cell mass of pre-implantationembryos during the blastocyst stage.3,4 They have the ability to differenti-ate into virtually all specialized cell types and thus are consideredpluripotent. They also have the ability to proliferate in an undifferentiatedstate, i.e. they have the ability to self renew. Since different human ESClines have been derived from different embryos, it is not surprising thatdifferent lines will exhibit different gene expression characteristics.14

Within this general category of stem cells there are in addition to thosederived from embryos, those called embryonic germ cells (ECGs). Theseare derived from the gonadal ridge of a fetus that is five to ten weeks old,5–15 and these are primordial germ cells that in vivo give rise to eggs orsperm in the adult.

3.2 Induced pluripotent stem (iPS ) cells

These iPS cells are the result of the transformation of an adult, somaticcell through reprogramming into a pluriopotent stem cell.16,17 Althoughpluriopotent, these cells are not necessarily identical to ES cells eventhough they are similar. The reprogramming initially has been throughretroviral transfection although there are a number of efforts in progressto carry out the reprogramming without the use of transfection. LikeESCs, iPS cells can lead to teratoma formation.


3.3 Adult stem cells

Adult stem cell populations have been found in many tissues of the humanbody. They are believed to be important to the repair mechanism intrinsicto many tissues and organs. They also in general are tissue specific; how-ever, there are some exceptions. One of the exceptions to the believedtissue specificity of adult stem cells is the mesenchymal stem cell.6,7,18,19

This type of adult stem cell is derived from bone marrow stroma. In fact,one could view bone marrow transplantation as the earliest cell-basedtherapy. In vitro the MSC can differentiate into a variety of cell types. Itis thus viewed as multipotent, but not pluriopotent or totipotent. Anotherexception to the general specificity of adult stem cells are the amniotic-fluid and placental derived stem cells.20 They have been shown to have thecapacity for self renewal like ESCs, can give rise to numerous cell types,and have been characterized as having properties somewhere in betweenthose of ESCs and adult stem cells. Finally, adult stem cells may be foundin adipose tissue21 and also in the umbilical cord.22,23

There clearly is much more that needs to be done to understand thecharacteristics of these different stem cell types and the factors involvedin determining the differentiation pathway down which a stem cell can bedirected. Just as the functional characteristics of a fully differentiated cellis orchestrated by a symphony of signals, the same can be said for thefate of a stem cell. This symphony includes soluble molecules, cell-cellcontact, and the substrate/extracellular matrix to which the cell is adher-ent. It also includes what is of particular interest to this author and that isthe role of physical or mechanical forces in modulating stem cell behav-ior. This includes the role of the physical force environment in theregulation or modulation of stem cell fate. As an example, we haveshown that mouse ESCs in an early state of differentiation and whenexposed to laminar flow and the associated shear stress exhibit an upreg-ulation of endothelial cell phenotypic markers.24 This suggests that suchphysical forces, acting as part of a stem cell’s microenvironment, canparticipate in the direction of the differentiation process, perhaps evenaccelerate it. Taking a different approach, it has been demonstrated thatmechanical stain inhibits the differentiation of human ESCs.25 Geometryis a different physical characteristic that can influence stem cell

6 R. M. Nerem

differentiation. This has been demonstrated for MSCs.26 Interestingly,there also are reports that the mechanical properties of the extracellularmatrix can influence stem cell fate.27 There also is the role of epigeneticsin the regulation of stem cell fate. An example of this epigenetic role isthe regulation of gene expression through histone modification or DNAmethylation.28–30 Thus, there are a variety of ways in which the microen-vironment can orchestrate stem cell fate, and we know very little abouthow to design the symphony of signals so as to optimize the outcome ofthis orchestration.

4. From Benchtop Science to Cell-Based Applications

If in fact stem cells are to be employed in a particular therapy or treatment,then one must address the translation of the benchtop research through aprocess that will result in the number of cells required for a specific appli-cation and one that will achieve regulatory approval.31,32 There are anumber of aspects that must be considered, and in December 2008Georgia Tech hosted a workshop on Stem Cell Biomanufacturing, bring-ing together a group of industry and academic researchers as participants.Some of the issues identified are as follows.

• The sourcing and isolation of the stem cell.• The monitoring of stem cell phenotype.• Possible inhomogeneity in the starting population.• The expansion and propagation of the cells.• The control of stem cell fate.• Methods to assess genetic and epigenetic stability.• Meaningful real time in-process assays.

All of this would need to be done with the quality control requiredfor regulatory approval. This leads one to the concept of an automatedcell processing facility. To develop such a facility would requireresearch that leads to a knowledge base for process design. This wouldinclude having the ability to measure process variables in real time andexperiments designed to determine functional relationships betweenprocess variables and what might be called product quality. There would


need to be robust strategies for the interrogation and evaluation of thevariables affecting the processing of cells. There also would need to bean implementation of closed loop control methods. All of this wouldneed to be supported by the use of multivariable statistical analysis todetermine the variables affecting product quality. In addition, mathe-matical models relating the product and the process variables wouldneed to be developed.

This of course means a different type of research on stem cells thanwhat today is largely appearing in the literature. If we are to translate allthe exciting advances taking place in our understanding of stem cells toapplications including patient therapies, however, this is what will berequired.

5. Concluding Comments

Tissue engineering, including replacement, repair, and regeneration,offers the hope that in the future we will be able to develop new therapiesand treatments. This will be particularly important for diseases andinjuries where currently there are no adequate treatments available. A crit-ical issue is that of cell source, and here the variety of stem cells availablehave the potential of providing the answer. For each type of stem cell,however, there are issues that need to be addressed. Also, beyond the basicscience there also will need to be research aimed at understanding how tooptimize the processing of stem cells. Only with the combination ofresearch on basic stem cell biology and research on stem cell processingwill it be possible to translate the basic benchtop science into future appli-cations and patient therapies.

Acknowledgments

The author acknowledges the support provided by the GeorgiaTech/Emory Center for Regenerative Medicine and his part-time visit-ing professorship at Chonbuk National University in Jeonju, SouthKorea. He also thanks his colleagues and students who have taught himso much.

8 R. M. Nerem

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2

Recent Advances and FuturePerspectives on Somatic Cell

Reprogramming

Kun-Yong Kim and In-Hyun Park

1. Introduction

Embryonic stem (ES) cells are pluripotent cells derived from the innermass of mammalian blastocysts. They have the capacity to self-renew,while maintaining pluripotency: the ability to generate all cell types of thethree germinal layers. Therefore, a variety of clinical applications havebeen proposed for ES cells, as well as in vitro studies of basic diseasemechanisms, screens for drug discovery, and tissue engineering for degen-erative diseases. However, ES cells are generic cells that are unrelated tothe patient requiring treatment and the usage of human embryonic stemcells continues to be a contentious ethical issue.

Embryonic development and cellular differentiation are presumedto be unidirectional processes and cells undergo a progressive loss ofpluripotency during cell fate specification. In the 1950s, classical exper-iments demonstrated that differentiated cells retain the geneticinformation required to revert to pluripotency1 and attempts were madeto generate pluripotent stem cells functionally equivalent to ES cells from

13

differentiated cells (reprogramming) — including by somatic cell nucleartransfer, fusion of differentiated cells with pluripotent cells, application ofpluripotent stem cell extracts to somatic cells, and direct in vitro adapta-tion of germ cells.2 Ultimately in 2006, Takahashi and Yamanaka showedthat enforced expression of four transcription factors, Oct4, Sox2, Klf4and c-Myc reprogrammed murine somatic cells to pluripotency.3 After thisdiscovery, reprogramming drew enormous attention not just from stemcell biologists, but also from clinicians, bioengineers, geneticists, anddevelopmental biologists. In this chapter, we review recent advances inthe reprogramming field and discuss the potential future applications ofinduced pluripotent stem (iPS) cells.

2. Nuclear Reprogramming

Briggs and King showed that nuclei from Rana pipiens blastula cells havethe ability to undergo normal cleavage and develop into completeembryos when transplanted into enclosed oocytes.1 This was the firstdemonstration that the oocyte cytoplasm contains factors that can repro-gram the nuclei of differentiated cells back to a pluripotent state that allowdonor cells to normally divide and develop into complete embryos. Thisfinding was extended by a study by Gurdon and colleagues, who reportedthat even fully differentiated intestinal cells from Xenopus could be repro-grammed by frog oocytes.4 Although the efficiency was very low, theseoocytes could give rise to adult animals indicating that the pluripotentstate could be reacquired. Somatic cell nuclear transfer (SCNT) was notconfirmed in other species until 1997, when Dolly the sheep was cloned.5

The discovery of reprogramming by SCNT led to further advances in theunderstanding of the epigenetic processes governing self-renewal anddifferentiation in murine and human ES cells.

Several groups demonstrated that fusing a somatic cell with an ES cellcould induce pluripotency.6,7 This reprogramming can be accomplishedwhen embryonal carcinoma (EC) cells, embryonic germ (EG) cells, or te-ratocarcinoma cells are fused with somatic cells. At the tetraploidy stage ofthe ensuing cell, the molecular program of the original somatic cell can beerased and epigenetically reprogrammed to that of an ES cells. With SCNTmethods, reprogramming to a pluripotent state occurs within a few days and

14 K.-Y. Kim and I.-H. Park

can be greatly enhanced by co-expression of pluripotency-associated genessuch as Oct4, Sox2, c-Myc, Klf4, Nanog, and Sall4. Yet, each of these meth-ods has clinical limitations. SCNT has the advantage of using intrinsicreprogramming machinery in oocytes, but it requires the donation of humanoocytes that can be in limited supply and raise ethical concerns. In somaticcell fusion, there are no ethical problems and no limitations on the supplyof cells, but the processes result in the formation of the undesired tetraploidthat is genetically unstable, and the overexpression of transcription factorsincreases the frequency of tumorigenesis in the resulting progeny.

In recent years, nuclear and cytoplasmic extracts from undifferentiatedcells have been tried to induce the pluripotency of differentiated somaticcells. Extracts from pluripotent EG, EC cells or ES cells that contain the reg-ulatory components needed for reprogramming can induce the pluripotencyof somatic cells.8 The reprogramming extracts reversibly permeabilize thesomatic cell and then induce transcriptional reprogramming, leading to epi-genetic modification of the somatic cells. Even interspecies reprogrammingvia cell extracts showed some success in de-differentiating somatic cells.The extracts of regenerated new limbs induced partial dedifferentiation ofc2c12-derived mouse myotubes into myoblasts and the extracts of Xenopuseggs induced the expression of the pluripotency genes Oct4 and Sox2 inmammalian somatic cells.9,10 When fibroblast cells were reversibly perme-abilized and transiently exposed to extracts from mouse EC cells, they leadto Oct4 biphasic activation. Incubation of extracts from mouse ES cells withreversibly permeabilized NIH3T3 cells induced partial reprogramming.This approach to inducing pluripotency using pluripotent cell extracts couldbe an excellent alternative to nuclear transfer reprogramming due to the factthat eggs are not required and the resultant cells are diploid. However, theintrinsic difficulty to continue the exposure of the somatic cells to pluripo-tent cell extract holds yet the successful derivation of completelyreprogrammed pluripotent stem cells.

Hence, a variety of studies have been proposed to better understand thereprogramming of pluripotent stem cells. However, the principal limita-tion to all the above technologies for cell reprogramming is that thegenerated pluripotent stem cells are unrelated to the patient who requiresclinical treatment, and the use of human ES cells remains a contentiousethical issue.

Reprogramming and iPS Cells 15

3. Reprogramming by Defined Factors

Reprogramming somatic cells by using a defined set of factors is a novelconcept and opens extraordinary possibilities for producing pluripotentstem cells without raising the ethical concerns associated with destroyinghuman embryos.5,6 while allowing for patients to be treated with theirown reprogrammed somatic cells. In their original report, Takahashi andYamanaka retrovirally transduced different combinations of 24 candidatereprogramming genes into mouse embryonic fibroblasts derived fromtransgenic mice containing a neomycin resistance gene knocked into theFbx15 locus.3 Cells that recovered neomycin resistance from the reactivationof the reporter gene were selected. They found that the retrovirus-mediatedintroduction of transcription factors was sufficient to reprogram mousefibroblasts back to a pluripotent state. Specifically, the combined intro-duction of the four transcription factors, Oct4, Sox2, Klf4, and c-Mycwere identified as sufficient to give rise to pluripotent cells, termedinduced pluripotent stem (iPS) cells.

Selected iPS cells showed properties very similar to murine ES cells inmorphology, proliferative characteristics, and expression of pluripotencymarkers. Moreover, iPS cells differentiated into all three germ layersin vitro, and were able to form teratomas when injected into nude mice,confirming their pluripotency. Although Fbx15-selected iPS cells formedchimeras, they did not result in germ line transmission, raising the concernthat iPS cells are not truly equivalent to ES cells. However, several studieshave reported that iPS cells selected using Nanog or Oct4 are moreepigenetically related to ES cells and can produce chimeras capable ofgerm line transmission.11,12 However, these iPS cells did not pass the moststringent pluripotency test: tetraploid complementation. Albeit negligible,the continuous expression of reprogramming genes seems to be responsi-ble for the limited differentiation potential of the iPS cells, since the iPScells produced by inducible lentiviral reprogramming factors succeeded togenerate full-term mouse after tetracomplementation.13,14

After the identification of murine iPS cells was reported, using thesame four transcription factors, Oct4, Sox2, Klf4 and c-Myc or combinedwith novel factors, Nanog and Lin28, three different groups isolatedhuman iPS cells.15–17 Human iPS cells from either combination of factors


shared defining characteristics with human ES cells, including geneexpression profiles, morphology, proliferation, patterns of DNA methyla-tion and histone modification, as well as telomerase activities. Human iPScells were also able to differentiate into three germ layers in vitro asembryonic bodies and to form teratomas after injection into immunedeficient mice.

4. Recent Advances in Reprogramming Methods

When the first iPS cells were isolated, many questions regarding the tech-niques to generate iPS cells were raised, such as how to identify iPS cells,how to derive iPS cells without potentially tumorigenic oncogenes, andhow to generate iPS cells without using retro/lentivirus. Over the lastthree years, there have been many advances that have begun to overcomethese hurdles.

Initially, Takahashi and Yamanaka created the first iPS cells using theneomycin resistant gene regulated by the pluripotent cell specific Fbx15locus and claimed that reprogramming was a very inefficient process.3

Selection tools seemed essential to identify the iPS cells, and Nanog orOct4 promoter-based drug resistant genes were used to help isolatemurine iPS cells.11,12,18 Lately, murine and human iPS cells were derivedby using the morphologies unique to ES cells.15–17 Furthermore, silencingof the retro- and lentivirus could be retrospectively used to identify thereprogrammed cells from the partially reprogrammed or transformedcells.19 The induction of the gene that mediates retroviral silencing in EScells, Trim28, seems to be responsible for retroviral silencing duringreprogramming in iPS cells.20

The usage of oncogenenic c-Myc and Klf4 in generating iPS cells is amajor hurdle for potential clinical applications. One of the four repro-gramming factors, c-Myc, is a well-established oncogene, and its continuedexpression and/or potential reactivation in iPS cell derivatives is not suit-able for future iPS cell therapies. Indeed, head and neck tumor formationhas been observed in iPS cell-derived chimeric mice possibly due to thereactivation of c-Myc.11 However, c-Myc is dispensable for reprogram-ming murine and human somatic cells, although its absence significantlyreduces the reprogramming efficiency.21 Some somatic cells highly express


endogenous reprogramming genes and may not need the expression of allreprogramming factors. Indeed, neural progenitor cells (NPCs) that highlyexpress endogenous Sox2, and can be reprogrammed without the ectopicexpression of Sox2.22 Furthermore, even introduction of Oct4 alone wasshown to be sufficient to generate iPS cells from NPGs.23,24 Similarly,meningiocytes and keratinocytes appear to be particularly prone to repro-gramming due to their relatively high endogenous expression of Sox2,Myc and Klf4.25,26 These results indicate that the reprogramming cells likeNPCs would require a minimal genetic modification in reprogrammingand result in most desirable safe iPS cells.

Limitation to the current methods in generating patient specific iPScells is the residual presence of the reprogramming factors in chromo-some. Retro/lentiviral integration into the genome carries a risk of tumorformation when random integration activates pathways for cell prolifera-tion or inhibits the tumor suppressor pathways. Using methods based onnon-integrating vectors or direct exposure to reprogramming proteins isdesirable. Stadtfeld and colleagues were able to generate iPS cells fromadult mouse fibroblast and liver cells using non-integrating adenoviralvectors.27 They demonstrated that adenoviral-mediated transient expres-sion of the exogenous reprogramming factors eliminated the risk ofinsertional mutagenesis. Okita and colleagues also successfully generatediPS cells without using any viral vectors but multiple transient transfec-tion of the reprogramming factors.28 Lately, oriP/EBNA1-based episomalvector was successfully used to generate human iPS cells.29 All the non-integrating vector methods provide a safer iPS cells but suffer from theextremely low reprogramming efficiency.

Usage of three or four transcription factors for reprogramming resultsin the high incidence of multiple integration of reprogramming factors intochromosome. Combining the reprogramming factors with picornaviral 2Asequences allowed the expression of multiple genes in one backbone.30,31

This method generated iPS cells through less number of retro- or lentiviralinsertion into the genome, as opposed to using separate viral vectors foreach reprogramming factor. Because the single viral copy may alsobe removed from the iPS cell genome after reprogramming (e.g. byloxP/Cre technology) the authors successfully generated safer iPS cells.32

Similarly, Kaji and colleagues developed a piggyBac transposon-based


vector expressing four genes enabling the creation of transgene-free iPScells following removal of the transposon.33 Despite these advancements,the concern lingers that all of these factors have links to tumorigenesis.

An alternative approach that allows the complete avoidance of the useof oncogenes altogether is to use small molecules instead. Shi et al. gener-ated iPS cells from NPCs by transduction of Oct4 and Klf4, and found thatsimultaneous treatment with G9a inhibitor, BIX-01294, remarkablyincreased the reprogramming efficiency.34 Subsequent studies haveconfirmed that epigenetic modification or the activation of self-renewingsignaling through small molecules can improve reprogramming.Additionally, the histone deacetylase (HDAC) inhibitor valproic acid (VPA)and Trichostatin A (TSA), as well as the DNA methyltransferase inhibitor,5-aza-cytidine have been shown to increase reprogramming efficiency.35,36

The Wnt signaling component WNT3a, or the L-channel calcium channelagonist Bayk8644 (BayK) also can increase the reprogrammingefficiency.22 In another study, the inhibition of mitogen-activated protein(MAP) kinase signaling and synthase kinase-3 (GSK3), allowed repro-gramming to occur, even in the absence of Sox2 and c-Myc.37 Recently,tumor suppressor pathways, including p53, Ink4a/Arf and p21, were shownto play a role as a barrier to reprogramming.38–41 These several signalingpathways are well known to facilitate the ES cell self-renewal or cellproliferation and seem to increase the reprogramming efficiency.

Ultimately, iPS cells have been generated without using vectors at all,but rather by directly introducing proteins into fibroblasts. Zhou andcolleagues purified recombinant reprogramming factors fused with thepoly-arginine (i.e.11R) protein transduction domain of the C-termini andwere able to generate iPS cells from mouse embryonic fibroblasts.42 Kimand colleagues established 293T cell lines stably expressing reprogrammingfactors fused with the poly-arginine tag and demonstrated the formation ofhuman iPS cells by exposure of the cell extract to fibroblasts.43

5. Future Perspectives on Reprogramming and iPS Cells

Dr. Yamanaka’s reports of generating iPS cells using defined factorsrevolutionized the approach to manipulating the cellular identity. Initiatedby the finding that fibroblasts became induced to the myogenic lineage by


expressing a given myogenic factor,44 dedifferentiation or reprogrammingto different cellular fates have been explored, including hematopoietic,pancreatic and cardiac lineages.45–47 Pluripotent iPS cells generated viareprogramming will have a broader applicability, since they can differen-tiate into any cellular lineage. They can be used to investigate the diseaseprogression in vitro, and to provide a platform to screening chemicals forgenetic disorders (Fig. 1). Recent advancement of high throughputsequencing, when combined with iPS cells, will allow us to investigate theearly development of genetically defined, personalized cells in vitro.48

After human ES cells were isolated, they have been used to makemodels of human diseases.49,50 Human ES cells genetically modified byeither overexpression or knock-down of genes of interest was proposed tomimic the early embryonic cells of human diseases in vitro. In vitro differ-entiation of ES cells were presumed to follow the early developmentalprogram of normal embryonic development.51 iPS cells derived directly


Fig. 1. Recent advances and future perspectives of reprogramming. Since the Yamanakalab reported the generation of iPS cells in murine fibroblasts, there has been an explosionof research on factor-based reprogramming. 1) iPS cells were generated without usingintegrating retro- and lentiviral vectors. Small molecules were identified that increasedreprogramming efficiency. 2) Reprogrammed iPS cells will be used to generate in vitrodisease model. 3) In vivo transdifferentiation will be a practical alternative to pluripotentstem cells for genetic or non-genetic degenerative diseases. Shown in red are challengesthat will lead to successful utility of the iPS cells in regenerative medicine.

from patients provide an in vitro model that will more closely mimic thepathology of the disease. Following are examples of the rigorous attemptsto generate human disease models using murine and human iPS cells.

Murine iPS cells have been used to illustrate the therapeutic potentialof iPS cells in vivo. For example, Hanna and colleagues showed that trans-genic mice engineered to have human sickle cell anemia could besuccessfully treated with hematopoietic progenitor cells produced fromautologous iPS cells and genetically repaired.52 Wernig et al. showed thatiPS cell-derived neuronal cells could integrate into fetal brains and ame-liorate the symptoms of rats with Parkinson’s disease.53 Since then, morestudies have reported the derivation of therapeutically relevant cell typesfrom iPS cells, including human insulin-secreting cells, and functionalmouse and human cardiomyocytes, as well as mouse endothelial cells thatsuccessfully treated mice with coagulation disorder hemophilia A.54–56

Human iPS cells from patients’ fibroblasts were generated to investi-gate human diseases in vitro. Our group has generated iPS cells derivedfrom a variety of genetic disorders, including adenosine deaminasedeficiency-related severe combined immunodeficiency (ADA-SCID),Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD)type III, Duchenne (DMD) and Becker muscular dystrophy (BMD),Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1diabetes mellitus (JDM), Down syndrome (DS)/ trisomy 21 and the car-rier state of Lesch-Nyhan syndrome.57 iPS cells generated from patientsof neuromuscular diseases, including amyotrophic lateral sclerosis(ALS) and spinal muscular atrophy (SMA), were shown to differentiateinto motor neurons, providing a novel in vitro motoneuron diseasemodel.58,59 A range of iPS cells from Parkinson’s patients was also gen-erated as an invaluable resource for studying the disease.60 These cells donot contain transgenic reprogramming factors in their genomes as thefactors have been excised by Cre recombinase from the integratedlentiviral vector. Recently, Lee and colleagues extended the iPS celltechnology to model autosomal recessive familial dysautonomia (FD)in vitro, and demonstrated the FD-related mis-splicing of IKBKAP andconcurrent defects in neurogenic differentiation and migration behavior.61

Most of iPS cells used to model human diseases still contained the retro-viral vectors in their chromosomes. With the advent of reprogramming


methods not relying on integrating virus, iPS cells are expected to bemore close to ES cells and make better disease models.43

Although it is highly hoped to utilize iPS cells in various applicationsmentioned above, there are several issues to be resolved (Fig. 1). First ofall, methods to generate clinically useful iPS cells should be improved.Non-integrating virus, direct protein transduction, and nuclear transferwill make genetically non-modified iPS cells. But, their reprogrammingefficiency is extremely low, and will not be practical to produce iPS cellsin a reliable manner. Improvement in viral vectors, or screening smallmolecules will be essential to optimize the reprogramming methods.62,63

In order to reach the final goal of regenerative medicine using pluripo-tent stem cells, it is crucial to make them acquire the desired lineagein vitro. Directed differentiation of pluripotent stem cells relied on the pre-vious knowledge of cell specification obtained from developmentalbiology.51 Treatment of cytokines, growth factors, chemicals, and smallmolecules have been attempted to differentiate pluripotent stem cells.Ectopic expression of lineage specific transcription factors is a reliableapproach to direct the differentiating cells into specific lineage.64,65 Cellsurface markers were used to isolate the cells of interests. When mouse EScell lines expressing fluorescent proteins of lineage specific markers areavailable, they provide an effective way to isolate pure cell population.The isolation of lineage specific cells from human ES cells was facilitatedby the transgenic human ES cell lines with lineage markers. Teratomaformed by the incompletely differentiated pluripotent stem cells continu-ously impedes the utility of cells differentiated from ES cells.Improvement of negative selection of partially or non-differentiated cells,or rigorous isolation of completely differentiated cells are required.In vivo transdifferentiation will be an alternative to iPS cells of great prac-tical importance. Most of the degenerative disorders show loss inparenchymal or functional cells in tissues: loss of endocrine beta cells intype I diabetes, dopaminergic neurons in Parkinson’s diseases andmotoneuron in ALS. Despite the degeneration of these cell types, the stro-mal or adjacent cells surrounding cells are still intact. When expressedwith a combination of genes important for the development of the samelineages of the degenerated cells, the surviving cells in the damaged tis-sue will change their cellular fate to the cells of the interest and will help


to recover the function. Given the great opportunity of manipulating cellfates, it becomes possible to conquer the devastating diseases, but thereare still much to overcome.

Acknowledgments

We thank Brian Adams for discussion and critical reading for the manuscript.

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3

Hematopoietic Stem Cells

Jennifer J. Trowbridge

1. Introduction

The mammalian hematopoietic system is a dynamic organ, containingmature cells that function in the processes of adaptive and innate immu-nity, blood clotting, and oxygen transport. These mature cells have alimited lifespan and are replenished by various blood lineage-specific pro-genitor cells, which in turn are replenished by hematopoietic stem cells(HSCs). Thus, it is the HSC population that serves as the foundation of thesystem, giving rise to all mature hematopoietic lineages throughout thelifetime of an organism. To perform this specialized function, HSCs havetwo concomitant properties that make them unique; the ability to differ-entiate to give rise to all mature hematopoietic cell types and the unlimitedability to divide to produce daughter HSCs that maintain the stem cellpool, a process termed self-renewal.

HSCs were the first stem cells to be identified, by Till, McCulloughand Siminovitch, in 1963.1,2 Over the last 46 years, a great number of ele-gant studies in the field of HSC biology have characterized the timeline ofemergence and locations of these cells during mammalian development,the combination(s) of cell surface antigens that can be used to identify

31

and/or prospectively isolate HSCs, and have developed advanced in vitroand in vivo assay systems to reliably evaluate the function of these cells.As our understanding of the biology of HSCs has progressed, so haveclinical therapies utilizing these specialized cells. The first successfulbone marrow (BM) transplants as treatment strategies for hereditaryimmunodeficiency and acute leukemias were performed in the late 1960sand early 1970s (reviewed in Ref. 3). Now, forty years later, transplanta-tion of HSCs is being developed and/or successfully applied towardhematological diseases, hereditary metabolism disorders, congenitalimmunodeficiency, diseases of the central nervous system, solid tumorsand lower extremity ischemic disease.4

Solutions to the immediate and future challenges faced in clinical HSCtransplantation will depend on developments in the field of tissue engi-neering, including methods to expand available sources of HSCs and todifferentiate pluripotent cell lines into HSCs for clinical utility. In thischapter, we will discuss available sources of HSCs (adult, fetal andembryonic), current and future clinical applications of HSCs, and thechallenges that can be addressed by tissue engineering.

2. Hematopoietic Stem Cell Sources

2.1 Adult

The first HSC source used in clinical therapy was adult BM, the site inwhich HSCs were first identified and isolated from.1 As clinical BM trans-plantation has been a reality for approximately 40 years, a large amountof clinical data has been amassed that clearly demonstrates the successesand limitations of this therapeutic strategy (Table 1). More recently, it wasdiscovered that HSCs can be mobilized from the BM into the peripheralblood by stimulation with hematopoietic growth factors5 or myelosup-pressive therapy.6 Mobilized peripheral blood (M-PB) has been usedsuccessfully in lieu of BM to reconstitute hematopoietic and immunefunction in patients, with the major advantages that it is a less invasiveharvesting method and typically results in shorter engraftment time, thusreducing complications.

One of the major limitations in allogeneic BM or M-PB transplantation,in which the donors are non-identical to the patients, is the availability of

32 J. J. Trowbridge

histocompatible donors. Typically, suitable donors are family membersfully matched with the patient in expression of human leukocyte-associatedantigens (HLA) or differ in one HLA antigen only.7 Establishing large,international donor volunteer registries has made progress toward a solu-tion for the lack of compatible donors. A second limitation/risk to considerin use of BM or M-PB for transplantation is the common complication ofgraft-versus-host disease (GVHD), where the immune cells from thedonor BM or M-PB recognize the patient’s cells as foreign and mount animmunologic attack. Treatment of GVHD involves immune suppression,potentially raising the risk of infection and, in cancer therapy, risk ofrelapse of disease.

2.2 Fetal

Umbilical cord blood (UCB), blood that remains in the umbilical cordafter childbirth, has been widely accepted as a rich source of HSCs. Since

Hematopoietic Stem Cells 33

Table 1. Sources of Hematopoietic Stem Cells.

Advantages Limitations

AdultBone marrow Greatest amount of historical Invasive harvestPeripheral blood clinical data Cannot expand HSCs

Rapid engraftment Requires histocompatibledonor

Risk of GVHDFetalUmbilical cord blood High concentration of HSCs Limited HSCs per unitPlacenta Low/no risk to isolate Cannot expand HSCs

Less stringent HLA Delayed engraftmentrequirements

Lower risk of GVHDImmediately available through

bankingEmbryonicEmbryonic stem cells Unlimited proliferative capacity No clinical dataiPS cells “Patient specific” Currently cannot reliably

produce HSCs

the first human UCB transplant performed 20 years ago,8 more than10,000 UCB transplants have been successful in both pediatric and adultpatients (reviewed in Ref. 9). The placenta itself has more recently beenshown in mouse10,11 and human12 to be a potent source of HSCs, however,this source has yet to be clinically utilized. Importantly, cord blood bankshave been established worldwide for the collection and cryopreservationof UCB for allogeneic transplantation. This bank represents an importantdistinction and advantage over the donor BM registry, that is, UCB can befrozen and banked for future thaw and immediate use while adult BMmust be freshly harvested for transplant (Table 1).

UCB, and potentially the placenta, represent valuable alternativesources for patients who require HSC transplantation but have no readilyavailable HLA matched donor, as this type of transplant permits a greaterdegree of HLA mismatching without an unacceptably high incidence ofGVHD.13 Patients and UCB units can differ in two HLA antigens, how-ever, the required cell dose must be more than 2.5 × 107 cells perkilogram of the patient.14 This caveat, the available cell dose per unit ofUCB, greatly limits the broader use of UCB transplantation in adultpatients. Recent developments to overcome this cell dose issue includetransplanting two UCB units per patient,15,16 intrabone injection of UCBcells, and ex vivo expansion of HSCs, which will be discussed furtherbelow.

2.3 Embryonic

Human embryonic stem (ES) cells, first derived by James Thomson et al.in 1998,17 have received a large amount of attention owing to their greatpotential in regenerative medicine. These cells are derived from the innercell mass of early human blastocysts, can be expanded and propagatedindefinitely in culture, and have the potential to differentiate to generatecells of any tissue type. Ethical controversy surrounding the use of humanblastocysts to derive ES cell lines has been bypassed by the discovery thatit is possible to derive induced pluripotent stem cells (iPS cells) from adulthuman fibroblasts.18,19 Methods to reprogram adult cells into embryonic-like cells have been reliably reproduced by many groups and offer thepotential of patient-specific cellular therapies. While the therapeutic

34 J. J. Trowbridge

potential of iPS cells is great, it remains unclear as to the precise similar-ity of these cells to human ES cells. As such, continuation of studiesutilizing both of these cell types, preferably in parallel, is ideal. A recentchange in the US federal policy to permit federal funding for a greaternumber of human ES cell lines will certainly accelerate momentum in thisfield.20

The ability to differentiate human ES and iPS cells into HSCs forclinical use would have unique advantages, including the ability to engi-neer these cells to be drug resistant, allowing more specific and effectiveadministration of chemotherapy in cancer treatment, and would permitestablishment of hematopoietic chimerism to promote immune accept-ance of other tissues derived from the same source of pluripotent cells.20

While in principle, using human ES or iPS cells as a cell replacementsource for HSCs and other lineages is a thrilling prospect, it is decep-tively simple. The feasibility of using these cells in a clinical setting isunclear and we do not yet understand how to differentiate these cells ina controlled and precise manner21 (Table 1). Clearly, human ES and iPScells are able to spontaneously develop into cell types representing allthree germ layers in vivo, as shown by teratoma formation, but the cellu-lar and molecular mechanisms controlling these processes areincompletely understood.21 Thus, attempts to direct differentiation ofhuman ES cells into HSCs in vitro have amounted to very limited successand heterogeneous results over the past 25 years.22 Certain human EScell-derived hematopoietic cells are observed to have HSC-like proper-ties, however, these cells do not represent functional HSCs capable oflong-term, clonal, multilineage hematopoietic reconstitution of mice orlarger animal models.23–26

While some groups have developed protocols to differentiate humanES cells into more mature hematopoietic lineages, including erythroid,27–30

myeloid31,32 and lymphoid cells,33–35 few have demonstrated that these pro-tocols actually direct cells to the hematopoietic fate as opposed to creatingconditions permissive to spontaneous differentiation or expansion of spon-taneously differentiated hematopoietic cells.21 Development of methods toreliably and efficiently create HSCs from human ES or iPS cells will rep-resent a significant leap towards their clinical use. This will depend ondevelopment of culture techniques to more closely mimic the in vivo


microenvironment needed to stimulate hematopoietic specification ofpluripotent stem cells, as discussed further below.

3. Applications

Clinical HSC transplantation has classically been utilized for replace-ment of hematopoietic cells that have otherwise been damaged by aparticular genetic condition, disease, or exposure to chemotherapy orradiation. A long history of clinical success in treatment of conditionssuch as hereditary immunodeficiency, leukemia/lymphoma, and aplasticanemia has been documented. Use of HSC transplantation to replaceother cell types, such as in cerebral palsy, type I diabetes, neurologicaldamage, endocrine disease, and spinal cord injury, has recently been pro-posed and debated (reviewed in Ref. 12). The possibility of using HSCsin non-hematopoietic cell replacement therapy was bolstered a number ofyears ago by several reports demonstrating remarkable regenerativepotential of HSCs in animal models of heart infarct,36 stroke,37 spinalcord injury38 and liver damage.39 A contrasting study has definitivelydemonstrated that transplantation of a single HSC was able to robustlyreconstitute the hematopoietic system of mice but did not contribute tonon-hematopoietic tissues including the brain, kidney, gut, liver or mus-cle.40 This work suggests that differentiation of circulating HSCs and/ortheir progeny into non-hematopoietic organs is an extremely rare event,if it occurs at all.

In resolving these seemingly conflicting reports, it is an interesting andtempting hypothesis to consider that the role of HSCs or their progeny inrepair of damaged organs is not necessarily due to direct differentiation,but rather the release of trophic factors that are able to stimulate repair. Infact, observed beneficial effects of UCB transplantation include reducedinflammation, protection of nervous tissue from apoptosis and nerve fiberreorganization.41 UCB transplantation for type I diabetes, cerebral palsy,brain injury and endocrine disease is in the process of being transitionedfrom the lab to the clinic, with numerous patients being treated in clinicaltrials (reviewed in Ref. 41). The clinical benefit of such therapies, and themechanism behind them, remain to be discovered. Certainly, clinical HSCtransplantation for hematopoietic replacement will continue to achieve

36 J. J. Trowbridge

greater and greater success as we improve transplant protocols andmethods to expand sources of HSCs.

4. Challenges for Tissue Engineering

There are a number of challenges faced in clinical HSC transplantationthat have great potential to be addressed and solved by tissue engineering,including methods to expand currently available sources of HSCs andmethods to differentiate pluripotent stem cells (human ES and iPS cells)into HSCs for clinical utility. One of the major issues that directly appliesto both of these applications surrounds the ideal method for evaluatingand enumerating HSCs. It has been well established in the field of HSCbiology that these cells are most rigorously defined functionally, throughdemonstration of their ability to self-renew and differentiate into all bloodlineages in vivo. The NOD/SCID mouse xenotransplant assay, wherebyhuman HSCs are evaluated for their ability to reconstitute the blood sys-tem of non-obese diabetic/severe combined immunodeficient mice, hasbecome widely accepted as a surrogate assay for human HSCs. Morerecently, it has become clear that mice with greater degrees of immunod-eficiency are more sensitive hosts for human HSC engraftment. Thus, it isdebated whether the xenotransplant assay is a true measure of human HSCfunction as opposed to a measure of immunologic escape from rejection.9

Primate studies suggest that the cells which contribute to NOD/SCIDreconstitution represent short-term repopulating cells rather than trueHSCs.42 Likely, novel methods developed to expand human HSCs asshown in mouse xenotransplant models will need to be confirmed inhigher-order primates prior to their use in clinical therapy.

4.1 HSC expansion

HSCs require a complex microenvironment to retain their stem cell prop-erties. In the BM, HSCs are surrounded by bone matrix and cells includingosteoblasts, mesenchymal stem cells, fibroblasts and adipocytes, each ofwhich produces various cytokines and growth factors. Ultimately, the goalof HSC expansion is to mimic this microenvironment in order to obtainsufficient numbers of HSCs to reconstitute long-term hematopoiesis from


adult, fetal, or embryonic sources (Fig. 1). Different culture medias,growth factors and supplements have been widely tested.43 Since the early1980s, many hematopoietic cytokines were reported to be able to expandHSCs in vitro without significant alteration of their primitive activity, how-ever, it is now clear by xenotransplant assay that traditional culture ofHSCs compromises their in vivo potential.4 In addition, early attempts tomove these preclinical results to patients failed to show improvements inengraftment time after HSC transplantation.44 Clearly, it is difficult to gen-uinely simulate the hematopoietic microenvironment in vitro and culture ofHSCs has historically introduced defects that promote apoptosis,45 disrupthoming of HSCs to the BM,46,47 and exhaust their self-renewal capacity.48

In consideration of novel systems and methods for HSC expansion,ex vivo HSC culture should be a closed bioprocess to avoid contaminationand should attempt to achieve the greatest level of expansion of HSCswith the lowest level of manipulation. Traditional static cultures (includingflasks and plates) may not be the most suitable, as they do not provide a

38 J. J. Trowbridge

Fig. 1. Current strategies to mimic the bone marrow microenvironment for the purposesof expanding HSCs ex vivo. Strategies include the use of modified cytokine cocktails/soluble growth factors, co-culture on supporting cell layers, and the use of bioreactors.HSC: hematopoietic stem cell, MSC: mesenchymal stem cell.

proper three-dimensional environment to achieve nutrient and oxygen dis-tribution.43 Recently, a static bioprocess consisting of two gas permeableculture bags, separated by a magnetic system to eliminate undesired cells,was shown to promote expansion of human HSCs.49 A number of othernew protocols for HSC expansion are in development, involving modifiedcytokine cocktails, co-culture methods and bioreactors (Fig. 1). Mostex vivo culture conditions include the cytokines interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF),and Flt-3 ligand (FLT3L).50 In terms of novel cytokines to add to thiscocktail, angiopoietin-like 5 (ANGPTL5) and insulin-like growth factorbinding protein 2 (IGFBP2) have recently been shown to expand humanUCB HSCs as assayed by NOD/SCID xenotransplant.51 Presently, severalgroups are testing tetraethylenepentamine (a copper chelator), histonedeacetylase inhibitors and DNA methylation inhibitors in human HSCculture systems, with promising results.52–54 In addition to cytokines andpharmacological inhibitors, many studies have defined key signalingpathways that regulate normal development and expansion of HSCsin vivo, including Notch, Wnt/β-catenin, Hedgehog, and BMP signaling(reviewed in Ref. 55). This knowledge will also be important to apply inthe development of protocols to expand HSCs ex vivo.

Co-culture of human HSCs with a supporting layer of cells provides analternative strategy to liquid culture-based expansion, in an attempt tomimic cellular networks, direct cell-cell contacts, and soluble regulatoryproteins found in vivo. In vitro and in vivo studies have shown that thesesupport cells (also called stromal cells) can provide signals to control pro-liferation, survival, and differentiation of HSCs.56 Mesenchymal stem cells(MSCs) provide the most popular source of supporting cells. MSCs can beisolated from a variety of fetal and adult tissues,55 and have been used inpreclinical models as a method to maintain HSCs in a primitive state.9

Finally, bioreactors offer a modern, tissue engineering-based approachto model the hematopoietic microenvironment with a relatively stableoxygen concentration, pH and nutrient supply. Diverse bioreactors withspecific characteristics have been designed for ex vivo HSC expansion,including stirred and perfusion systems, rotating wall vessels, and thosethat minimize shear stress (reviewed in Ref. 43). With the growing rangeof clinical applications of HSCs and lack of efficient tools to expand them,


novel developments in ex vivo culture and tissue engineering offer greatpromise for the future of clinical HSC expansion.

4.2 Differentiation of pluripotent stem cells to HSCs

As highlighted above, the differentiation of human ES or iPS cells intoHSCs remains a technical challenge. In mouse ES cells, this challenge hasbeen met with some success through overexpression of the genes Cdx4and HoxB4,57,58 which permit derivation of a limited number of functionalHSCs (Fig. 2). Unfortunately, however, this strategy does not directly

40 J. J. Trowbridge

Fig. 2. Protocol for generating mouse HSCs from pluripotent stem cells, includinginduced expression of Cdx4 and HoxΒ4, and co-culture on the supportive stromal cell lineOP9.57,58 ESC: embryonic stem cell, EB: embryoid body, GFP: green fluorescence protein.

apply to human ES cells. Current progress in differentiation of HSCs fromhuman ES cells have relied on co-culture with supportive cells,24,25 for-mation of embryoid bodies,23 or these methods in combination. Clearly,the limited success of the current differentiation strategies indicates thatkey signals necessary for full developmental maturation of cultured ES oriPS cells into HSCs must be missing.

New experimental approaches toward hematopoietic differentiation frompluripotent stem cells are desperately needed. These may include use ofsome the methods described above for HSC expansion, however, there areseveral distinctions from HSC expansion studies that need to be taken intoaccount. One important consideration is the potential biological differencebetween human ES and iPS cells, which may be reflected in their ability todifferentiate into HSCs and/or the signals required to induce this. It has beensuggested that iPS cells, as they are derived from adult tissues, have a greatercellular memory and as such may be more amenable to directed differentia-tion into HSCs or other specific cell types.59 A second, critical considerationis that residual undifferentiated ES or iPS cells pose a risk of teratoma for-mation in patients. Thus, differentiation protocols need to be highly efficientin formation of HSCs, or must include steps to purge residual undifferenti-ated ES or iPS cells from HSCs. In emphasis of this point, it has been foundthat unfractionated mouse ES cells, following differentiation by co-culturewith OP9 stromal cells and injection into the fetal liver of monkeys, causedteratomas in the thoracic or abdominal cavities of all recipients.60

The ability to generate fully functional human ES or iPS-derivedHSCs capable of long-term multilineage reconstitution of animal modelsand ultimately patients remains a challenge and will depend upon devel-opments in tissue engineering as well as further understanding of theintrinsic gene regulation and extrinsic environmental cues that controlthese developmental processes in vivo.

Acknowledgments

Thank you to Dr. Stuart Orkin for mentorship, and Drs. Jonathan Snowand Marc Kerenyi for critical review. Financial support from theLeukemia and Lymphoma Society, Children’s Leukemia ResearchAssociation and Elsa U. Pardee Foundation.


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4

Mesenchymal Stem Cells for TissueRegeneration

Ngan F. Huang and Song Li

1. Introduction

In the past two decades, significant progress has been made in the field ofstem cell research. An important finding is that adult stem cells harborgreater regeneration potential and plasticity than what was previouslythought. This discovery has led to tremendous interest in developingmethods to direct stem cell differentiation into lineages for the therapeu-tic delivery into diseased or dysfunctional tissues. Among the adult stemcells, mesenchymal stem cells (MSCs) are a promising therapeutic cellsource due to the ease of isolation, high proliferative capacity, and multi-potency.1 MSCs can be found in numerous tissues of the adult mammaland can be harvested in a reproducible manner. Since large quantities ofthese cells can be cultured in vitro, it is feasible for MSCs to be delivereddirectly in vivo or incorporated into tissue engineered constructs beforetransplantation. These two approaches have been explored for treating awide range of diseases or traumatic events, including myocardial infarc-tion, peripheral arterial disease (PAD), spinal cord injury and skin wounds.

49

By developing robust methods of differentiating MSCs into therapeuticcells of interest and organizing the cells into functional three-dimensionaltissues, it may be possible to fulfill the potential of MSCs for clinical use.This review aims to provide an overview of some therapeutic applicationsfor MSCs in tissue engineering and regenerative medicine.

2. MSC Sources and Phenotype

MSCs can be generally defined as adherent and elongated cells that residein mesenchymal tissues and can self-renew as well as produce progenywith more specialized function. MSCs have been observed and purifiedfrom numerous origins, including bone marrow, adipose tissue, skeletalmuscle, blood, liver spleen and dental pulp.2–8 Among them, bone marrowand adipose tissue are perhaps most characterized origins of MSCs, andthis review will focus on MSCs derived from these two sources.

2.1 Bone marrow MSCs

Although MSCs account for only 0.01% among total nucleated cells in thebone marrow, they have over million-fold expansion capability and mul-tilineage differentiation potential.1,9 Bone marrow MSCs are easilyharvested by aspiration from the iliac crest. Phenotypically, there is nounique marker that specifically identifies bone marrow MSCs.Consequently, MSCs are characterized based on the positive expression ofnumerous cell surface antigens such as CD29 (integrin β1), CD44 (recep-tor for hyaluronic acid and matrix proteins), CD105 (endoglin), andCD166 (cell adhesion molecule).10 On the other hand, they do not expressmarkers typically associated with hematopoietic lineage, such as CD14(monocyte surface antigen), CD34 (hematopoietic stem cell surface anti-gen) and CD45 (leukocyte surface antigen).1 Due to the differences incharacterization methods, the International Society for Cellular Therapyrecommended the designation of these cells as multipotent mesenchymalstromal cells and proposed the following minimal criteria for MSCdesignation: adherence to plastic dishes; phenotypic expression of CD105,CD73 and CD90; lack of surface molecule expression of CD45, CD34,CD14 or CD11b, CD79α or CD19 and class II major histocompatibility

50 N. F. Huang and S. Li

complex antigen (HLA-DR); and differentiation capacity into osteoblasts,adipocytes and chondroblasts in vitro.11

Based on the cell surface antigens, bone marrow MSCs can be isolatedusing fluorescence activated cell sorting (FACS) or magnetic activatedcell sorting (MACS). Other methods to purify MSCs include Percoll gra-dient centrifugation and their selective adherence onto tissue-culturetreated Petri dishes. To maintain their proliferative capacity, the purifiedbone marrow MSCs can be expanded in culture media containing definedserum-free components (i.e. StemPro® MSC Serum Free Medium,Invitrogen, Carlsbad, CA) or pre-screened fetal bovine serum. Underthese growth conditions, bone marrow MSCs can be cultured for morethan eight passages.

2.2 Adipose-derived stem cells (ASCs)

According to the nomenclature established by the International FatApplied Technology Society, ASCs are adipose-derived MSCs that areadherent in plastic culture dishes.12 These cells were first reported byresearchers who showed that cells dissociated from collagenase-treatedhuman adipose tissue gave rise to multipotent cells that can differentiateinto the cells of adipose tissue, cartilage and bone.13 These cells were laterconfirmed to give rise to other cell types, including endothelial cells(ECs), smooth muscle cells (SMCs) and cardiomyocytes.14–16 To isolateASCs, adipose tissue derived from liposuction is digested with collage-nase and then centrifuged to separate the stromal population in the lowerlayer of the pellet from the adipocytes in the upper layer.17 Like bone mar-row MSCs, ASCs can be purified based on the expression profile ofsurface marker antigens using FACS or MACS. ASCs can be maintainedin defined serum-free media (i.e. MesenPRO RS Media, Invitrogen, CA)as well as serum-containing media.

Although ASCs and bone marrow MSCs have greater than 90% simi-larity in immunophenotype,18 there are some reported differences insurface antigen expression. For example, CD14 and HLA-DR wasreported to be absent in bone marrow MSCs, but have been identified inearly passage human ASCs at low frequency.19 Furthermore, ASCs appearto have temporal changes in immunophenotype with subsequent

Mesenchymal Stem Cells for Tissue Regeneration 51

passaging.19 However, these differences in immunophenotype could alsobe attributed to differences in species or the methods of isolation, purifi-cation, or detection.

3. Differentiation of MSCs in vitro

For tissue engineering and regenerative medicine applications, one strat-egy is to differentiate the cells in vitro into lineages of interest beforedelivering them in vivo for therapeutic treatment. Bone marrow MSCsand ASCs have been shown to differentiate into a variety of lineages,including myogenic, osteogenic, chondrogenic, and adipogenic lineages.20

A number of strategies to direct their differentiation have been used,including the use of soluble factors, mechanical stimulation, extracellularmatrix (ECM) factors and genetic engineering approaches, which arebriefly discussed below.

One of the most commonly used strategies to induce differentiation isby the addition of soluble factors, such as growth factors and small mole-cules. Using soluble factors, bone marrow MSCs and ASCs have a highpropensity to differentiate into cells of mesenchymal lineage, includingbone, adipose tissue and cartilage. Osteogenic differentiation canbe induced by culturing the cells in the presence of dexamethasone, ascor-bic acid, and β-glycerophosphate, whereas adipogenesis is enhanced by1-methyl-3-isobutylxanthine, dexamethasone, insulin, and indomethacin.1

Chondrogenic growth factors include transforming growth factor-β3(TGF-β3) and bone morphogenetic protein-6 (BMP-6).21 Besidesosteogenic, adopogenic, and chondrogenic lineages, MSCs and ASCshave been shown to differentiate towards other lineages at lower yields.For example, platelet-derived growth factor (PDGF) and TGF-β3stimulate smooth muscle phenotype,22 whereas 5-azacytidine treatmentinduced the formation of cardiac-like cells that expressed cardiac markersβ-myosin heavy chain, desmin and α-cardiac actin.23

Besides soluble factors, mechanical stimulation is another potent reg-ulator of cell behavior and function. Physiologically, mechanicalstimulation plays an integral role in cell phenotype. For example, bloodvessels experienced fluid shear stress and cyclic strain due to the pulsatileflow generated by the beating heart, and bone is subjected to compressive


loading from gravitational forces. Due to the importance of mechanicalfactors for physiological maintenance, the role of mechanical factors onMSCs and ASCs differentiation has become an area of research interest.We and others have shown that uniaxial strain promotes smooth muscledifferentiation of bone marrow MSCs when cell orientation was restrictedby microgrooves.24,25 In contrast, for chondrogenesis, the application ofcyclic mechanical compression to human bone marrow MSC embeddedin biodegradable hyaluronan–gelatin composite scaffolds promotessignificant upregulation of chondrogenic markers type II collagen andaggrecan.26

Another method of inducing MSC differentiation is using ECMs,which are biological scaffolding proteins that provide structural integrityas well as molecular cues that regulate cell behavior and function.27,28 Thebiophysical and biochemical cues transmitted by the ECM to cellsinclude matrix rigidity, matrix patterning and matrix composition.Physio-logically, matrix rigidity varies throughout different tissues of thebody, from soft tissues of the brain to hard tissues in the bone. For exam-ple, Engler et al. showed that physiological rigidity dictated the humanbone marrow MSC differentiation.29 When cultured on polyacrylamidegels of varying rigidities, the MSCs differentiated to osteogenic lineageon rigid matrix (34 kPa), but soft (0.1–1 kPa) gels promoted neurogene-sis. Besides matrix rigidity, the spatial pattern of the ECM also regulatesMSC differentiation by restricting cellular shape. For example, small(1024 μm2) micropatterns of fibronectin stimulated human bone marrowMSCs to differentiate towards adipogenic lineage and retain a roundedmorphology, whereas large (10,000 μm2) islands supported osteogenicdifferentiation and adherent morphology.30 Finally, the matrix composi-tion may also influence MSC differentiation. There is evidence thatMatrigel could enhance neuronal differentiation of MSCs.31 For example,porcine bone marrow MSCs cultured in three-dimensional patches ofpolyethylene glycol (PEG)-modified fibrin showed EC phenotype.32

These studies suggest that the ECM plays a dynamic role of modulatingMSC behavior and phenotype.

Besides soluble, mechanical, and ECM factors in the microenviron-ment, intracellular factors also play a role in inducing differentiation.MicroRNAs (miRs) are short endogenous nucleotide RNAs that


post-transcriptionally regulate gene expression. In addition to modulatingbiological processes such as cell proliferation or cell death, they haverecently been shown to direct stem cell fate lineage by negatively regu-lating gene expression.33,34 For example, overexpression of miR-21increased adipogenesis of human ASCs by decreasing TGF-β signalingpathway member TGFBR2 at the mRNA and protein levels.35 Conversely,inhibiting miR-21 reduced adipogenesis but led to an increase in TGFBR2protein levels. Another group demonstrated that miR-148b, miR-27a, andmiR-489 modulate osteogenesis in human bone marrow MSCs, even inthe absence of osteogenic inducing media.36

Together, these results suggest that MSC and ASC differentiation arecontrolled by multiple microenvironmental cues and intracellular signal-ing. Further studies to elucidate the mechanism of microenvironmentalfactors on differentiation will be critical for directing cell differentiationwith high purity for tissue engineering and regeneration purposes.

4. Tissue Engineering and Regeneration Using BoneMarrow MSCs and ASCs

MSCs and ASCs are promising stem cell candidates for the repair orregeneration of diseased tissues, and they have been frequently utilized inin situ or in vitro tissue engineering approaches to repair various tissues,4

including the repair and regeneration of blood vessels, bone, teeth, skele-tal muscle, teeth, and spinal cord (Fig. 1). For the in situ approach, thecells and other factors are delivered in vivo, and the therapeutic potentialof the cells is dependent on the interactions of cells and host environment.For the in vitro approach, cells and matrix/scaffolds are used to engineerfunctional tissue constructs prior to implantation. Here we illustrate thesedifferent approaches of tissue engineering for the regeneration of theheart, blood vessels, skin and cartilage (Table 1).

4.1 In situ tissue regeneration

4.1.1 Cardiac repair

Cardiovascular disease remains as one of the leading causes of mortality inthe United States. Over 70 million people in the US are symptomatic from


or at risk of cardiovascular disease.37 Stem cell-based approaches to repairor regenerate the heart after myocardial infarction are promising. The goalof stem cell therapy is to regenerate cardiac muscle, enhance angiogenesisand ultimately improve cardiac function. The methods of delivering thetherapeutic cells to the heart include systemic delivery, regional coronaryinfusion, or local myocardial injection.38 In one study, autologous swinebone marrow MSC were directly injected into the infarcted heart, andMSCs differentiated towards myogenic lineages as early as two weekspost-injection. Four weeks after cell delivery, the extent of aneurismal thin-ning and contractile dysfunction were significantly reduced in thecell-treated group.39 Besides direct injection, regional and systemic infu-sion of bone marrow MSCs has also been demonstrated. When99mTc-labeled rat bone marrow MSCs were transfused to infarcted rathearts either into the left ventricular cavity or by intravenous delivery, the


Fig. 1. Application of MSCs for tissue regeneration.

intravenous delivery approach resulted in cell localization predominantlyto the lungs and to a lesser degree to the heart.40 In contrast, regional deliv-ery produced better retention of cells within the heart, especially in theinfarct region, and a lower uptake in the lungs.

Besides delivery of naive cells, MSCs and ASCs have also been genet-ically modified to improve cell survival or therapeutic effect. In ratmodels of myocardial infarction, the local injection of rat bone marrowMSCs overexpressing prosurvival factor Akt1 led to reduced intramy-ocardial inflammation, along with restoration of normal systolic anddiastolic function.41 In another study, rat bone marrow MSCs that over-expressed anti-apoptotic gene bcl-2 were injected into infarct region. Thistreatment increased cell survival, decreased infarct scar and promotedneovascularization to assess their survival, engraftment, and functionalimprovement after myocardial infarction.42 These results suggest thatgenetic modification of MSCs can improve the therapeutic outcome.


Table 1. Overview of Tissue Engineering and RegenerationApplications Using Bone Marrow MSCs or ASCs.

Organ/Tissue Treatment References

Heart Cell delivery 40, 41, 63Cell delivery in ECM 46, 64

Blood vessel Cell delivery 47–49Cell delivery in ECM 22, 50–52

Skeletal muscle Cell delivery 65, 66Cell delivery in ECM 67, 68

Bone Cell delivery 69, 70Cell delivery in ECM 71, 72

Cartilage Cell delivery 73, 74Cell delivery in ECM 61, 62

Skin Cell delivery 75, 76Cell delivery in ECM 55, 56

Teeth Cell delivery 77, 78Cell delivery in ECM 79, 80

Spinal cord Cell delivery 81, 82Cell delivery in ECM 83, 84

Another approach to cardiac repair is to co-inject therapeutic cells withECMs. ECMs alone such as collagen, fibrin, alginate, and matrigel canenhance neovascularization in the infarcted heart or attenuate infarct scarthinning.28,43–45 The rationale behind the co-injection of cells with ECMs isthat the ECMs may provide therapeutic enhancement of cardiac functionand angiogenesis, while also providing structural support to localize thetransplanted cells to the site of delivery. For example, we showed that co-injection of human bone marrow MSCs with fibrin significantly enhancedneovasculature formation following chronic myocardial infarction.46

4.1.2 Peripheral vascular repair

PAD is typically due to atherosclerotic occlusive disease of the peripheralarteries of the limbs, causing symptoms such as intermittent claudication,painful ischemic ulcerations, and limb-threatening gangrene.37 A featureof PAD is dysfunction or damage to the vascular endothelium, thediaphanous layer of ECs that exerts control over vascular reactivity,remodeling and angiogenesis. Cell-based approaches to restore or regen-erate the endothelium so as to enhance the angiogenic response toischemia hold promise for the treatment of PAD.

MSCs and ASCs have been examined in the setting of vascular repairfor PAD, with the goal of generating and incorporating new functionalvessels into existing vessels to remodel them. In one study, mice thatunderwent hindlimb ischemia, an experimental model of PAD, weretreated with adductor muscle injections of murine bone marrow MSCs,mature ECs or culture medium.47 The animals treated with MSCs demon-strated significant improvement in limb blood perfusion and attenuationof muscle fibrosis after 21 days, compared to the other treatment groups.The cells did not appear to be incorporated into the mature collateral ves-sels, but appeared to increase the levels of VEGF and basic fibroblastgrowth factor (bFGF) in the adductor muscle, suggesting a paracrinemechanism of enhancement. Besides bone marrow MSCs, ASCs havealso shown therapeutic benefit for the treatment of hindlimb ischemia.When human ASCs were delivered into immunodeficient mice either at oneor seven days after ligation of the femoral artery, the cell-treated animalsdemonstrated significant improvement in blood perfusion after 21 days.48


In a comparative study of the therapeutic efficacy between human bonemarrow MSCs and ASCs, equal numbers of cells were injected intramus-cularly into the ischemic limb of immunodeficient mice.49 Controlanimals received saline injection only. Two weeks after cell transplanta-tion, laser Doppler blood perfusion imaging showed significantly higherimprovement in the ASC-treated group, compared to the bone marrowMSC and control groups. To explore the mechanism of the difference intherapeutic effect, the authors reported that ASCs expressed higher levelsof matrix metalloproteinases (MMPs) than bone marrow MSCs. The ther-apeutic effect of ASCs was mitigated when ASCs were transfected withMMP3 or MMP9 siRNA prior to transplantation into the ischemic limb,suggesting that MMP3 and MMP9 were involved in the proangiogeniceffect of ASCs. In summary, these studies demonstrate that MSCs andASCs significantly improve limb blood perfusion and are promising can-didates for treatment of PAD.

4.2 In vitro tissue engineering

4.2.1 Tissue engineering of blood vessels

Bypass surgeries are often used to treat obstructed coronary and peripheralarteries. However, synthetic vascular grafts with small diameter (<6 mm)have high failure rate because of frequent clogging. Consequently, atissue-engineered vascular graft is a promising solution to this problem.MSCs can differentiate into SMCs and secrete growth factors to recruitECs, and thus are a potential cell source for constructing vascular grafts.Interestingly, vascular grafts seeded with bone marrow MSCs haveimproved patency, which is attributed to the antiplatelet adhesion propertyof heparan sulfate proteoglycans on the surface of bone marrow MSCs.50

By engineering biochemical and mechanical factors (i.e. matrix pro-teins, soluble factors, and cyclic strain) in a bioreactor, the proliferationand differentiation of bone marrow MSCs can be controlled in vasculargrafts.22 In vivo studies have also shown that bone marrow MSCs can dif-ferentiate into SMCs and promote endothelialization.51 However, theelectrophysiological profile of bone marrow MSC-differentiated SMCsappeared to be different from that of mature SMCs, suggesting that only


partial differentiation of MSCs into SMCs were achieved. Interestingly,vascular grafts seeded with bone marrow mononuclear cells also havehigh patency.52 Whether bone marrow-derived MSCs or stromal cells andASCs can replace SMCs and ECs as a cell source for vascular graft con-struction need further investigation.

4.2.2 Tissue engineering of biological skin equivalentsfor wound repair

Skin wounds result from various conditions, including burns, traumaticaccidents, or disease. Standard treatments for wound repair include autol-ogous or cadaveric skin transplantations, but these methods face thepotential risks of donor site morbidity and transmission of diseases. Analternative approach is tissue engineered skin equivalents for replacingthe damaged skin. Physiologically, skin is comprised of a stratified andkeratinized epidermis that physical protects the body, and below the epi-dermis is the dermal layer that provides strength and support.53 For thetreatment of wound injuries or disease, tissue-engineered skin replace-ments provide an off-the-shelf alternative that can minimize the potentialcomplications of disease transmission or tissue harvesting. The desirableskin equivalents should quickly adhere to the wound, mimic the physio-logical function and mechanical properties of normal skin, acceleratewound healing, and resist immune rejection.54

Tissue engineering of skin equivalents using bone marrow MSCs andASCs are promising. In a porcine wound healing model, engineered skinconstructs composed of autologous bone marrow MSCs seeded in collagen-glycosaminoglycan polymer scaffolds were grafted onto partial thicknesswounds.55 After four weeks, the wound contraction was measured as themovement of wound edges towards the center of the wound. In compari-son to the no graft treatment group, the cell-seeded scaffolds contracted to57% of the original area and the acellular scaffold group contracted to51%, which was significantly better than the no treatment control groupthat contracted to 20% of the original area. Histologically, the MSCs per-sisted in the epidermis and dermis up to four weeks, suggesting that thecells migrated from the scaffold to the neo-epidermis and dermis. In com-parison to the acellular scaffold group after four weeks, the MSC-treated


group showed significant enhancement of vascular density. This data sug-gested that MSC-seeded biologically scaffolds could reduce woundcontraction and improve neovascularization. In addition to promotingneovascularization, ASCs also participate in wound healing by differenti-ating into vascular lineages. Engineered constructs consisting of humanASCs cultured in decellularized cadaveric dermal matrix were graftedonto full thickness excisional wounds of immunodeficient mice for up tofour weeks.56 The results showed that ASCs delivered in the dermal matrixpersisted locally to the site of delivery, improved wound healing, and dif-ferentiated into endothelial and epidermal lineages. Together, thesestudies suggest that engineered skin grafts containing bone marrow MSCsor ASCs accelerate wound healing, improve neovascularization, and maydifferentiate into vascular and epidermal lineages.

4.2.3 Cartilage tissue engineering

Articular cartilage plays an important physiological function of lubricat-ing between diarthrodial joints and distributing mechanical loads. Due tothe avascular nature of articular cartilage, any damage or disease can bedifficult to heal because of limited regeneration capacity. Current treat-ments to promote cartilage repair include subchondral abrasion,microfracture, transplantation of osteochondral plugs and autologouschondrocyte transplantation, but these approaches do not successfullyrestore long lasting healthy cartilage.57,58 As a result, tissue engineering isa promising alternative. MSCs and ASCs are candidate cell sources forengineered cartilage because of their high expansion ratio and propensityto differentiate towards chondrogenic lineages.

A number of studies have investigated the role of engineered cartilagetissue constructs using MSCs or ASCs. In one study, the chondrogenicpotential of human ASCs was tested in three-dimensional ECMs consist-ing of alginate, agarose or gelatin.59 In the presence of chondrogenicmedia containing TGF-β, the content of cartilage matrix such as hydroxy-proline and sulfated glycosaminoglycans significantly increased with timefor all three ECMs. To test their in vivo differentiation potential, humanASC-like cells were grown in alginate gels and transplanted subcuta-neously into nude mice for up to 20 weeks.60 Histological analysis


demonstrated that the ASCs formed cartilage and maintained their pheno-type without evidence of hypertrophy. Cartilage formation was verified byhistological stains for cartilage ECM as well as by gene expression andWestern blot characterization for collagen II, SOX9, cartilage oligomericprotein and the cartilage-specific proteoglycan aggrecan.

In addition to synthetic or naturally-derived ECMs for cartilage repair,decellularized cartilage ECM is another potential candidate because italready contains the structural and functional elements of native cartilage.To test the efficacy of decellularized ECM for the repair of cartilagedefects, acellular cartilage-derived matrix was generated by decellulariz-ing bovine articular cartilage, grinding the matrix into fine powder, andthen remolding the matrix into porous three-dimensional cylindrical con-structs.61 Rabbit bone marrow MSCs were seeded into the decellularizedECM and then transplanted into full thickness osteochondral defects inrabbits for up to 12 weeks. Based on histological assessment, the cell-treated group had significantly better cartilage quality at 12 weeks,compared to the acellular treatment group. Furthermore, the cell-treatedgroup showed significant improvement at 12 weeks, compared to sixweeks, which suggested temporal changes in chondrogenic repair. In arelated study using ECMs derived from human cadaveric joints andcanine bone marrow MSCs, the cell-seeded constructs were deliveredsubcutaneously into nude mice for four weeks.62 The results demonstratedthat the engineered constructs formed ectopic cartilage-like tissue,with evidence of collagen II deposition and positive safranin O staining.In summary, these studies illustrate the potential of cartilage tissueengineering using bone marrow MSCs or ASCs.

5. Future Directions

In summary, bone marrow MSCs and ASCs are candidate cell sources fortissue engineering and regenerative medicine because of their ease andreproducibility of isolation, high proliferation capacity and ability to differ-entiate into therapeutic cell lineages. However, substantial barriers will needto be overcome before these cells become a standard treatment for clinicalcare. These challenges include determining the optimal conditions for cellexpansion, enhancement in cell viability and function in vivo, elucidation


of the mechanisms underlying their repair and regeneration ability, andimmune acceptance for allogeneic transplantation. Nevertheless, someclinical trials are already underway (http://www.clinicaltrials.gov). Asthese limitations become resolved, it is anticipated that bone marrowMSC and ASC therapy will fulfill their promise of clinical efficacy.

Acknowledgments

This work was supported in part by grants from the National Institutes ofHealth: HL953552 (a postdoctoral fellowship to N.F.H.) and HL083900(to S.L.).

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5

Delivery Vehicles for DeployingMesenchymal Stem Cells

in Tissue Repair

Michael S. Friedman and J. Kent Leach

1. Introduction

Cell-based therapies are a promising alternative to organ transplantation.Numerous cell sources are under investigation for their efficacy inpromoting or contributing to the repair and regeneration of human tissues.Over the past decade, MSCs have attracted much attention in the cell ther-apy and regenerative medicine fields. Unlike more mature cells, MSCscan be expanded through several passages without loss of differentiationpotential, have multi-lineage potential, possess potent anti-inflammatoryand immunomodulatory properties, and can be isolated from numeroustissues throughout the body.

While the systemic injection of MSCs has shown some promise as atreatment for immune-mediated disease, their use in tissue regenerationapplications has met with little clinical success. Recent work indicatesthat MSCs fail to engraft long-term, and that any therapeutic benefit

71

derived from MSCs is through transient tissue residence and secretion oftrophic factors. However, many of these studies fail to integrate cellulartherapy with tissue engineering approaches for deploying MSCs in theappropriate physiologic context with the relevant microenvironmental andmechanical cues. In this chapter, we will discuss some of the biomaterialsthat have been utilized to deploy mesenchymal stem cells in tissue repairand regeneration applications.

2. Delivery of MSCs for Repairing Cardiovascular Tissues

Cardiovascular disease is the leading cause of death in the United States,and treatment of vascular disease accounts for a large proportion of healthcare expenditures. There is a vast shortage of viable cardiac tissues andgraft segments available for transplantation due to disease and morbidity.Cell-based approaches to engineering cardiovascular structures representan exciting alternative to traditional treatment options. In the paragraphsbelow, we will summarize several recent investigations that havedeployed MSCs using various biomaterials to produce new blood vesselsand repair damaged myocardium.

2.1 Engineering long-lasting blood vessels

The presence of a robust vasculature is critical to supply nutrients andcirculating factors to developing and engineered tissues, as well as thosetissues undergoing repair. The feasibility of engineering microvascularnetworks in vivo has been shown using human umbilical vein endothelialcells (HUVECs), human microvascular endothelial cells (HMVECs), andmore recently using cells differentiated from human embryonic stemcells.1 However, these leaky vessels are rapidly pruned and remodeledwithout stabilization by pericytes. MSCs are an ideal cell populationto promote anastamosis and long-term stability due to their secretionof trophic factors, potential for differentiation toward the myogenicphenotype, and in vivo identity and function as pericytes2,3 (Fig. 1). Thepromise of MSCs has ushered in an exciting era of implanting co-culturesor accessory cells to form new tissues using injectable and implantablematerials.

72 M. S. Friedman and J. K. Leach

Hydrogels derived from natural or synthetic polymers are a preferredmaterial for implanting cellular populations that facilitate neovasculariza-tion due to their mechanical properties and injectable capacity. Hydrogelscomposed of natural materials are advantageous, as they commonlydegrade into easily metabolized degradation products. However, there issignificant lot-to-lot variability during their preparation, potentiallyconfounding their widespread use. Moreover, a substantial percentage ofthe patient population may have established immunogenicity to someanimal-derived proteins (e.g. collagen), potentially stimulating an unde-sirable response to the cell carrier. Synthetic polymers address thelimitations associated with immunogenicity, availability, and variability,but may require the incorporation of binding domains to enable celladhesion or advanced chemistries to attain the desirable degradationparameters. Regardless of the biomaterial, it is critical to utilize a sub-strate that enables long-term cell survival, remodeling of the gel overtime, and the alignment of capillaries to facilitate anastomosis with thehost vasculature.

Matrigel is a solubilized basement membrane preparation extractedfrom the Engelbreth-Holm-Swarm mouse sarcoma and is rich in extracel-lular matrix constituents such as laminin, collagen IV, and heparan sulfateproteoglycans. Bone marrow- or cord blood-derived MSCs, together with

Delivery Vehicles for Deploying MSCs in Tissue Repair 73

Fig. 1. The implantation of co-cultures of endothelial cells and mesenchymal stem cellsfor enhancing angiogenesis. MSCs secrete trophic and proangiogenic factors (left) thatstimulate endothelial cell proliferation (center) and stabilize newly formed capillaries(right).

bone marrow-derived endothelial progenitor cells (EPCs), were entrappedin Matrigel and implanted into the dorsum of rats.4 This co-culture yieldedan extensive network of human blood vessels after one week and formedfunctional anastomoses with the existing vasculature. The implantedendothelial progenitor cells were restricted to the luminal aspect of thevessels, while mesenchymal progenitor cells were adjacent to lumens,confirming their role as perivascular cells. Importantly, the engineeredvascular networks remained patent at four weeks in vivo. Similar resultswere found by deploying murine or human MSC-type populations,together with endothelial cells, in fibronectin-type I collagen gel implants.5,6

Implantation of the endothelial-mesenchymal co-cultures consistentlyresulted in stable vessels that persisted in vivo, some for up to one year.However, those implants containing only endothelial cells quicklyregressed. The implant material facilitated cell adhesion, proliferation,and arrangement into capillary vessels. Unlike implants formed fromMatrigel or collagen that are limited by potential immunogenicity, hydro-gels formed of fibrin can be produced from autologous blood samples.Mechanically robust fibrin hydrogels exhibited significant increases invascular invasion when used to deploy MSCs due to the proteolytic degra-dation of the matrix by MSC-secreted metalloproteinases.7 Syntheticpolymers have also been used to implant MSCs with an eye towardneovascularization. Differentiated MSCs entrapped within cylindersformed of poly(ethylene glycol) diacrylate enabled robust neovascular-ization and contributed to the formation of highly vascularized fibrouscapsules representative of soft tissue.8

Blood vessels must be produced over several length scales, rangingfrom 3–8 μm capillaries to vascular grafts with diameters exceeding6 mm. MSCs have been deployed on various biomaterials to assist in theformation of tissue engineered vascular grafts (TEVGs) and support graftsthat are endothelialized in vivo. Bone marrow-derived MSCs wereembedded within the walls of nanofibrous mesh TEVGs produced fromelectrospun poly-L-lactic acid (PLLA) by seeding a flat film and rolling itaround a mandrel to produce a vessel-like structure9 (Fig. 2). Graftsimplanted for up to 60 days in the common carotid artery of athymic ratsfacilitated efficient cell recruitment and organization, significant ECMsynthesis and self-assembly, and excellent long-term patency. Gong and


Niklason seeded MSCs in mesh scaffolds formed of poly(glycolic acid)(PGA), and cells were cultured under optimized conditions in pulsatileperfusion bioreactors to induce differentiation toward the myogenicphenotype.10 This graft was readily endothelialized when seeded andmaintained in culture over eight weeks. After culture, MSCs expressedsmooth muscle cell actin, a hallmark of the myogenic lineage, and MSC-seeded grafts possessed significantly more collagen content and elevatedburst pressures compared to earlier protocols. These data suggest thatMSCs provide a viable alternative to autologous smooth muscle cells toaddress the reduced production of vessel-strengthening collagen with age


Fig. 2. (A) Nanofiber mesh formed by electrospinning of poly(glycolic acid) (imagecourtesy of Randall Janairo and Song Li, University of California, Berkeley). (B) Fibringel observed with confocal reflectance microscopy (image courtesy of Ekaterina Kniazevaand Andrew Putnam, University of Michigan). (C) Polymeric sponge formed by gasfoaming/particulate leaching of poly(lactide-co-glycolide).

in TEVGs. Numerous other proteins and synthetic polymers have beenelectrospun into materials for bioresorbable vascular grafts (reviewed inRef. 11), but the contribution of MSCs toward vessel formation has notbeen examined in depth. MSCs have also been entrapped in natural mate-rials and induced toward the myogenic phenotype in culture. Multipotentadult progenitor cells were incorporated in fibrin vascular molds, culturedin the presence of an optimized inductive cocktail for three weeks, andtheir response to stimulation and mechanical load were characterized.12

Cells within the molds aligned circumferentially as desired, expressedsmooth muscle cell-specific genes and surface markers, and generatedcontractile force when chemically stimulated.

2.2 Implanting MSCs to repair damaged myocardium

Myocardial infarction (MI) and subsequent heart failure represent themain cause of death in industrialized nations. Left untreated, the loss ofviable myocardium resulting from MI results in continued expansion ofthe initial infarct area and left ventricle (LV), finally leading to heartfailure. Since cardiomyocytes rarely proliferate or differentiate afterinjury, transplantation of stem cells is now increasingly recognized as aneffective method to repair the infarcted myocardium.13 The mechanism ofMSC contribution toward myocardial repair is presently debated in theliterature, as is the ideal route and timing of delivery. Intravenous deliveryof MSCs is highly inefficient in light of the pulmonary first pass effect,14

thereby necessitating the targeted delivery of these cells. Intramyocardialinjection of MSCs has been reported to improve cardiac function andresting blood flow and likely attains higher engraftment efficiency thansystemic delivery. However, MSCs are stimulated by the properties of thesurrounding diseased myocardium and may still migrate away from theinjection site, thereby providing opportunities for instructing the behaviorof these cells through materials-based delivery strategies.

Fibrin is a biodegradable, fast gelling matrix, which easily entrapsand enables the survival, adhesion, and proliferation of MSCs. Themechanical properties of this biopolymer can be tailored by modulatingthe fibrinogen concentration and thrombin concentration independently.However, there is a trade-off between mechanical properties and cell


viability, with higher concentrations of each component facilitating rapidgelation of a high-strength product but failing to promote optimal cellviability and growth. To address these limitations, Zhang et al. modifiedfibrinogen with a benzotriazole carbonate derivative of polyethyleneglycol (PEG) to increase the number of crosslinks with adjacent fibrinmonomer molecules, thereby increasing mechanical properties whilemaintaining the viability of encapsulated cells.15 PEGylation increasedthe storage modulus by nearly 40% without significantly increasing thegelation time. When MSCs were mixed into the gel, cells proliferatedfaster than MSCs grown on tissue culture plastic, migrated out of the gelwhen stimulated, and expressed cell surface markers indicative of differ-entiation toward the endothelial lineage in the absence of cytokinetreatment. Liu et al. entrapped autologous porcine MSCs in a fibrin geland applied the patch to the LV anterior wall following MI.16 After21 days, LV contractile performance was improved in conjunction withincreased neovascularization, likely as a function of improvedMSC engraftment following migration from the gel and the localizedproduction of trophic factors that stimulate host cell migration into theischemic site.

MSCs have been implanted on the wall of ischemic myocardium usingcomposite materials designed to capitalize on the benefits of each con-stituent. Poly-glycolide-co-caprolactone (PGCL) is a synthetic compositematerial that possesses elasticity, suggesting that it could be employed toengineer a patch for mechanically dynamic environments such as theheart. PGCL scaffolds were seeded with syngeneic bone marrow mononu-clear cells and sutured onto the epicardial surface of rats seven daysbefore induction of MI.17 Animals treated with cell-seeded PGCL patchesor PGCL alone exhibited significant reductions in LV remodeling associ-ated with heart failure, while cell-seeded constructs were associated withincreased neovascularization. These data suggest that PGCL contributedmechanical integrity to limit LV dilation, while the addition of cells stim-ulated repair of the damaged myocardium. The potential of MSC-seededbiopolymer composites to promote cardiac repair is still unknown, but thisbiomaterial represents a promising candidate that possesses elasticmaterial properties and can promote the cardiomyogenic differentiationof MSCs.


3. Delivery Vehicles for Deploying Stem Cells inSkin Regeneration

Chronic skin wounds including those associated with diabetes, affect5.7 million patients and cost the United States healthcare system anestimated US$20 billion annually.18 Approximately 40,000 burn patientsare admitted to US hospitals every year, and the use of skin grafting totreat burn patients represents a US$1 billion market.18 Not surprisingly,single modality therapeutic approaches such as growth factor treatmentusing platelet derived growth factor, have met with little success particu-larly in light of the complex pathophysiology of chronic and acute dermalwounds.19 More recently, multifactorial approaches utilizing stem cellsand tissue engineering approaches have been applied to treating acute andchronic skin wounds. We will discuss some recent approaches that utilizebioengineered scaffolds to deploy keratinocyte stem cells, fibroblasts,MSCs, and adipose stem cells (ASCs) to restore structure, function,reduce scarring, and improve the cosmetic appearance of skin.

3.1 Cultured epithelial autograft

The use of autograft, allograft, and xenograft skin transplants by Hinduswas described in Sanskrit texts and dates to around 3000–2500 B.C.20

While such approaches are commonly used in current medical practice,extensive burns and diabetes-associated pathology limit the use of auto-grafting in acute and chronic dermal wounds. Allografts can only be usedas temporary cover due to immune rejection, and also carry the risk of dis-ease transfer.20 One of the first cell and tissue engineering approachesdeveloped to overcome some of the difficulties associated with auto andallografting was the development of keratinocyte culture to generatecohesive sheets of stratified epithelium referred to as cultured epithelialautograft (CEA).

Epicel® (Genzyme Biosurgery) is a commercialized CEA product thatis generated in two to three weeks using bioreactor technology.20 Severalgroups have also evaluated matrices such as fibrin, plasma, collagen,chitosan, hyaluronic acid and polymers such as polyurethane, Teflon®

film, and polyhydroxyethyl methacrylate for delivery of pre-confluent


keratinocytes to wound sites.21 However, CEAs are mechanically fragile,blister, and ulcerate due to the poor formation of a basement membrane andthe absence of an underlying dermis. More recent approaches to skin regen-eration deploy multiple cell types in more complex, three-dimensionalconstructs to more closely mimic a complete dermal-epidermal structure forrepair and regeneration of deeper wounds.

3.2 Dermal substitutes

Dermal substitutes may be cellular or acellular, but lack an epidermalcomponent. As opposed to CEAs, which can take weeks to culture, dermalsubstitutes are supplied as off-the-shelf products. Dermagraft® (AdvancedBioHealing) is a dermal substitute consisting of metabolically activeneonatal foreskin fibroblasts on a polyglactin mesh.22 The foreskin fibrob-lasts deposit an ECM comprised of collagens, glycosaminoglycans, andgrowth factors onto the mesh that is then cryopreserved. Dermagraft® hasbeen FDA approved for use in full thickness diabetic foot ulcers.Alloderm® (LifeCell) is manufactured from donated skin that is decellu-larized to prevent an immune rejection response, and subsequentlyfreeze-dried. Although it is only FDA approved for breast reconstructionand hernia procedures, it has been used in research settings for treatingfull thickness dermal wounds.23

Several dermal substitutes have also been described in a laboratorysetting. In a pig model of dermal regeneration, autologous fibroblastswere deployed in a collagen gel containing alpha elastin hydrolysate.24

Dermal fibroblasts were seeded onto this scaffold and cultured for tendays. The fibroblasts were applied to a full-thickness wound and coveredwith a split-skin mesh graft. The scaffolds seeded with the highest numberof fibroblasts showed significantly improved healing relative to acellularconstructs.24 In an in vitro model, HUVECs were overlayed onto a dermallayer generated by dermal fibroblasts. The dermal layer was shown to sup-port the formation of vessel-like structures by HUVECs in a hepatocytegrowth factor (HGF) dependent manner.25 In a similar model, MSCs weread-mixed with HUVECs and incorporated into the dermal layer.26 TheMSCs were shown to further stabilize and enhance the formation of thesevessel-like structures.


3.3 Bilayered skin substitutes/living skin equivalents

Living skin equivalents (LSEs) approximate the structure of skin, con-taining both an epidermal and dermal component in which different typesof stem cells can be deployed. Similar to dermal equivalents, this categorycontains an array of tissue engineered constructs for deploying stem cellsthat are either commercially available or described by individual labora-tories. Apligraf® was the first bilayered, allogeneic skin substituteapproved for treating chronic wounds.22 Apligraf® is derived from livingneonatal foreskin fibroblasts seeded onto bovine type I collagen to gener-ate a dermal layer. Neonatal foreskin keratinocytes are subsequentlyseeded onto the dermal layer to generate a functional epidermis. In clini-cal studies evaluating chronic wound healing, patients treated withApligraf® were more likely to heal faster and more completely relative tothe standard of care.22

Integra Bilayer Matrix Wound Dressing and Integra DermalRegeneration Template are FDA approved for the treatment ofchronic wounds and burns, respectively. Both products have a dermalcomponent comprised of cross-linked bovine tendon collagen and sharkchondroitin 6-sulfate, and a pseudo-epidermal component comprised ofsilicon.23 Neither product contains a cellular constituent, a native ECM, orthe growth factors present in other bilayered skin substitutes, dermalsubstitutes, or CEAs. Relative to Alloderm® and Dermagraft®, Integrademonstrates very little construct remodeling.23 The deployment of MSCswithin Integra demonstrated significantly increased neovascularizationand growth factor incorporation relative to the biomaterial withoutMSCs.27

Several laboratories have developed alternative delivery vehicles fordeploying stem cells in skin regeneration applications. A LSE comprisedof autologous keratinocytes and fibroblasts deployed in clotted plasma gelhave been successfully used for the treatment of burns. In an in vitromodel, ASCs have been used in place of fibroblasts to generate a self-assembled dermal layer in the presence of ascorbate.28 After merging threeof these dermal layers, a keratinocyte layer was overlayed to yield a bilay-ered LSE. ASCs have also been differentiated in vitro to adipocytes andincorporated into a tri-layered LSE with an adipose containing fascia.28


3.4 Other vehicles for deploying stem cells

MSCs deployed in collagen gels generate a dermal-like substitute that issimilar to collagen gels containing dermal fibroblasts.29 Autologous MSCsdeployed in fibrin gels were shown to promote healing of large skinwounds associated with skin cancer resection, as well as healing ofchronic foot ulcers (duration more than one year) associated with dia-betes.30 In a porcine model of skin wound healing, platelet rich plasma(PRP) has been used as a delivery vehicle for ASCs.31 In this model, ASCswere shown to increase vessel formation, while PRP with ASCs wasshown to improve the cosmetic appearance of the dermal wounds.31 In aporcine partial-thickness burn model, MSCs were seeded onto freeze-dried collagen-glycosaminoglycan scaffolds and cultured for two days.32

The skin equivalents were raised to the air-liquid interface for two days togenerate an epidermis. Pigs treated with the MSC-seeded constructsdemonstrated significantly increased wound contraction, as well assignificantly increased vascular density at four weeks.

4. Biomaterials for Implanting MSCs for RegeneratingOsteochondral Tissues

MSCs are under intense investigation for use in cell-based approaches torepair skeletal defects. Although these cells can be readily induced to dif-ferentiate toward both the chrondrogenic and osteoblastic lineages inculture, their direct participation in the formation of each tissue has beenlimited when delivered to the tissue site. The systemic or local injectionof MSCs without an appropriate carrier or vehicle fails to provide the nec-essary structure and framework for cells to populate tissue defects. Aswell, many cells migrate away from the defect site or undergo apoptosisor necrosis when placed in the harsh environment. In an effort to improvecell survival and participation in the repair of cartilage and bone defects,extensive efforts are ongoing to develop biomaterials that bridge thedefect site and provide a platform for instructing the differentiation ofthese progenitor cells toward the desired phenotype. We will brieflyreview recent efforts in the development and application of biomaterialsto implant MSCs for the formation and repair of cartilage and bone.


4.1 Deploying MSCs for cartilage formation

Cartilage is an avascular tissue with low cellularity and a limited capacityfor self-repair, thereby making it an ideal candidate for cell-basedapproaches toward tissue engineering. MSCs have generated significantinterest in cartilage formation as an alternative to autologous chondro-cytes. Chondrogenesis with MSCs generally involves induction withtransforming growth factor-β (TGF-β ) and a 3D culture environment(e.g. micromass, cell pellet, seeded within biomaterials). With regard tothe 3D culture environment, the degree of chondrogenesis is scaffolddependent,33 thereby providing substantial motivation for careful bioma-terial selection for the delivery of MSCs for cartilage repair. Theapplication of biomaterial systems enables more extensive control overcell behavior than the popular cell pellet approach and provides a strategyfor limiting cell migration away from the defect site. A host of natural andsynthetic materials have been used for MSC chondrogenesis (Table 1),and these materials are broadly divided into substrates that are eitherinjectable or implantable.


Table 1. Commonly Used Biomaterials for Implanting MSCs to Repair Bone andCartilage.

Biomaterial Animal Model Example References

Natural polymersAlginate Rat, rabbit 50, 53Collagen Rabbit, rat 54Fibrin Equine 54Hyaluronic acid Rabbit 35Silk fibroin Rat 55

Synthetic polymersPoly(α-hydroxy esters) Mouse, rabbit 48, 53Poly(ε-caprolactone) Swine 37Poly(ethylene oxide) Mouse 56Poly(propylene glycol) fumarate Rabbit 57

BioceramicsHydroxyapatite Rat, rabbit, mouse 46, 48Tricalcium phosphate Sheep 38

Hydrogels may be used as injectable scaffolds due to their ability to filldefects of any shape and size. Hydrogels have a high water content,support the transport of nutrients and waste, mimic many characteristicsof the extracellular matrix, and have tailorable mechanical propertiesupon physical or chemical crosslinking. Furthermore, MSCs can behomogeneously suspended within these materials, where encapsulatedcells generally retain a rounded morphology that may induce a chondro-cytic phenotype. Equine articular cartilage defects of the femoropatellarjoint treated with fibrin-encapsulated MSCs exhibited enhanced tissuerepair after 30 days characterized by greater tissue formation andincreased presence of type II collagen, yet these improvements were lostcompared to cell-free fibrin after eight months.34 Liu et al. reported thatMSCs transplanted in hyaluronic acid hydrogels resulted in firm, elasticcartilage that filled the entire defect after 12 weeks, and implanted con-structs were well integrated with host cartilage.35 The encapsulation ofMSCs in hydrogels formed of synthetic polymers releasing TGF-β exhib-ited increased type II collagen distribution and cartilage formationcompared to cell-containing gels alone.36 These findings suggest thatchondrogenesis of MSCs in situ may require induction with TGF-β inorder to realize the full potential of this approach.

Implantable materials such as sponges and meshes facilitate precisecontrol over material morphological properties compared to hydrogels,and these substrates enable the generation of mechanically stable con-structs in vitro for subsequent in vivo implantation. However, thesematerials suffer from difficulties with filling irregularly shaped defectsand maintaining contact along the entire defect periphery, each of whichfacilitates host cell migration and inhibits the formation of weak fibroustissue. In light of the responsiveness of MSCs to mechanical cues,whether from the substrate or mechanical loading, the properties of theimplant will have a profound contribution toward the resulting phenotypeupon implantation. In a recent study, circular poly(ε-caprolactone) (PCL)scaffolds cut from electrospun nanofibrous mats were seeded with MSCsand implanted into full-thickness defects in the femoral condyle of mini-pigs.37 Six months after implantation, MSC-seeded scaffolds exhibited themost complete repair within the defects compared to materials seededwith autologous chondrocytes or cell-free scaffolds. Similarly, autologous


ovine MSCs seeded on β-tricalcium phosphate (β-TCP) implants andimplanted into full thickness articular cartilage defects yielded hyaline-like tissue that was indistinguishable from the adjacent normal cartilage.38

Defects treated with cell-free implants contained fiber-like tissue andexhibited incomplete repair after six months.

4.2 Deploying MSCs for bone formation and regeneration

Bone autograft has been used for many years as the standard of care forbone regeneration applications such as the repair of non-union fractures,and in ectopic ossification applications such as lumbar fusion. Intrinsic toautograft are the trinity of factors that are critical for optimal bone regen-eration: an osteoconductive matrix to allow for new bone and vascularingrowth, osteoinductive factors to induce new bone formation, andosteogenic cells to deposit and mineralize a bone matrix. The limitationsof autograft, namely tissue availability, low fusion rates, and significantmorbidity, have motivated the pursuit of alternative materials for bridgingbone defects and stimulating bone repair. These materials include allo-geneic cancellous bone chips, collagen sponges, synthetic polymerimplants, and various hydrogels. Such materials have been described asalternatives to autograft and demonstrate osteoconductive properties, yetfor the most part, they lack osteoinductive factors as well as osteogeniccellular components.39 In an effort to provide an osteogenic cellular con-stituent, several groups have combined stem cells from different sourceswith these materials. In this section, we will describe the materials thathave been used in combination with different stem cells as alternatives tobone autograft in bone regeneration or ectopic/orthotopic ossificationapplications.

4.2.1 Cancellous bone chips and demineralized bone matrix(DBM )

Bone allograft (cancellous bone chips) and DBM are currently usedto treat fractures and in lumbar fusion procedures. They are sold as anoff-the-shelf product that possesses osteoinductive activity elicited byendogenous growth factors that remain associated with the ECM. Most


manufacturers offer cancellous bone chips as a decellularized product, butsome offer metabolically active cellular cancellous products that areprocessed and cryopreserved in a manner that maintains the viability ofendogenous stem cells (Trinity Evolution, Orthofix). For many years,bone marrow aspirate, containing a heterogeneous mixture of endogenoushematopoietic stem cells, MSCs, and endothelial progenitors, has beenused in combination with cancellous bone chips and DBM to generateosteoconductive grafts with augmented osteogenic activity. Cancellouschips loaded with bone marrow were shown to enhance the healing ofnon-union fractures in the clinical setting.40 In a posterior segmentallumbar fusion model in the dog, cancellous bone chips in combinationwith DBM and clotted bone marrow were shown to significantly improvethe success rate.39 Importantly, clotted bone marrow alone demonstratedno beneficial effect.

4.2.2 Collagen sponges and collagen composites

Collagen sponges, such as those found in the INFUSE Bone Graft(Medtronic), and collagen composites, such as Healos (collagen/hydroxya-patite, Depuy), Mozaic, and Vitoss (collagen/β̃ TCP, Integra and Orthovitarespectively), are osteconductive materials that lack both an intrinsicosteoinductive component as well as an osteogenic cellular component.Collagen sponges are highly compressible, while collagen-ceramic com-posites possess more robust mechanical properties. Both types ofscaffolds incorporate the native cell adhesion domains associated withcollagen. Recently, stem cells alone or in combination with growth factorshave been added to these materials to enhance their performance in boneregeneration and ectopic/orthotopic ossification applications. In a ratmodel of lumbar fusion, bone marrow aspirate was shown to enhancelumbar fusion mediated by a BMP-2 soaked collagen sponge.41 By con-trast, platelet rich plasma did not augment lumbar fusion when used withthe INFUSE kit. In a similar model of lumbar fusion, ASCs transducedwith an adenoviral BMP-2 vector and loaded into a collagen spongedemonstrated enhanced lumbar fusion relative to BMP-2 alone.42

Interestingly, ASCs loaded in a collagen scaffold generated little ortho-topic bone and failed to promote lumbar fusion. In an orthotopic,


xenogeneic model of bone formation, undifferentiated ASCs and MSCsimplanted in Healos persisted in vivo, while in vitro differentiatedASCs and MSCs did not.43 In a similar model, in vitro differentiated ASCsloaded into a honeycombed collagen scaffold demonstrated increasedbone formation relative to undifferentiated ASCs.44

4.2.3 Calcium phosphate and hydroxyapatite ceramics

Calcium phosphate ceramic scaffolds such as Copios bone void filler (cal-cium phosphate, dibasic, Zimmer) have been used in combination with anumber of different stem cell sources to augment new bone formation.While ceramic scaffolds are osteoconductive, they lack osteoinductivefactors and do not contain endogenous osteogenic cells. Macroporousbiphasic calcium phosphate (MBCP) was used as a scaffold in a rat modelof a radiation-induced defect of the long bones.45 Bone marrow aspirate,MSCs, or ASCs were loaded into these scaffolds and the scaffolds wereplaced in the defect area. In this model, scaffolds loaded with bone mar-row aspirate but not MSCs or ASCs, demonstrated significantly greaterbone formation than empty scaffolds, indicating that cultured cells wereinferior to fresh isolates. Compared to the low specific surface area (SSA)of synthetic β-TCP, high SSA materials have been developed that demon-strate levels of osteogenic differentiation similar to low SSA materials.Calcium-deficient hydroxyapatite (CDHA) has an SSA of 20–80 m2/g andbelongs to the high SSA ceramic group.46 In a rabbit tibial osteotomymodel, CDHA scaffolds loaded with MSC or with PRP demonstratedsignificantly increased bone volume relative to empty scaffolds.46 Therewas no added benefit when MSC and PRP were combined, suggesting thatCDHA is enhancing bone formation through stimulating MSC differenti-ation as a function of substrate rigidity or composition.

4.2.4 PLGA and PCL based scaffolds

Similar to ceramic scaffolds, PLGA and PCL based scaffolds are osteo-conductive but do not have any osteoinductive properties. Both PLGA andPCL have good biocompatibility profiles and biodegrade at rate that isdependent on their composition. Different stem cell populations have


been seeded on these scaffolds to provide an osteogenic component forbone regeneration applications. In a rabbit critical-sized femoral defectmodel, porous PLGA scaffolds were loaded with the osteoinductive fac-tor dihydroxy vitamin D3 (Calcitriol), MSCs, or Calcitriol with MSCs andplaced in the defect.47 Only scaffolds containing Calcitriol showed a bonyunion at nine weeks. Scaffolds incorporating MSCs had greater amountsof pre-mineralized matrix (osteoid) present, but Calcitriol was requiredfor successful union. In a mouse ectopic bone formation model, ASCswere loaded in a PLGA/hydroxyapatite composite in the presence orabsence of BMP-2 and implanted subcutaneously.48 Scaffolds with ASCsalone had negligible amounts of mineral, while scaffolds loaded withBMP-2 alone had significant amounts of mineral. ASC loaded scaffoldswith BMP-2 demonstrated the greatest amount of bone formation. In asimilar model using porous, honeycombed PCL-TCP composite scaffolds,the osteogenic capacity of fetal bone marrow MSCs, umbilical cord MSCs(UC-MSCs), adult MSCs, and ASCs was compared.49 The stem cells wereloaded on scaffolds, differentiated with dexamethasone and ascorbatein vitro, and subsequently implanted. Scaffolds loaded with fetal MSCsand adult MSCs demonstrated the highest levels of bone formation, whileadipose MSC loaded scaffolds demonstrated the least.

4.2.5 Hydrogels

Hydrogels are appealing candidates as scaffolds given their similarity tonative ECMs and high degree of biocompatibility. Moreover, these mate-rials enable highly efficient incorporation of cells and inductive factors tostimulate bone formation. In a rat critical-sized cranial defect model,ASCs alone, or ASCs genetically modified with an adenovirus to overex-press BMP-2 were loaded into an alginate hydrogel and implanted into thedefect.50 Only scaffolds with BMP-2 expressing ASCs demonstratedclosure of the critical-sized defect at 16 weeks. In a rat calvarial defectmodel, BMP-2, MSCs, or MSCs with BMP-2 were loaded into hyaluronicacid based hydrogels and implanted in the defect.51 While MSCs demon-strated poor adhesion to the scaffolds in vitro, scaffolds seeded with MSCsand BMP-2 demonstrated robust bone formation four weeks after implan-tation. Scaffolds seeded with MSCs alone showed slightly less bone


formation, while scaffolds seeded with BMP-2 alone showed the leastamount of bone formation. In an ectopic bone formation model, anMPEG-PCL in situ forming gel was used as a scaffold for implantingASCs.52 Only scaffolds loaded with ASCs demonstrated in vivo boneformation, while scaffolds loaded with ASCs and an osteogenic supple-ment had the highest levels of bone formation.

5. Conclusions

MSCs have tremendous potential to contribute to the repair of human tis-sues. In order to capitalize on the promise of these multipotent cells todifferentiate to many phenotypes, secrete tissue-inducing factors, and sup-press inflammatory responses, new approaches for deploying these cellsmust be developed and optimized under conditions that approximatehuman physiology. Furthermore, the selection of the delivery vehicleenables control over the mechanical properties, degradation time, cell dis-tribution, cytokine, growth factor, and morphogen gradients, and can eveninstruct cell fate. The examination of these systems under physiologicalconditions and in clinically relevant animal models will increase ourunderstanding of how MSCs contribute to the development and repair ofhuman tissues. Importantly, the results of these studies will provide newinformation on the efficacy of these materials to support the long-termsurvival and performance of MSCs in tissue repair and regeneration.

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6

Stem Cells for Cardiac Tissue Engineering

Jennifer L. Young, Karen L. Christman and Adam J. Engler

1. Cell Therapies for Myocardial Infarctionand Heart Failure

As a leading cause of death in the United States, congestive heart failure(CHF) post-myocardial infarction (MI) has incited the need to developnovel techniques that prevent muscle wall scarring and ventriculardilation, symptoms which contribute to impaired heart function. CHFis typically induced from a MI in which a major coronary vessel becomesoccluded, preventing appropriate oxygen exchange in the muscledownstream of this blockage. Ensuing cell death in the myocardiumis counterbalanced by increased secretion of collagen, which forms afibrotic scar to maintain wall integrity. As a consequence of this, ventriclecontraction is impaired and results in negative left ventricular (LV)remodeling and dilation of the heart.1 CHF-induced impairment results ina high morbidity rate as the body has limited capacity to replace damagedtissue and restore function. Although recent studies have reported on theability of the heart to repair itself after injury via progenitor cardiac stemcells (CSCs) and homing of hematopoietic stem cells (HSCs),2–4 intrinsiccardiac regeneration is limited by extremely slow cell turnover in the

95

myocardium,5 and turnover only further slows in older patients whereCHF predominates. Over the past two decades, novel strategies havetaken many routes and used many cell types to treat MI and CHF. Here wepresent a succinct review of three common regenerative medicine andtissue engineering strategies: cellular cardiomyoplasty, cardiac patches,and injectable scaffolds (Fig. 1). Though numerous variations have been

96 J. L. Young, K. L. Christman and A. J. Engler

Fig 1. Approaches to cell-based therapy in the heart. Following a myocardial infarction,cells in liquid solutions of saline or cell culture medium (cellular cardiomyoplasty), and incombination with a biomaterial scaffold (cardiac patch and injectable scaffold) have beenexplored to prevent progressive left ventricular remodeling that leads to left ventriculardilation and heart failure. Each technique has its own pros and cons as listed here, yet allapproaches have had some success in animal models.

attempted for these techniques, we will limit our discussion to theclinically-relevant cell types used, their mechanistic characterizationin vitro, and subsequent in vivo results.

Before presenting these methodologies however, it is perhaps helpfulto categorize the clinically used cell types today. Since its introduction inthe 1990s, cellular cardiomyoplasty, where somatic cells are injected inliquid solutions of saline or cell culture medium into the post-MI heartwall, has gained much attention as one of the first cell-based methodsproposed for repairing, replacing and restoring function to the injuredheart. Early studies in animal infarct models showed implanted somaticcells survive, prevent further ventricular dilation, and confer some level ofimproved myocardial output.6–8 Despite some promising results in animalmodels, clinical trials with autologous somatic cells using various injec-tion methods, while not showing adverse patient effects, have producedmixed results for improvement of global contractility,9–11 with the mecha-nisms behind improved cardiac function in these studies remainingunknown. One significant shift in the technique to better determine itsefficacy has been to use adult stem cells as they are regarded as the opti-mal source since they are easily obtained, readily expanded in vitro,non-immunogenic, and can be an autologous cell source.

Hematopoietic stem cells (HSCs) are bone marrow-derived cells thatcan regenerate myeloid and lymphoid lineage cells of the blood, thoughthere is some evidence that these cells are multipotent and can becomenon-blood derived lineages.12 Their ability to differentiate into cardiomy-ocytes in vitro and in animal models with infarcted myocardium, however,has proven less than successful.12–14 Mesenchymal stem cells (MSCs),named after their ability to differentiate into mesodermal lineages such asadipose, muscle, bone, etc.15 are also multipotent stem cells which can beeasily isolated from the bone marrow where they are relativelyabundant.16 These cells lack several cell-surface markers enabling them tobe immune privileged,17 and in culture, these cells have been shown todifferentiate into beating cardiomyocytes by the addition of thedemethylating agent, 5-azacytidine.18 It should be noted that though cellsproduced by this method exhibit some genotypic and phenotypic charac-teristics of adult cardiomyocytes, they lack the presence of contractileelements.

Cardiac Tissue Engineering 97

With this supporting evidence, human MSCs were once thought to bethe most clinically relevant source for myocardial cell therapy. Somestudies have reported the ability of MSCs to marginally restore functionand structure to animal infarct models by differentiating intocardiomyocytes and inducing angiogenesis19 though it was later demon-strated that improved cardiac function was likely due to paracrine effectsof the transplanted cells14,20 and not functional restoration. Others suggestthat implanted MSCs fused with resident cardiomyocytes and thus do notactually differentiate into functioning cardiac muscle cells.13,21 Increasinginfarct cellularity, which increases wall thickness and thus reduces wallstress, is also postulated to result in the observed functional effects.22

Although the mechanisms underlying improvements in heart functionhave yet to be identified, the results of cellular cardiomyoplasty in animalmodels have prompted several human clinical trials. Both skeletalmyoblasts and human MSCs were injected into the myocardium post-MI,and little to no improvement was observed.23–25 Thus it appears that injec-tion of cells alone into injured myocardium may not be sufficient torestore function and structure due to some mechanism blocking theirregenerative capacity. In fact, recent evidence which we will discuss nextappears to indicate that MSCs in the infarct differentiate not into musclebut into cells that form small calcified lesions, typical of MSC-derivedosteoblasts26 (see Fig. 2C).

2. Cellular Cardiomyoplasty Revisited: The Influence of in vitro Mechanics

Cells are highly responsive to their surroundings, and when presentedwith a disease microenvironment, their function is likely different fromwhat is observed in a healthy setting. In healthy tissue, forces, integrins,and the surrounding fibrous scaffold called the extracellular matrix (ECM)exist in a balance termed “dynamic reciprocity.” Thus altered cellsprogrammed by this damaged environment may not be able to sufficientlyregenerate tissue function or may even worsen the cell niche.27 The incon-clusive results from MSCs used in this treatment could be explainedin part by improper epigenetic regulation as dictated by the aberrantsurrounding ECM. Indeed as cells necrose in the heart wall, fibroblasts



Fig 2. Cellular cardiomyoplasty. (A) MSCs grown on hydrogels of defined stiffnessdevelop cell morphologies reflective of neural, myocyte, and osteocyte lineages.(B) MSCs were injected into infracted hearts (left; with inner ischemic and outer borderzones highlighted in shades of blue) where the elasticity of the wall was significantlychanged downstream of the occlusion site as shown in the right panel using atomic forcemicroscopy. (C) Histological analysis of the heart wall shows that 29 days after injecting105 MSCs, hearts showed massive calcifications (black deposits; left image) thatoriginated from injected EGFP+ MSCs.

secrete a substantial amount of collagen to form a scar in the infarct area.This is initially a compensatory mechanism to resist wall deformation andrupture.28 However, as noted by Berry and co-workers, this fibrotic scar isthree- to four-fold stiffer than normal muscle, as measured using anatomic force microscope (AFM),29 and is accompanied by a variety ofventricular remodeling.

Resistance to deformation from a force, i.e. the “stiffness” or more for-mally the elasticity (E, measured in Pascals, Pa), provided by ECM forcells to contract against, has an important role in development and func-tion. Tissue-specific matrices have distinct mechanical properties thathelp to direct developing cells to a specific lineage.15 It has been demon-strated that MSCs plated onto collagen I-coated polyacrylamide (PA) gelsthat mimic the elasticity of the brain (0.1 – 1 kPa), muscle (∼ 8–17 kPa),and bone (<30 kPa), give rise to neurocytes, myocytes, and osteocytes,respectively30,31 (Fig. 2A). Such responses are also seen with fibronectinbut not with laminin-1 and collagen IV-coated gels,31 suggesting that cellsare highly responsive to their physical environment when ligating appro-priate integrins. If placed into an abnormally rigid environment in vivo,e.g. one that is three- to four-fold more stiff than normal myocardium andfull of collagen I,29 such results would imply that MSCs would respond bybecoming osteoblast-like rather than muscle-like (Fig. 2B). Though thereare a variety of soluble factors that may attenuate this process, much ofthat is likely absent in this infarcted niche.

Such observations also highlight the glaring differences between the in vivo environment, in vitro mimics (e.g. PA gels), and traditional myocytecultures from the past several decades, i.e. cells grown on collagen-coatedrigid glass and tissue culture polystyrene. For the rigid substrate, three-step myofibrillogenesis32 is halted at the initial pre-myofibril step inisolated myotubes as shown by Griffin and co-workers;33 instead ofmature fibrils, they find that large stress fibers appear and myotubes areoverly adherent to the rigid substrate.33 Conversely, many labs have stud-ied myofibrillogenesis in culture, with cells grown either (1) in syncytia,where clustered cells can contract against one another rather than the rigidmatrix, or (2) on glass coated with thicker collagen layers, effectivelymaking the surface more compliant. For example, when cultured oncollagen gel-coated glass, with elasticity closer to muscle than rigid


substrates,34 precardiac mesoderm was able to undergo myofibrillogenesis32

and could beat rhythmically in similar collagen gels.35 Detailed analysis ofcardiomyocyte behavior as a function of matrix stiffness has shown thatmature36 and neonatal myocytes37 require a compliant niche that is as softor softer than healthy muscle for sustained contraction and properexpression of sarcoplasmic/endoplasmic reticular calcium ATPase, calciumstorage and transients, and traction stresses. Yet until now the relatively lowrigidity of the in vivo environment for the myocardium has largely beenunderappreciated.

When returning to the infarct example, it is thus not surprising thatsomatic cells as well as stem cells are regulated by the mechanical contextof their environment. While the highly ischemic region in the center of theinfarct may lack sufficient blood supply for a cell therapy alone to work,stiffness within the borderzone, which is only 50%–200% higher thannormal,29 likely prevents MSCs from restoring function there as well sinceno MSC-derived cardiomyocytes were found. As illustrated in Fig. 2,MSC-treated infarcted hearts sometimes show limited recovery29 andabnormal differentiation,26 agreeing with in vitro data demonstrating thatMSCs become osteogenic on gels with E of ∼30–50 kPa.30,31 Surprisingly,osteogenic lesions are found throughout the infarct, indicating that at leastmodest cell engraftment occurred throughout a dominant fraction of boththe highly ischemic necrotic core and the modestly ischemic borderzone.However, clinical studies using both similar marrow-derived stem cells,presumably with the same abnormal differentiation characteristics, andother somatic cells appear to demonstrate that there is still some improve-ment in global function as cells either are directly injected or home to theinjury site; observed improvement includes 6%–9% increase in LV ejec-tion fraction, reduced end-systolic LV volumes, and enhanced perfusionin the infarcted area four to six months after cell transplantation.9–11,38

Such discrepancies between studies directly measuring elasticity andimplicating problems with differentiation versus those that demonstratemodest functional improvement may likely be the result of differences invariable such as time-post infarct for cell injection, injection cell number,cell preparation protocols, spatial distribution of injections, etc. With dif-ferences in time-post infarct for cell injection in particular, cells seeing apre-formed rigid scar versus a matrix that is actively remodeling could


present environments that are either already rigid or one that is still softbut actively remodeling, respectively. Cells injected into the latter sce-nario may have a better chance of remodeling the environment, but a moredetailed examination is likely required.

Though more appropriate cell types for this therapy may exist, such asmore recently discovered cardiac stem cells, these data imply that it is notpossible to rely solely on direct injection of cells to remodel the matrix asit provides too few cues in the proper direction to promote the formationof appropriate cardiomyocyte lineages. Instead, two alternate approacheshave been proposed which involve delivering cells in epicardial sheets orin injectable scaffolds.

3. Tissue Engineering Approach: Utilizing Biomaterial Scaffolds

While there has been some success in animal models with cellularcardiomyoplasty, and numerous clinical trials are currently ongoing, thistechnique is plagued by limited cell retention and transplant survival.39,40

Injection into an ischemic environment and anoikis (cell death from theabsence of cell-matrix interactions) are two major factors attributed topoor cell transplant survival. To overcome both of these challenges, tissueengineering approaches are currently being examined. In this case, cellsare given a temporary extracellular matrix through the use of a biomater-ial scaffold, unlike the typical cellular cardiomyoplasty approach wherecells are delivered in only a liquid solution. Biomaterial approaches alsohave potential for growth factor delivery, which can further promote cellsurvival and reduce the ischemic environment.

3.1 Cardiac patches

Myocardial tissue engineering can be categorized into in vitro engineer-ing and injectable approaches (Fig. 1).41,42 In vitro engineered myocardialtissue, involving the classical tissue engineering approach of seeding cellson a scaffold and culturing the construct prior to implantation, was thefirst to be examined in the myocardium.43,44 In addition to utilizing pre-formed scaffolds, soluble scaffolds have been pre-mixed with cells and


shaped into the appropriate construct. Moreover, temperature responsivepolymer coated substrates have been employed to generate cell monolay-ers or sheets, which retain their secreted matrices.45,46 These can besubsequently combined to form a cardiac patch over the infarcted region.By providing an extracellular matrix for transplanted cells, cardiacpatches have been shown to increase cell survival, induce neovasculariza-tion, attenuate negative LV remodeling, and preserve cardiac function inpre-clinical models.42 While improvements in contractility are theoreti-cally possible with stem cell-derived cardiomyocytes, such effects to dateare likely through paracrine mechanisms.

Clinical results with this approach are sparse to date, yet results froma non-randomized Phase I clinical trial with a cell seeded collagen scaf-fold are encouraging at least in terms of safety and feasibility.47

Mononuclear bone marrow cells isolated from the patient were seeded ona porous collagen matrix (7 × 5 × 0.6 cm) in the operating room and thensutured onto the epicardium following a single off-pump coronary arterybypass graft surgery (OP-CABG). In chronic infarcts, with an average ageof approximately eight months, this cardiac patch increased wall thick-ness, limited negative LV remodeling, and improved diastolic function;however, patients also received injections of the bone marrow cells inautologous serum into the infarct and borderzone.

While in vitro engineering of a patch to cover up damaged myocardialtissue is a viable strategy for cardiac repair, there are limitations with thisapproach. For instance, as with other vascularized tissues, the develop-ment of sizable constructs in vitro is a major challenge. Secondly, thesepatches can only be applied to the epicardial surface and thus do notdirectly treat the infarct. Implantation of a patch would also require aninvasive surgical procedure, unlike injectable, percutaneous approaches.However, there are some distinct advantages that this in vitro approachoffers. For instance, organization and alignment of transplanted cells canbe uniquely achieved.48,49 While this is likely not critical for cells that arefunctioning strictly through paracrine effects, it would be important forcell types that can undergo cardiomyogenesis. New approaches to cardiacpatch design include the combination of prosurvival and angiogenicgrowth factors, and pre-vascularization on the omentum, which is a blood-vessel enriched membrane.50 This approach improved subsequent patch


integration with host myocardium, leading to preservation of LV volumeand ejection fraction, and has potential for the use with other cell types.A caveat however with this study was that LV volume and cardiac func-tion were not statistically different than acellular patches alsopre-vascularized on the omentum. More sophisticated biomimetic scaf-folds are also providing potential advances in the field. Protocols fordecellularizing myocardium51,52 have opened up the possibility of creatinga cardiac patch with a scaffold that contains many of the biochemical andmechanical cues that match original myocardial ECM composition andpotentially its mechanical properties, e.g. 10–20 kPa,29 as well. To thatend, synthetic scaffolds have also been engineered to match theanisotropic properties of healthy myocardium, albeit only right ventricularmyocardium has been successfully mimicked to date.53

3.2 Injectable scaffolds

Given the push towards more minimally invasive surgeries that requireless recovery time and reduce the chances of infection, injectableapproaches to myocardial tissue engineering, which could be deliveredminimally invasively through a catheter, are particularly attractive.Moreover, an injectable therapy could be delivered throughout the infarctwall and borderzone, and not solely to the epicardium as with cardiacpatches.

Injectable scaffolds for in situ myocardial tissue engineering can beutilized as either acellular or cellular treatments.42 In the first approach,material is injected into the infarct alone, and can serve to increase cellmigration into the infarct area, including neovascularization, to thickenand support the LV wall, or both. As cell therapy, injectable scaffolds canbe employed to increase cell transplant survival over the typical cellularcardiomyoplasty approach. An acellular approach may reach the clinicsooner since it has the potential to be an off-the-shelf treatment withoutthe added complications that cells bring, including the appropriate source,the need for in vitro expansion, or potential disease transmission.However, cellular therapies are currently in clinical trials, despite poorcell survival as with the typical cellular cardiomyoplasty technique.Injectable scaffolds have the potential to increase cell transplant survival,


with potential functional benefit, and as such are often viewed as animprovement over previously discussed methods.

With the knowledge of these pros and cons, it is critical to definedesign criteria associated with an ideal injectable scaffold for cell deliv-ery in the myocardium. First, an injectable myocardial scaffold shouldpromote neovascularization to reduce the ischemic environment, promotecell adhesion, survival, and maturation in the case of progenitor or stemcell delivery, and be injectable through a catheter. In terms of cell adhe-sion and survival, material choice and composition are especiallyimportant and should likely mimic both biochemical composition, whichincludes a complex mixture of proteins and polysaccharides, and mechan-ical properties of the native myocardial ECM which they are attemptingto replace. To date, several materials have been examined for injectableapproaches to myocardial tissue engineering, including fibrin,54,55

collagen,56 Matrigel,57–59 alginate,60,61 and self-assembling peptides42

among others. Collagen has been utilized to promote neovascularization,56

thicken the infarct wall and improve LV geometry,62 and deliver cells intothe myocardium;63 however, neutralized collagen gels rapidly at evenroom temperature. Matrigel, both alone57,58 and in combination withcollagen,59 has been shown to facilitate cell transplantation. Yet, clinicaltranslation of Matrigel is very unlikely since it is a matrix derived frommouse sarcoma, and is known to promote tumor growth in vitro andin vivo. Alginate, which is a polysaccharide derived from seaweed, hasbeen utilized mostly for thickening and supporting the LV wall;60,61 how-ever, as a regenerative scaffold, alginate is likely not the best choice sinceit is known to have poor cell adhesion,64 although it can be modified tocontain cell adhesion peptides.65 Self-assembling peptides can formnanofibrous networks inside the myocardium, which promote cell andvessel ingrowth.66 Yet, these matrices were not capable of improving celltransplant survival.67 Of all of these materials, the only neutralized mate-rial to be injected via catheter is alginate, which is being explored as anacellular therapy.68 Thus, while a material may be injectable via a syringe,this does not necessarily translate to percutaneous, minimally-invasivedelivery. In fact, despite increased survival in small animal models withinjectable scaffolds, no materials have advanced into clinical trials forenhancing cell therapy in the myocardium.


The ECM is known to play a role in almost every cell process includ-ing attachment, differentiation, morphogenesis, and whether a cellproliferates or apoptoses,15,29–31,33–37 and thus, the typical goal of tissueengineering scaffolds is to mimic the in vivo ECM for a particular tissue,mainly because of the importance of cell-matrix interactions. Recently, aninjectable, in situ gelling scaffold derived from decellularized ventricularmyocardium was tested in vivo,69 which retains many of the originalbiochemical cues of native cardiac ECM. This material has also beenrecently injected via an endocardial, percutaneous approach in a porcinemodel, which is the preferred delivery method for cell therapy in themyocardium.70 While the biochemical aspects of the myocardial ECMhave been effectively mimicked, no injectable materials have beendesigned to mimic the mechanical properties, whose importance has beenhighlighted above and likely plays an important role in determining trans-planted cell fate.

3.3 Material selection

Considering the key role of cell-ECM interactions in affecting cell behav-ior, and the tissue specificity of the ECM, a scaffold that possesses theappropriate ventricular ECM cues would be ideal for myocardial tissueengineering. However, few materials for cardiac tissue engineering haveinvolved this design approach. Neither naturally derived or syntheticmaterials possess all of the necessary attributes for a scaffold, whether fora cardiac patch or an injectable scaffold; each has their pros and cons.

Protein and natural-based hydrogels are especially applicable in tissueengineering strategies due to their ability to better mimic the in vivomicroenvironment by exhibiting familiar ECM components and structuralorganization. Cells can thus better respond to the signals provided byfamiliar components in their surrounding matrix. Additionally, thesemolecules are generally biocompatible, thereby reducing concerns overpotential toxicity. Synthetic biomaterials on the other hand can providebetter mechanical properties and can be more easily manipulated thannatural polymers.

Currently no ideal materials exist in terms of mimicking both bio-chemical and mechanical properties of the myocardium. Advancement in


this field will likely be achieved by the rational design of materials toappropriately mimic the desired properties of the native ECM. This willlikely change depending on the intended mechanism of therapy: structuralvs. paracrine vs. regeneration. Furthermore, designing materials with clin-ical translation in mind is a necessity. For example, although a materialmay be injectable via syringe, it does not necessarily translate to a mini-mally invasive approach. Design criteria must therefore includetranslation to man from the very beginning with small animal studies.

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7

Cardiovascular System: Stem Cellsin Tissue-Engineered Blood Vessels

Rajendra Sawh-Martinez, Edward McGillicuddy, Gustavo Villalona,Toshiharu Shin’oka and Christopher K. Breuer

1. Introduction

Cardiovascular disease (CVD) is the leading cause of death in the world.Ischemic heart disease, cerebrovascular disease and peripheral vasculardisease account for 13 million deaths annually.1 Additionally congenitalheart disease, which represents the most common birth defect affectingnearly 1% of all live births, remains a leading cause of death in the new-born period. In the United States alone, the American Heart Associationestimates the cost of treating CVD in 2009 to be US$475.3 billion.2

For end-stage cardiovascular disease in adults, or in children withcongenital cardiovascular anomalies, surgical reconstruction remains thetreatment of choice. Unfortunately there is a relative paucity of autolo-gous tissue available for reconstructive surgeries, necessitating the use ofsynthetic materials.

Biomaterials available in the cardiovascular surgeon’s armamentariuminclude autologous vessels and prosthetic materials. Autologous tissue issuperior to prosthetic materials such as Goretex®, as prosthetics are more

115

prone to thrombosis, infectious complications, neointimal hyperplasia,and accelerated atherosclerosis.3–6 In an adult patient undergoing multiplecardiac or peripheral vascular procedures, however, suitable autologoustissue may be scarce due to the diffuse nature of atherosclerosis.Additionally, in children undergoing surgery for complex congenital heartdefects, repairs may be extra-anatomic and require more autologous tissuethan is available. Clearly, there is demand for improved, biocompatiblevascular conduits in these populations.

The great promise and hope of vascular tissue engineering is the devel-opment of biological substitutes that restore, maintain or improve tissuefunction. The origins of vascular tissue engineering can be traced back toAlexis Carrel who received the Nobel Prize in medicine for his early workin vascular biology. This established the basic principles that provided thefoundation for vascular surgery. Later in his career while working incollaboration with Charles Lindbergh at the Rockefeller Institute, some ofthe early pioneering work in cell culture was performed. In their manu-script entitled “The Culture of Whole Organs,” Carrel and Lindberghpostulated that cell culture would one day be used to grow entire organs.7

However it was not until Weinberg and Bell’s seminal paper in 1986 thatLindberg and Carrel’s prediction became a reality for vascular tissue.8 Thissearch led to many advances in our basic understanding of tissue interac-tions, including the critical importance of cellular and matrix interactions,the modulation of cellular recruitment, growth and differentiation, as wellas a deeper appreciation for the challenges that are faced in attempting toapply these discoveries clinically. In this chapter we aim to guide the readerthrough the major milestones of vascular tissue engineering while high-lighting the importance of continued scientific investigations aimed atreducing the tremendous burden of cardiovascular disease.

1.1 Historical overview

The first report of the use of synthetic vascular grafts dates back to 1952when Voorhees et al., used Vinyon N cloth tubes as arterial interpositionsin dogs.9–12 Prior to this report, scientists were focused on using nativearteries as conduits.9,12 In the ensuing years, other synthetic materialsaimed at passively transporting blood with minimal reaction were

116 R. Sawh-Martinez et al.

developed, leading to the clinical success of polyethylene terephthalate(Dacron) and expanded polytetrafluoroethylene (ePTFE).

Major achievements in creating an ideal conduit have driven the con-tinued search for the best strategy to create fully functioning blood vesselsubstitutes. In 1986, Weinberg and Bell’s landmark publication describedthe use of bovine cells with rat collagen gel to create an artificial bloodvessel model. In 1998, Shinoka et al., first described the use of abiodegradable synthetic scaffold with ovine cells, demonstrating long-term autologous implantation in a low pressure pulmonary arterysystem.13 Also in 1998, L’Heureux et al., published their creation of anengineered graft that used human cells and was tested as a short-termxenogeneic implant in a high pressure arterial bypass model.14 In 1999,Nicklason et al. and Shum-Tim et al. described the creation of an arterygrown in-vitro using autologous porcine/ovine cells on a biodegradablepolymer.15,16 In their studies, Niklason et al. used small diameter grafts,and studied their vessels under arterial pressures in the short-term.Shum-Tim et al., developed large diameter grafts, studied under lowerpressures with long-term evaluation of function.

In 2001, Shin’oka et al., reported the first clinical use of a tissue-engineered vascular construct. The vascular graft was implanted in afour-year-old girl to reconstruct an occluded pulmonary artery, after aprior Fontan procedure.17 In another major milestone for vascular tissueengineering, in 2007 L’Heureux et al. used their sheet-based tissue engi-neered blood vessel clinically as replacements for failing arteriovenousshunts.18 Despite these tremendous achievements, the field has yet toreport the clinical use of a tissue-engineered vascular construct as a fullyarterial interposition in humans. Further, the mainstay therapy for vascu-lar reconstruction continues to be autologous arteries and veins. In thefollowing pages we will detail these early successes, discuss the applica-tions of vascular tissue engineering in clinical trials, and describe thefrontiers of our technology.

2. Critical Elements of an Artificial Blood Vessel

Synthetic vascular grafts were developed for patients with insufficientautologous tissue for bypass grafting. These grafts have reasonable safety

Stem Cells in Tissue-Engineered Blood Vessels 117

profiles, satisfactory surgical handling characteristics (i.e. suture retention),and are available “off-the-shelf” for use as large caliber bypass grafts.Currently available synthetic vascular conduits, however, have severalsignificant limitations. These grafts have no growth potential, thus manypediatric patients will “outgrow” the graft and require re-operation. Re-dooperations are associated with an increased risk of complications anddeath. Bioprosthetic materials such as gluteraldehyde-fixed xeno- or allo-grafts used for grafting are prone to ectopic calcification, resulting in poordurability. Both prosthetic and bioprosthetic materials are prone to infec-tion, putting the patient at risk for sepsis, graft rupture, distal septicemboli, as well as re-operation for explantation of graft material. Lastly,the use of synthetic grafts as small caliber bypass grafts is limited as theyare prone to thrombosis and neointimal hyperplasia, likely from a lack ofbiocompatibility and an inability to repair and remodel.

Tissue-engineered vascular conduits address the shortcomings ofcurrently available vascular conduits. The ideal tissue-engineered graftpossesses excellent surgical handling characteristics. An experiencedsurgeon must be able to handle the graft, modify it as necessary for thepatient’s anatomy, perform anatosmoses using standard surgical techniqueand instrumentation, and obtain hemostasis immediately following implan-tation. Because of the morbidity and mortality associated with longeroperative and anesthetic time, the surgeon must perform these anastomosesin a timely fashion. Optimally, tissue-engineered grafts would be available“off-the shelf” (similar to prosthetic grafts), and require minimal manipu-lation other than seeding the cell on the substrate the day of surgery.

In addition to satisfactory surgical handling techniques, the idealtissue-engineered vascular graft is biocompatible. The polymerized scaf-fold or decellularized matrix should degrade over time, leaving intactvascular neotissue that provides the structural integrity for the conduit.The degradation of scaffold materials results in a completely biocompati-ble structure that is not prone to infection or ectopic calcification, anddoes not require immunosuppression. Finally, the ideal tissue-engineeredgraft possesses the intrinsic ability to grow with the patient, obviating theneed for re-operations in the pediatric population.

The most important characteristic of a tissue-engineered vascular graftis, however, recapitulation of vessel form and function. Put simply, grafts


implanted in the arterial and venous circulation should mirror as closelyas possible the native artery and vein. The ideal arterial interposition graftpossess a functional, confluent, non-thrombogenic endothelium and athick smooth muscle laden tunica media that can accommodate mean arte-rial pressure and constrict appropriately to ensure perfusion to end-organsduring low flow states. Thus, the intrinsic mechanical strength of theseeded scaffold should be higher and the degradation time longer in arte-rial grafts than in venous grafts where mechanical integrity is less criticalbut adequate compliance is necessary.

3. Approaches to Creating TEBVS

3.1 The first artificial artery: Weinberg and Bell

The first reported successful construction of a tissue-engineered vasculargraft came from Weinberg and Bell in 1986. Prior work had established theuse of synthetic materials as vascular conduits, albeit not for small diametervessels (< 6 mm diameter), and autologous tissue had become the mainstayof vascular repair. Building on previous reports of partial vascular constructs,including in vitro growth of endothelial cells and models of the vascular wallin mock circulatory loops, this seminal paper reported culturing bovinevascular cells to seed a collagen matrix in an tubular mold.8,19

Specifically, bovine smooth muscle cells were combined with collagenusing a casting culture medium to create a tubular lattice. After a week ofculture, a synthetic Dacron sleeve was placed on the outer surface of theconstruct, and seeded with fibroblasts to create a neo-adventitia. Afteranother two weeks, the grafts were seeded with endothelial cells and leftin a rotational culture (1 rev/min) for one week. This procedure provideda template for the creation of a tubular cellular structure that demonstratedextracellular matrix deposition of collagen, alignment of smooth musclecells and a confluent endothelial cell layer, in a construct that had burstpressures of up to 323 mmHg when reinforced with added layers ofDacron. This led the authors to describe the use of their graft as a modelfor the study of the biological properties of blood vessels, rather than as apotentially clinically useful construct. Despite these shortcomings,Weinberg and Bell laid the foundation and provided a roadmap for


scientists to develop artificial blood vessels, as their publication markedthe birth of vascular tissue engineering.

3.2 First clinical use of a TEBV

Congenital cardiac anomalies, a diverse spectrum of defects, result in sig-nificant perinatal morbidity and mortality. Untreated single ventricleanomalies are associated with 70% mortality in the first year of life. Thetherapy of choice is surgical reconstruction. Without surgery, survival ofthis cohort into adulthood is rare. Initially described in 1971, the Fontanoperation separates pulmonic and systemic blood flow; subsequentlydecreasing the incidence of chronic hypoxia and high output cardiacfailure. This suite of procedures requires a synthetic graft, commonlyPolytetrafluoroethylene (PTFE, or Gore-Tex®). As mentioned above,PTFE has several limitations, including potential for infection, thrombosis,and ectopic calcification.20–22 Furthermore, PTFE does not grow, resultingin patients “outgrowing” the graft and requiring re-operation, or sufferingcomplications related to intentional graft over-sizing.

The fabrication and seeding of biodegradable polymer scaffolds foruse in humans was an outgrowth of work performed in large animalmodels. Precursors to the clinical study included the creation of a tissue-engineered heart valve by Zund and others.23–26 This valve constructconsisted of biodegradable polyglycolic acid fibers serially seeded withfibroblasts and endothelial cells. These tissue-engineered valvular con-structs were implanted in the pulmonary valve position in juvenile lambs.Functional performance of the grafts was satisfactory on ultrasound, andhistological analysis revealed appropriate cellular architecture. Theseresults were validated by a larger study with acellular valve controls, inwhich the seeded scaffolds demonstrated superior functionality.27

The natural extension of tissue-engineered heart valves was thecreation of tissue-engineered vascular conduits. These tubular constructscould be fabricated from the same scaffold materials and cellularelements, with the additional advantage of less complex biomechanicalconstraints.16,28,29

Although both tissue-engineered heart valves and blood vesselsdemonstrated mechanical integrity and vascular neotissue formation


in vivo, the original seeding models relied on time-consuming expansionof endothelial cells and smooth muscle cells in vitro. This made experi-ments time consuming and vulnerable to culture contamination, inaddition to limiting the practicality of human use as each patient wouldrequire multiple procedures.30 Attention was turned to other sources ofseeded cells, with a focus on cells that could be procured on the day ofsurgical implantation of tissue-engineered grafts. Seeking an abundantcell source that did not require ex vivo tissue culture expansion,Matsumura and colleagues seeded polymer scaffolds with autologousbone marrow and implanted these constructs into the inferior venacava of dogs.31,32 Not only did these grafts remain patent, histologicalanalysis revealed cell populations elaborating vascular endothelialgrowth factor (VEGF) and expressing endothelial or smooth muscle cellmarkers.31

The first reported use of a tissue-engineered graft in humans occurredin 1999, in a four-year-old girl with a single right ventricle and pulmonaryatresia.17 At the age of three she had undergone pulmonary artery angio-plasty and the Fontan procedure. Subsequent angiography revealed totalocclusion of the right intermediate pulmonary artery. A 2 cm segment ofperipheral vein was explanted and cells expanded ex vivo. Cells wereseeded onto a caprolactone–polylactic acid copolymer scaffold,reinforced with woven polyglycolic acid. The occluded pulmonary arterywas successfully reconstructed with the tissue-engineered graft. No post-operative complications were reported. On follow-up angiography, thetransplanted vessel was patent with no signs of aneurismal dilation.

Promising large animal study data and the first successful humanapplication of a tissue-engineered graft provided the impetus for a largerclinical study. From May 2000 to December 2004, 25 patients (mean age5.5 years) with single ventricle physiology were implanted with tissue-engineered grafts at Tokyo Women’s Medical University. Extra cardiactotal cavopulmonary connection (EC TCPC) conduits (a connectionbetween the vena cava and the pulmonary arteries) for surgical correctionof single ventricle physiology represented an ideal hemodynamic startingpoint for tissue-engineered grafts in humans. In this system, high flowrates minimize thrombotic risks while relatively low pressure minimizeswall tension on the conduit. This pilot study evaluated two types of


scaffolds, PCLA-PGA (n = 11) and PCLA-PLA (n = 14). Autologous bone marrow (5 mm/Kg BW), aspirated from the patient’s ilium under generalanesthesia, was used to seed the polymer scaffold and the construct wasincubated for two hours in media prior to implantation. All 25 patientssurvived to hospital discharge.33 Duplex ultrasonography, computedtomography, magnetic resonance angiography, and cineangiography wereutilized for graft surveillance (Fig. 1). All grafts remained patent, and noaneurismal dilation was detected with any imaging modality.34 Of note,


Fig. 1. Three-dimensional computed tomography reconstruction of in vivo TEBV. Tissueengineered vascular graft in a 13-year-old with single ventricle physiology. The graftconnects the superior vena cava to the pulmonary arteries. Three-dimensional recon-structed computed tomography of the heart, great vessels, and tissue engineered vasculargraft demonstrate a widely patent graft with no evidence of stenosis, thrombosis, oraneurismal dilation.

six patients developed silent graft stenosis, and four underwent successfulballoon angioplasty. Four patients died during follow-up (mean follow-up5.8 years). The causes of death, however, were not related to tissue-engineered graft dysfunction; all four patients had imaging demonstratinga widely patent graft prior to death. Of the 21 surviving patients, 18 wereclassified as New York Heart Association (NYHA) Class I (no impairmentof physical activity) and 3 were classified as NYHA Class II (mild impair-ment of physical activity).

Although the Tokyo Women’s study remains a landmark in tissueengineering, this data represents a single institutions experience andpatients were not randomized. Additionally, although autologous bonemarrow cells remain an attractive cell source for tissue-engineered graftseeding, no histological specimens exist from this clinical trial (autopsiesare not usually performed in Japan, and most patients are still alive). Thefate of seeded bone marrow, the optimal seeded cell number, and the roleof postoperative anticoagulation remains to be determined.

3.3 Sheet-based tissue engineering

In the continuing search for an ideal conduit to repair damaged humanvessels, L’Heureux et al. employed a new methodology termed TissueEngineering by Self-Assembly (TESA) to produce human vessels in vitro.TESA channels the ability of cells of mesenchymal origin to secrete andassemble large amounts of ECM in various geometries. Sheet-based tis-sue engineering (SBTE), a variation of TESA, employs sheets of livingcells and the natural ECM they synthesized to produce tubular structuresand create vessel-like structures. In developing SBTE, L’Heureux et al.sought to overcome the need to use synthetic material for structural sup-port of biological grafts as was the case for Weinberg and Bell.8,14,35 In thisapproach only cellular components were employed in the creation of theirartificial blood vessel.

The first TEVG produced by SBTE contained only human skin fibrob-lasts cultured from dermal specimens removed during reductive breastsurgery; smooth muscle cells and endothelial cells were isolated fromnewborn umbilical veins or adult saphaneous veins. Nonetheless, it had aburst pressure of 2594 ± 501 mmHg.14,36 Cell sheets were produced in


culture medium supplemented with 50 μg/ml of sodium ascorbate, peeledoff from culture flasks and tubularized around inert tubular support cylin-ders. After a maturation period of at least eight weeks, the sheets becomecohesive tubes that can be seeded with endothelial cells. The endotheliallayer of the constructs expressed von Willenbrand factor immunohisto-chemical staining and demonstrated ac-LDL uptake; smooth muscle cellsstained positively for alpha-smooth muscle actin and desmin. Fibroblastsdid not stain for smooth muscle cell markers, but expressed vimentin andsynthesized elastin, which organized in circular arrays. The ECMcontained laminin, fibronectin, chondroitin sulfates and various collagensubtypes. Further research demonstrated that these artificial blood vessels retained concentration dependent contractile properties, responsiveto various vasoconstricting (histamine, bradykinin, ATP) and vasodilating(sodium nitroprusside, SIN-1, forskolin) agents with and without inhibitory agents.36

In the following years, the model was simplified to exclude smoothmuscle cells and was shown to be feasible using skin fibroblasts andvenous endothelium isolated from elderly patients with cardiovascular dis-ease. Preclinical studies in immunodeficient nude rats, evaluated for up to225 days, demonstrated patency rates of 85% without aneurism as abdom-inal aorta interpositional grafts. To study the in vivo development of thesegrafts in a more representative biomechanical environment, the tissue-engineered blood vessels were implanted in immunosuppressedcynomolgus primates. Explants at six and eight weeks demonstrated patentvessels with no signs of luminal narrowing or aneurismal formation.Alpha-actin positive smooth muscle cells and proteoglycan expression wasalso observed in both models.36,37

In 2007, L’Heureux and McAllister reported the second clinical use ofvascular tissue-engineered constructs when they described the implanta-tion of their tissue-engineered vascular graft in three patients undergoinghemodialysis treatment (Fig. 2).18 These dialysis patients had a history ofpreviously failed access grafts. After 24 patient-months of follow-up forthis cohort, only one of the grafts used for dialysis access had a thrombo-genic failure, secondary to low postoperative flow rate and moderatedilatation of the graft. McAllister et al. then expanded their cohort to tenpatients in total, drawing from two centers, in Argentina and in Poland.38


Three out of the ten implanted grafts failed within the safety phase of theirstudy, and one patient required surgical re-intervention to maintainpatency at 11 months. These results are consistent with the expected fail-ure rates with native hemodialysis access grafts (fistula) in such high-riskpatient populations. Overall, after six months, the artificial graftsimplanted as hemodialysis access grafts had a 60% primary patency ratewhich is superior to that of the standard of care synthetic graft (ePTFE).

3.4 Stem cells for vascular tissue engineering

During the past decade, significant attention has been turned to the use ofstem cells in tissue engineered vascular grafts. Stem cells and endothelialprogenitor cells can differentiate into vascular lineages and thus have thepotential to repair vascular systems. Despite their obvious potential in clin-ical practice, there still remain many controversies regarding how EPCsactually enhance endothelial repair and neovascularization.39 Additionally,because of the limited expansion ability of EPCs, expansion of sufficientEPC populations for therapeutic angiogenesis remains a significantimpediment for most investigators. On the other hand, embryonic stem


Fig. 2. The tissue-engineered blood vessel preoperatively (A), at three Months afterimplantation (B, computed tomographic angiography). VA: venous anastomosis, AA:arterial anastomosis. Reproduced with permission from Ref. 18. Copyright © 2007Massachusetts Medical Society. All rights reserved.

cells have an extensive self-renewal activity and can be expanded withoutlimit, thus ES cell-derived endothelial cells may be feasible as a novel cellsource for therapeutic angiogenesis.40,41

Nourse and colleagues used VEGF to induce differentiation of func-tional endothelium from human embryonic stem cells.42 ContinuousVEGF treatment of embryonic stem cells resulted in a four- to five-foldenrichment of CD31(+) cells but did not increase endothelial proliferationrates, suggesting a primary effect on differentiation. CD31(+) cells puri-fied from differentiating embyroid bodies upregulated ICAM-1 andVCAM-1 in response to TNF-α, confirming their ability to function asendothelial cells. Collagen gel constructs containing the human embry-onic stem cell derived endothelial cells and implanted into infarcted nuderat hearts formed dense networks of patent vessels filled with host bloodcells. Thus Nourse et al. demonstrated the ability of human embryonicstem cell derived endothelial cells to facilitate neovascularization oftissue-engineered constructs.

In addition to driving ES cells into an endothelial lineage, investiga-tors have turned to embryonic stem cell derived smooth muscle cells as apotential cell source in cardiovascular tissue engineering.43,44 NADPHoxidase (Nox4) over-expression in embryonic stem cell culture resulted inincreased smooth muscle cell marker production, whereas knockdown ofNox4 induced a decrease in production. Moreover, Nox4 was demon-strated to drive smooth muscle differentiation through generation ofH2O2.

45 Thus Nox4 expression maintains differentiation status and func-tional features of stem cell-derived smooth muscle cells, highlighting itsimpact on vessel formation in vivo. It is clear that embryonic stem cellsand endothelial progenitor cells will be important in vascular tissue engi-neering in the future, and endothelial cell and smooth muscle cells derivedfrom embryonic stem cell culture will require further characterization.

Autologous, readily available, bone marrow mononuclear cells(BMC) in both human and animal models promote angiogenesis, earlyendothelialization, and contain multi-potent cells that can also form partof the growing neovessel.46 In trying to develop a TEVG, Matsumanaet al., reported initial success with the use of harvested vessel wall cellsafter isolation and culture. They found that this was a time consumingprocess that required previous hospitalization for vessel harvesting and


that in half of the cases they were not able to obtain sufficient cells on theday of surgery. As a result, this group turned to BMC as the cell sourcefor their constructs, removing the impediments of long-term cell cultureand contamination, and more importantly, providing sufficient cells forthe seeding of biodegradable scaffolds.34,47–49 In the first human clinicaltrial with long term follow-up,50 25 TEVG were implanted using autolo-gous BM derived mononuclear cells with no evidence of aneurysmformation, graft rupture, graft infection, or ectopic calcification; fourpatients had graft stenosis and underwent successful percutaneous angio-plasty. These initial findings demonstrated the feasibility and safety ofthis technique and provided evidence for the use of BMC for the con-struction of tissue engineered vascular graft. (Clinical studies describedin detail in section 3.2.)

4. Conclusion

The history of reconstructive medicine and surgery dates back centuriesas physicians and scientists have continually sought to restore function todamaged tissue. Vascular tissue engineering began its path towards clini-cal utility with the construction of the first blood vessel model byWeinberg and Bell. In the ensuing years, several major breakthroughs inbiological vascular graft production have led this young field into its firstsuccessful clinical applications. Vigorous research continues, as theseclinical trials are in their early phase; it is a matter of time before they areattempted in the United States under FDA guidance.

In the last 30 years we have witnessed the success, and the variouspros and cons of the methodologies used to create neovessels. The firstgeneration of tissue-engineered arterial grafts represent a significant stepforward, but several challenges remain to be addressed. Approaches thatrely on bioreactors require significant culture time, preclude “off theshelf” availability and place the conduit at risk for contamination. Also,the cell source remains challenging, as adult human SMC’s have limitednumber of passages in vitro, precluding the development of a well-developed tunica media. Finally, standardized imaging algorithms forimplanted grafts will provide a more structured analysis of graft patency,dilation, and/or stenosis.


Undoubtedly, new approaches and techniques have yet to be devel-oped. Modern technology in polymer fabrication or assembly may yield amore ideal scaffolding material. Advances in cell culture techniques mayallow for faster production of artificial tissue. Ultimately, a deeper under-standing of the signaling cascades involved in cellular interactions inneotissue development will be critical to the construction of third genera-tion grafts. Modification of the genetic and molecular constitution of cellsmay lead to the development of tissue with selective maturation proper-ties. Employing particle release technology, grafts may be constructed thatwould elute growth factors, cytokines or other molecular signals that mayrecruit host cell development onto scaffolding material, doing away withthe need for cell seeding altogether.

As this technology develops, successful clinical applicability will beparamount to future implementation. The focus in the field remains onresponsible development of modern tools to combat disease that affect alarge portion of the population. Potential widespread use for TEBVs will bedependent on patient safety, and functionality of TEBVs that is as least equalto currently used synthetic grafts. Through landmark accomplishments andcontinued clinical achievements, we near the promise of artificially con-structing neotissues that replicate the function of the human body.

Acknowledgments

The authors would like to thank Nicolas L’Heureux, Ph.D. and the editorsfor their careful attention and helpful critiques.

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8

Stem Cells for Vascular Regeneration:An Engineering Approach

Laura E. Dickinson and Sharon Gerecht

1. Introduction

With advances in tissue engineering and the ever-rising number ofvascular diseases in western countries, vascular regeneration hasemerged as a major research focus. Cardiovascular disease is the numberone cause of death worldwide1 and is on the rise in developing countries,with atherosclerosis alone accounting for more than half of the deaths inwestern countries. Current treatments for vascular disease include aregimented schedule of statins or surgical methods to physically clear orbypass diseased blood vessels. But these methods only provide tempo-rary relief or simply delay the onset of complications. Recently, cellulartherapies have emerged as a promising approach to vascular disease.Stem and progenitor cells are capable of differentiating into vascularlineages and thus have the potential to repair injured vasculature.Delivery of stem or progenitor cells could induce angiogenesis, theformation of blood vessels from the existing vasculature, and the regen-eration of diseased tissues.2

135

In human vascular tissue, blood vessels are composed of an internalmonolayer of endothelial cells (ECs) surrounded by a thicker external shellof smooth muscle cells (SMCs). SMCs provide structural support to stabi-lize endothelial cells and also regulate vascular functions during vesselgrowth, such as modulating blood flow and maintaining vasoconstriction.3

During angiogenesis, remodeling of the extracellular environmentsurrounding blood vessels occurs to allow the migration and integration ofECs and SMCs.4 For successful vascular regeneration, ECs and SMCsneed to integrate with each other and with the host vasculature. Thus far,one of the major obstacles in vascular regenerative medicine is derivingcell populations in vitro that can be translated in vivo for patient therapy.Biocompatible scaffolds can provide microenvironments conducive to cellattachment, differentiation, and integrated three-dimensional (3D) vascularformation. This chapter discusses current approaches to vascular regenera-tion and therapies using stem cells (SCs): it will explore various cellsources and their potential for vascular regeneration and the engineeringapproaches to vascular differentiation, focusing on biomaterials for theformation of 3D vascular structures.

2. Cell Sources

2.1 Embryonic stem cells

Embryonic SCs (ESCs) are derived from the inner cell mass of blasto-cysts of the developing embryo; are pluripotent, capable ofdifferentiating into every somatic cell type; and have unlimited capacityfor self-renewal. In vivo, the extracellular microenvironment presentsinstructive biochemical cues that govern sequential cell fate decisionsand differentiation. In vitro, spontaneous differentiation of human ESCsbegins with the formation of embryoid bodies (EBs).5 This occurs afterthe withdrawal of factors that maintain ESCs in their undifferentiatedstate. Culturing human ESCs in suspension results in cell aggregation,differentiation, and the formation of EBs,5 which recapitulate limitedaspects of embryonic development and contain a variety of spontaneouslydifferentiated cell types (including integrated vasculature networks)(Fig. 1A).6–8 However, since EBs contain various cell types, spontaneous

136 L. E. Dickinson and S. Gerecht

differentiation of ECs and SMCs from EBs is not efficient. To achievea purified culture of vascular cells, desired progenitor cell types areisolated from EBs and supplemented with exogenous growth factorsor specific extracellular matrix (ECM) components to further guidedifferentiation.

Stem Cells for Vascular Regeneration 137

Fig. 1. Vasculature structures along embryoid body formation. (A) Confocal microscopyanalysis of a vasculature at different time points of EB development reveal progress inarrangement of PECAM1+ cells and the pick in arrangement of CD34+ cells after fourweeks of development. Reprinted from Ref. 8 with permission from John Wiley & Sons,Inc. (B) hESC-derived ECs display cobblestone morphology, VE-cad at cell-cell junctionsin immunofluorescence, and cord formation in Matrigel. Human ESC-derived SMCsexhibit spindle-shaped morphology, and highly express SM markers α-SM actin and SM-myosin heavy chain. α -SMA+ cells surround ECs and form microvessels that carry mouseblood when Matrigel containing hESC-derived ECs and SMCs was transplanted into nudemice. Scale bar = 50 μ m (Reprinted from Ref. 9 with permission from Lippincott Williamsand Wilkins.)

Differentiated hESCs contain a population of vascular progenitor cellswith the ability to differentiate into both endothelial-like and smoothmuscle (SM)-like cells.9 Vascular progenitor cells, isolated from EBs atday 10 by the expression of the specific endothelial/hematopoietic markerCD34, are selectively induced to differentiate into either endothelial orSM-like cells when cultured in supplemental medium containing vascularendothelial growth factor (VEGF) or platelet-derived growth factor(PDGF), respectively (Fig. 1B).9 VEGF is a pro-angiogenic factor knownto stimulate angiogenesis both in vitro and in vivo, and induce the endothe-lial and hematopoietic differentiation of hESCs.7 ECs derived from EBsform vascular structures both in vitro and in vivo.6,7 When ECs and SM-likecells embedded in Matrigel scaffolds were implanted in severe combinedimmunodeficient (SCID) mice, Ferreira et al. reported the formation ofmicrovasculatures that functionally integrated with the hosts (Fig. 1B). Asboth endothelial and SMCs are integral in blood vessel formation, implant-ing both hESC-derived cell types may provide more effective vascularregenerative therapies.10

Spontaneous and induced differentiation of ESCs can occur through3-dimension EB formations, as well as in 2-dimension adherent cultures.Endothelial progenitor cells have been isolated from EBs and culturedtwo-dimensionally for final differentiation into vascular cell lineages.9

Two-dimensional differentiation protocols incorporate cell feeder layersor ECM components that provide specific secreted biochemicalmolecules or proteins known to promote SC attachment and enhance dif-ferentiation. Various ECM components, such as collagen or fibronectin,have been implicated for their role in guiding differentiation. Thesemacromolecules provide cell adhesion sites and influence cell fatethrough integrin-mediated signaling events. Collagens are the mostabundant ECM component. Specifically, collagen IV is implicated inmesodermal differentiation.7,11 Undifferentiated ESCs cultured oncollagen IV substrates present an efficient mesodermal differentiationmodel.11 Enhanced differentiation to ECs or SMCs is observed withsupplementation of VEGF or PDGF, respectively.7

Co-culturing pluripotent SCs with terminally differentiated cellsprovides an interactive environment that allows the exchange of secretedcytokines and regulatory signals necessary for cell fate decisions. Culturing


differentiating SCs with specific stromal cell lines to promote vasculardifferentiation has been extensively studied. Human ESCs have been dif-ferentiated in vitro into hematopoietic precursors on 2D mouse bonemarrow (BM) cells and endothelial feeder cells. Co-culturing hESCs withBM or yolk sac ECs can lead to differentiation into early hematopoieticprecursors with the potential to differentiate into mature ECs.12 ECs andSMCs derived from hESCs already demonstrate regenerative potential infacilitating neovascularization and improving blood flow in vascularinjury models,10 and therefore are a promising source for cell-based regen-erative therapies.

The successful differentiation of SCs to ECs or SMCs is characterizedby cellular morphology, function, and the presence of specific cellularmarkers and proteins. ECs display a cobblestone morphology andexpress specific endothelial markers, such as vascular endothelialcadherin (VE-cad), platelet EC adhesion molecule-1 (PECAM1), CD34,vascular endothelial growth factor receptor 2 (VEGFR2), and vonWillebrand factor (VWF). Functional ECs also have the capacity to formcapillary-like structures in vitro; to express endothelial nitric oxidesynthase (eNOS), a protein that generates nitrous oxide in blood vesselsand regulates vascular function; and to uptake vascular EC labelingreagent Dil-Ac-LDL.13 SMCs, on the other hand, have a spindle-shapedmorphology and exhibit the functional ability to contract/relax inresponse to pharmacological agents. In addition, SMCs express specificmarkers restricted to contractile MCs; α -SM actin, SM myosin heavychain, calponin, caldesmon, SM22, and smoothelin.14

2.2 Mesenchymal stem cells

Mesenchymal SCs (MSCs) are multipotent SCs derived mainly from BMor adipose tissue. Unlike hESCs, MSCs are easy to isolate and expand,and differentiate into cell types of mesodermal lineages, includingvascular cells.15 MSCs have demonstrated the ability to regenerate vesselintegrity at sites of vascular injury and to enhance angiogenesis throughthe release of pro-angiogenic factors.16 Supplemental VEGF and basicfibroblast growth factor (bFGF), known to mediate proliferation andmigration of MSCs,17 can successfully induce differentiation of BM


MSCs into vascular endothelium-like cells.18 MSCs have been shown todifferentiate in vitro into SMCs and to form blood vessel walls similar tonative vessels,19 making them another potential cell source for vascularregeneration applications and therapies.

2.3 Endothelial progenitor cells

Progenitor cells are another viable cell source for vascular regenerationtherapies. Endothelial progenitor cells (EPCs), first isolated from humanperipheral blood in 1997,20 are capable of differentiating into matureECs in response to such stimuli as growth factors, cytokines, andmechanical shear stresses.21,22 EPCs are capable of mobilizing from BMto ischemic tissue to facilitate neovascularization.20 This process isinitiated by hypoxia, which induces the production and secretion of pro-angiogenic factors, such as VEGF, and recruits EPCs to sites of injury.Isolation of EPCs from additional sources (beside the peripheral circu-lation and BM) has been demonstrated, including umbilical cord blood,liver tissue, or vascular walls themselves.23–26 Unlike pluripotent SCs,progenitor stem cells differentiate into a predetermined cell type andhave a finite capacity for self-renewal. However, EPCs have the benefitof providing patient-specific therapies since these cells can be isolatedfrom individual patients.

2.4 Induced pluripotent stem cells (IPSCs)

Human somatic cells, such as fibroblasts, transfected with stem-cell asso-ciated genes can be reprogrammed (induced) to pluripotent SCs (IPSCs)that exhibit the essential characteristics of hESCs.27,28 IPSCs maintain thedevelopmental potential to differentiate into cell types from all three germlayers and could allow the generation of patient-specific pluripotent cellsfor regenerative medicine.27 Recently, human IPSCs have been differenti-ated into both ECs and mural cells by culturing on OP9 murine BM cells,the same method used to differentiate hESCs.29 The ECs and mural cellsdifferentiated from IPSCs displayed nearly identical properties to thosefrom hESCs, indicating the potential of IPSCs for vascular regenerationtherapies.



3. Engineering Vascular Differentiation

Many different approaches have been employed to differentiate organizedvascular structures from embryonic and adult SCs, both in vitro andin vivo. As discussed above, instructive biochemical cues, such as growthfactors or ECM components, provide culture environments that guide andregulate the efficient differentiation of SCs to vascular cell types.However, engineering microenvironments that incorporate the biome-chanical or biophysical cues observed during angiogenesis also directvascular differentiation. Herein we briefly discuss how mechanical stim-ulation or surface topography is utilized to induce SC differentiation toendothelial and smooth muscle cell phenotypes.

3.1 Mechanical stimulation

In addition to growth factors, mechanical forces are also known to regu-late cellular functions and to induce lineage-specific differentiation of SCsin vitro. Mechanical stimuli that mimic the physiological environments ofblood vessels have been shown to accelerate the endothelial maturation ofEPCs and to induce differentiation of murine ESCs.21 Reproducing theshear stress that cells experience during blood flow in arteries stimulatescellular differentiation. By exposing ESC cultures to a laminar flow, cellsproliferate and differentiate into vascular ECs,30 whereas cyclical straininduces vascular SMC differentiation.31 Under a pulsatile flow loadingsystem that simulates both hydrodynamic shear stress and circumferentialstrain, as would be experienced in human veins, ESCs differentiated intovascular wall cells, including both endothelial-like and SM-like cellssimultaneously.32 Another recently engineered system subjected hMSCsgrown on tubular silicone substrates to shear flow, radial flow, andpulsatile pressure to induce the expression of ECs and SMCs.33

3.2 Surface topography

The vascular microenvironment is a topographically complex milieu,providing the structural platform necessary for tube morphogenesis. Manyof the cellular interactions within this environment occur in the micron


and sub-micron regime. In fact, synthetic micro- and nanoscale substrateshave been shown to influence the cellular functions of ECs and SMCs viacontact guidance, and induce their organization into vascular struc-tures.34,35 Human EPCs, when cultured on submicron line gratingsubstrates, align in the direction of the features and are capable of form-ing supercellular band structures compared to hEPCs grown on flatsubstrates.34 The addition of Matrigel induced hEPCs to form extensivecapillary networks with longer average tube lengths than hEPCs merelygrown on flat substrates (Fig. 2A). This indicates that hEPCs, as well asECs and SMCs, can potentially be stimulated by topographical cues toform vascular structures in vitro.

4. Three-Dimensional Space

Reconstructing 3D environments that mimic biochemical and biophysi-cal cues in vivo for vascular differentiation and functional regeneration isa developing field of research. In hESC biology, the surrounding 3Dmicroenvironment influences cellular function and differentiationthrough interactions with the ECM components and neighboring cells.The ECM is highly involved in vascular development and regenerationby providing the scaffold necessary to stabilize the organization of ECsto functional endothelium within its integrated fiber and proteinnetworks,36 while also presenting instructive biochemical cues to regu-late and support cell proliferation, migration and differentiation.37 TheECM provides structural support for the vascular endothelium by actingas a scaffold to stabilize the organization of ECs into blood vessels, aswell as support cell proliferation, migration, and survival.38 Therefore,ex vivo vascular engineering generally involves the use of scaffoldswhich are designed to provide 3D structural and logistical templates fortissue development, to control the cellular microenvironment, and toprovide the necessary molecular and physical regulatory signals. Thus,the development of a 3D scaffold compatible with vascular stem andprogenitor cells will provide an experimental approach to direct vasculardifferentiation and assembly.


4.1 Biomaterials for vascular differentiation

Biomaterial scaffolds provide a 3D support to mimic the ECM and pro-mote cell attachment and growth and are of synthetically biodegradablematerials, natural materials, or a composite of both. Indeed, biomaterialshave been fabricated into porous, fibrous, or hydrogel scaffolds; each withdistinct properties, as summarized in Table 1. A fibrous scaffold mimics the

Fig. 2. Vascular differentiation and assembly. (A) (i) Human EPCs cultured on nanoto-pographical substrates align in supercellular band structures when compared to EPCscultured on flat substrates. Morphological differences are evident through expression ofendothelial cell phenotypes, VECAD and PECAM1 (scale bar = 50 μm), as well as capil-lary tube formation (scale bar = 200 μm). On flat substrates, EPCs form a low density ofunorganized capillary tubes, but form extensive networks of organized structures withlonger average capillary lengths on nanosubstrates (ii). (Reproduced from Ref. 34 withpermission from © Wiley-VCH Verlag GmbH & Co. KGaA.) (B) Differentiation of hESCsinto vascular lineages. Cell sprouting from hESC colonies observed after 48 hours in HAhydrogels with VEGF-containing media. Elongating cells are positive for vascular α -SMactin and endothelial CD34. (Reprinted from Ref. 41 with permission from © 2007National Academy of Sciences, USA.)

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. E. D

ickinson and S. Gerecht

Table 1. Biomaterials for Vascular Regeneration.

Natural/Polymer Synthetic Reason for Use Advantages Disadvantages Results

Alginate Natural Gels under Low toxicity Loss of mechanical Allows efficient EB formation;polysaccharide gentle conditions stiffness over time induces vasculogenesis

when ionically in EBs39

crosslinkedDextran Natural Functional groups Biodegradable; Not enzymatically Enhances vascular

polysaccharide make it amenable biocompatible degradable in vivo; differentiation ofto modifications slow tissue hESCs and maturation

integration of EPCsHyaluronic acid Natural ECM component, Biocompatible, Poor mechanical Supports hESC propagation;

polysaccharide ubiquitous during biodegradable properties and stimulates EC proliferationdevelopment; cell attachmentregulatesangiogenesis

Fibrin Natural protein Involved in blood Biocompatible; produced Poor mechanical Supports BM progenitorclotting, in situ from patients (no properties cell differentiation andcapillary formation potential inflammatory vessel formation

responses)

(Continued )

Stem C

ells for Vascular Regeneration

145

Table 1. (Continued )

Natural/Polymer Synthetic Reason for Use Advantages Disadvantages Results

Poly (l-lactic Synthetic Provides mechanical Supports 3D structures Promotes 3D acid) (PLLA) stiffness vascularization in vivo

Poly(lactic- Synthetic Facilitates cellular Quickly degrades into Promotes in vivo ingrowth ofco-[glycolic and tissue ingrowth easily metabolized vascular tissue, cell adhesion,acid]) (PLGA) products and proliferation

Poly (glycolic- Synthetic Elastomeric material Biocompatible Promotes tissue ingrowth,co-sebacate) that mimics ECM integrated vascularizationacrylate in vivo, hESC encapsulation,(PGSA) proliferation

Polycapro-lactone Synthetic Long-term degradation Biocompatible; Supports EC adhesion,supports in vivo biodegradable into proliferation, and spreadingapplications fragments eliminated

by macrophages;high elasticity54

3D — three-dimensional, BM — bone marrow, EB — embryoid body, EC — endothelial cell, ECM — extracellular matrix, EPC — endothelial progenitor cell, hESC — human

embryonic stem cell.


fiber network of the native ECM (composed predominantly of collagenand elastin), while porous scaffolds are produced with macroscopicvoids that enable cellular interaction.39 Hydrogels are water-swollen,crosslinked polymers fabricated from synthetic or natural biomaterialsthat provide structural support and can direct differentiation of hESCs toform vascular networks in vitro.40,41 Because their capacity to absorb andretain large amounts of water mimics the structural properties of the ECM,hydrogels have been investigated for their potential as drug deliverydevices, tissue engineering scaffolds, and cell culture substrates.42

Furthermore, hydrogels can be engineered specifically for vascular regen-eration applications by optimizing controllable mechanical, degradation,and structural properties via compositional modifications, crosslinkingratios, etc.

Ideal scaffolds have been identified as having the following properties:

(1) porous and permeable to allow cell growth, migration, and interac-tion, as well as the transport of nutrients,

(2) biodegradable to match in vivo/in vitro tissue growth with nontoxic,easily eliminated by products,

(3) biocompatible and with a surface chemistry suitable for promotingcell attachment, proliferation, and differentiation,

(4) mechanically favorable to support cellular organization, and(5) shapeable, to conform to different geometries and sizes.43

These properties are controllable in scaffold design to generate a constructthat can support and guide the regeneration of vascular tissue eitherin vitro or in vivo.

4.1.1 Natural biomaterials

Biomaterials derived from ECM components or plants are naturally bioac-tive, biocompatible, and biodegradable. Alginate, one such naturallyderived biomaterial, is a polysaccharide with homopolymeric blocks of1,4-linked β-D-mannuronic and α-L-gluronic residues and is isolated fromthe cell walls of brown seaweed. Alginate polymers can be ionicallycrosslinked to form a hydrogel by addition of divalent cations, calcium,


and barium, and have been extensively studied as 3D scaffolds compatiblewith many cell types for culture and encapsulation.44 In fact, 3D porousalginate scaffolds promote efficient EB formation with a high degree ofcell proliferation and differentiation when used to culture undifferentiatedhESCs.39 In fact, EB formation is predominantly restricted to scaffoldpores, resulting in highly vascularized EBs containing microvascularnetworks and tube-like structures. This indicates that the physicalconfinement exerted by the porous alginate scaffold is sufficient to inducevasculogenesis in differentiating EBs. However, alginate is not enzymati-cally degradable in mammals and therefore poses potential barriers fortissue integration with host vasculature.

Another biomaterial, dextran, is a natural, branched polysaccharidesynthesized from sucrose using lactic acid bacteria, such as Streptococcusmutans, and is composed of α-1,6-linked D-glucopyranose residues.Pendant functional groups, such as –OH, make dextran amenable to chem-ical modifications for greater flexibility in the formulation ofdextran-based hydrogels. Modifying dextran-based hydrogels with celladhesive ligands, such laminin- or fibronectin-derived Arg-Gly-Asp(RGD) peptides, increases the potential for using dextran hydrogels for tis-sue engineering applications by enhancing cell adhesion and survival.45

Recently, bioactive dextran-based hydrogels were shown to enhance thevascular differentiation of hESCs. Ferreira et al. showed that encapsulated,undifferentiated hESC aggregates differentiated and formed EBs in RGD-modified dextran-based hydrogels with microencapsulated VEGF. At day10, encapsulated EBs showed increased expression and localization of theendothelial markers CD34 and PECAM1, along with the formation of aprimitive vascular network. When the cells were removed from the dextranhydrogel encapsulation and cultured in vascular differentiation media,specific proliferation occurred along the vascular lineage.40 This demon-strates the potential of functionalized dextran hydrogels to derive largequantities of ECs for vascular regeneration from hESCs. Dextran-basedhydrogels can also function as 3D scaffold materials for potential in vivotransplantation. Bifunctional dextran, modified with methacrylate andaldehyde with incorporated gelatin, mimics the complex composition andtopography of the ECM. These novel dextran-modified hydrogels promoteEC adhesion and support the spreading and proliferation of SMCs.46

One of the chief components of the ECM is the glycosaminoglycanhyaluronic acid (HA), a non-sulfated linear polysaccharide consisting ofglucuronic acid and N-acetylglucosamine residues. HA is involved inmany hESC cellular processes, including adhesion, morphogenesis,proliferation, and motility in vivo.47 The regulation of these cellularevents is attributed to the interaction of HA with the cell surface recep-tors CD44 and receptor for HA Mediated Mobility (RHAMM), whichtransduce intracellular signals. CD44 is known to mediate HA-inducedcell proliferation and survival pathways, while RHAMM is involved inHA-induced cell motility, signaling, and differentiation via extra- andintracellular pathways.47 RHAMM also has been implicated in the main-tenance of hESC pluripotency, viability, and cell cycle control.48

Undifferentiated hESCs have been found to express high levels of HA,CD44, and RHAMM in culture. Similarly, the developing embryo is sur-rounded by a high concentration of HA during early embryogenesis,which decreases with the onset of differentiation.41 In fact, the inner cellmass cells of the developing embryo are embedded in HA-rich 3D matri-ces that regulate their self-renewal and differentiation.49 In addition to arole in embryonic development, HA regulates angiogenesis by stimulat-ing EC proliferation, migration, and sprouting.50 HA hydrogels wererecently investigated to determine their effect on hESCs in vitro. Theywere discovered to create a microenvironment conducive to the propaga-tion of undifferentiated hESCs.41 When encapsulated in 3D hydrogelscomposed entirely of HA, colonies of hESCs maintained their undiffer-entiated state of self-renewal and preserved their normal geneticintegrity. EB formation from hESC-HA released cells, indicates thathESCs cultured in HA hydrogels retain their full differentiation potential,and can switch to vascular differentiation with the introduction of angio-genic factors. The addition of VEGF to the differentiation medium in HAgels induced cell sprouting and elongation within 48 hours. The presenceof specific endothelial and SM markers, CD34 and α-SM actin, confirmedthat these cells are indeed of vascular lineages (Fig. 2B). These HAhydrogels could be used as a method to enhance the propagation ofhESCs being further differentiated into vascular/endothelial lineages forregeneration applications. Already, HA-based scaffolds have provento be a promising biomaterial for vascular regeneration applications.HA-based scaffolds have been instrumental in developing tissue


engineered vasculatures in vitro and orchestrating the vascular remodelingevents necessary for small artery reconstruction in vivo.51

Fibrin is the major structural, fibrous protein involved in blood clot-ting and in remodeling the ECM during wound healing. Capillary growthin situ frequently occurs in a fibrin-rich extracellular environment. Whenadult BM progenitor cells are cultured in 3D fibrin matrices in vitro,vascular structures develop that express endothelial markers (VWF,CD34, VEGFR-2) and are surrounded by mural cells expressing α-SMactin.52 Not only do fibrin scaffolds support vascular differentiation andregeneration in vitro, but implanted fibrin gels seeded with BM-derivedECs and SM progenitor cells form vessels that integrate with nativevasculature.53

4.1.2 Synthetic materials

Alternatively, synthetic materials have also been investigated for vascularregeneration applications. Advantages of using synthetic materials includethe manipulation of tunable physical parameters, such as mechanicalintegrity. Many polyester-based synthetic polymers are being investigatedbecause of their mechanical strength, biocompatibility, and degradationinto easily metabolized products, such as glycolic acids, which are elimi-nated as carbon dioxide and water.54

Poly(lactic acid), which is produced through the polymerization of lac-tic acid, has a high mechanical strength in the form of poly(L-lactic acid)(PLLA). Synthetic materials can be modified with ligands to enhance thecellular adhesion of anchorage-dependent cells, like EPCs. PLLA scaf-folds grafted with RGD peptides support the in vitro growth andendothelial functions of EPCs, as well as promote vascular regenerationin murine wound models in vivo.55 Fibers of PLLA interwoven withpolyglycolic acid (PGA) form a biodegradable scaffold conducive to engi-neering microvessels in vitro.56 EPC-derived ECs co-seeded with SMCson this bipolymeric (PGA-PLLA) scaffold spontaneously formed capil-lary and microvascular-like structures. The porous architecture of thesescaffolds provide a favorable environment for microvessel formation, andECs cultured with SMCs formed 76 ± 35 vessels/mm2.56

PGA-based copolymers, such as poly(lactic-co-glycolic acid) (PLGA),are also used for vascular regeneration applications. PGA is copolymerized


with PLA, and can create various forms of PLGA, depending on the gly-colide to lactide ratio. Shaping biomaterials into geometries that mimicvasculature is another technique to promote vessel formation.Cylindrically processed PLGA scaffolds promote the in vivo ingrowth ofvascular tissue and facilitate the adhesion and proliferation of SMCs, withseeded ECs forming lumens, demonstrating the benefits of PLGA as a scaf-fold material for vascular regeneration studies.57 Immobilization or coatingof bioactive molecules can further enhance scaffolds for vascular regener-ation. Incorporating the angiogenic factor VEGF into PLGA scaffolds forits controlled release induces the differentiation of human MSCs intoECs.58 Composite scaffolds have been generated to optimize the propertiesof different materials and have been shown to effectively enhance vascularformation. Biodegradable scaffolds consisting of a 50/50 blend of PLLAand PLGA have been fabricated to direct differentiation and to organizehESCs into tissue-like structures.59 PLLA provides the mechanical stiffnessto support 3D structures, and PLGA degrades quickly, facilitating cellularingrowth. PLLA/PLGA scaffolds coated with Matrigel or fibronectin pro-mote the cell attachment and survival of early differentiating hESCs (EBs).When supplemented with growth factors, such as retinoic acid, transform-ing growth factor B (TGF-B), activin A, or insulin-growth factor (IGF), 3Dvascular network formation — in addition to liver, cartilage, and neural tis-sues — is observed. When transplanted into SCID mice, the constructsintegrated with the host vasculature, indicating the potential of polymerscaffolds for directed in vitro 3D differentiation and in vivo transplanta-tion.59 In later studies, hESC-derived ECs cultured with mouse myoblastsand embryonic fibroblasts formed large vessels within the PLLA/PLGAcomposite constructs.60 The embryonic fibroblasts promoted stabilizationof the vessel structures by differentiating into SM-like cells (confirmed bySM actin immunofluorescence staining) and co-localizing around thehESC-derived ECs. Implantation into SCID mice improved the vascular-ization and survival of the skeletal muscle constructs, as confirmed byintegration with the host vasculature.60 In vitro vascularization of con-structs with successful in vivo integration is the major obstacle in tissueengineering, and many studies have attempted to solve this problem.

Synthetic elastomeric materials such as poly (glycerol sebacate) (PGSA),may better mimic the ECM of native blood vessels and are being investi-gated for potential use in vascular regeneration. Photocurable PGSA forms a


uniform, porous, and flexible scaffold suitable for cell encapsulation andhESC culture. Undifferentiated hESCs seeded in PGSA scaffolds prolif-erated and formed EBs within one week. Biocompatibility, tissueingrowth, and integration with the host vasculature were observed aftertransplantation, indicating the potential of PGSA as a promising materialin vascular regeneration.61

Other synthetic polymers that have been investigated for vascularregeneration include poly(ethylene glycol) (PEG) and polycaprolactone.PEG is a polymer of ethylene oxide, and PEG-based hydrogels have beenused to encapsulate SCs and direct their differentiation.62 PEG is an inher-ently hydrophilic material, but modification with adhesive peptidesenhances cell adhesion and interaction with PEG-based scaffolds.63 Arecently developed synthetic PEG-based hydrogel incorporates crosslinkedmatrix metalloproteinase (MMP)-sensitive peptides, matrix-bound RGDpeptides, and the bioactive peptide thymosin β4 (Tβ 4) to more accuratelymimic the natural collagenous ECM of vascular networks in vivo.64

Incorporating MMP-sensitive peptides produced a hydrogel matrix capa-ble of cell-mediated proteolytic degradation and remodeling,65 essential forvasculogenesis, while incorporating Tβ 4 in the PEG-based hydrogelallowed EC adhesion, migration, and vascular-like organization in vitro.

Polycaprolactone, another biodegradable polyester, has already beenapproved by the US Food and Drug Administration for vascular regenera-tion. Using polycaprolactone has the major advantage of slowly degradingin vivo into fragments that are easily eliminated by macrophages.54 Tubularnanofiber polycaprolactone scaffolds support EC adhesion, proliferation,and spreading, proving to be a viable option for vascular grafts using SCderived ECs.66 A scaffold composed of polycaprolactone-polylactic acidcopolymer, reinforced with polyglycolic acid and seeded with vein cells,was successfully transplanted into a four-year-old patient and successfullyreconstructed her pulmonary artery.67

4.1.3 Shaped scaffolds

For vascular regeneration and remodeling, scaffolds of a tubular structureare desirable to direct vascular assembly and structure formation. Tubularscaffolds have been engineered using electrospinning techniques, whichproduce ultrathin synthetic fibers of a mesh, imitating fibrils of ECM.68


Soletti et al. recently developed and tested a bilayer poly(ester-urethane)urea-based scaffold obtained by combining electrospinning anda phase separation technique. The bilayer scaffold presented two concen-tric layers, with a highly cellularized, porous internal layer mimicking thetunica media and a fibrous, external electrospun layer as the adventitiallayer. Studies found that the mechanical properties of the scaffold werephysiologically consistent and comparable to native vessels, and muscle-derived SCs cultured on these scaffolds under dynamic conditionsmaintained a high cell density.68 Recently, functional vessel walls havebeen engineered in vitro by optimizing these factors. Scaffolds coatedwith the ECM matrix protein fibronectin and subjected to cyclic straininduced human MSCs to differentiate into SMCs and form small-diameterhuman vessel walls.19

Acknowledgments

We would like to acknowledge funding from the AHA-Scientist Develop-ment grant, and a March of Dimes-O’Conner Starter Scholar award (forS.G). LED is an IGERT trainee and a National Science FoundationGraduate Fellow.

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9

Stem Cells and Wound Repair

Sae Hee Ko,* Allison Nauta,* Geoffrey C. Gurtnerand Michael T. Longaker

1. Clinical Burden of Wound Healing

Wound healing and the approach to acute and chronic wounds are clinicalconcerns that are not always adequately addressed by current therapeuticinterventions. The ability to heal wounds can complicated by multiplefactors, including — but not limited to — vascular disease, diabetes,chronic disease, smoking, infection, radiation therapy, and immunocom-promise. Scars forming after injury are problematic in that they can bedisfiguring and limit the functional integrity of the dermis.

Diabetes has become both a national and global health concern, as theCDC estimates approximately 23.6 million people or 7.8% of the UnitedStates population currently have the disease, diagnosed or undiagnosed.1

In 2006, diabetes was the seventh leading cause of death in the UnitedStates, and in 2004, approximately 71,000 non-traumatic lower-limbamputations were performed in diabetic patients.1 In 2003, the number ofdiabetic patients worldwide was estimated at 197 million, and this number

159

*These authors contributed equally to this work.

is projected to increase to 366 million by 2030 due to increased longevity.2

Limb ulcerations, infections, Charcot neuroarthropathy, and amputationsare estimated at billions of dollars per year in healthcare costs. Non-heal-ing lower extremity diabetic ulcers account for approximately 25%–50%of all hospital admissions in the diabetic population and are responsiblefor the majority of resultant amputations.3

Current therapeutic interventions focus on controlling factors impli-cated in poor wound healing. To limit infection, patients are givensystemic antibiotics, and chronic diseases, such as diabetes, are medicallycontrolled. Devitalized tissue can provide nutrition for the wound’s bacte-rial load and impair defenses. Under circumstances where there is anabundance of debris, debridement can be used to clean the wound bed andallows for reduction of pressure and evaluation of tracking and tunneling.Debridement can be accomplished through a variety of methods: surgical,biological, enzymatic, autolytic, or mechanical. Debridement must alwaysprecede the application of topical agents and dressings.4

Various dressings and topical agents are used to prepare the woundbed. Tissue moisture balance is important to maintain in order to promotegranulation tissue and autolytic processes and prevent either dessicationor the accumulation of excess fluid. Infection, which can be local, ascend-ing, or systemic, must be identified and treated with topical agents andsystemic antibiotics. Dressings can be classified as passive, providingonly occlusive function, or active and interactive, modifying the woundenvironment physiology.5

Despite these current therapeutic approaches in clinical practice,wounds continue to underheal, overheal, and scar. Growth factortherapy has shown great promise in treating wounds. For example,becaplermin gel, a DNA recombinant platelet-derived growth factor, hasbeen shown to stimulate the mechanisms of wound healing.6,7 Othergrowth factors currently under investigation include VEGF, fibroblastgrowth factor, and keratinocyte growth factor.4 However, more studiesare needed in addressing the unmet need for effective wound repair andregeneration. As such, stem cell-based therapy is promising as a novelway to approach wounds and shift the balance from scarring toregeneration of tissue.

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2. Physiology of Wound Healing

When tissue is injured, organisms naturally protect themselves againstwater loss and the invasion of microorganisms by initiating wound healing.There are three main clinical outcomes when human adult tissue encoun-ters injury. First, the tissues can heal slowly and inadequately. Second, thetissues can overheal and deposit and overabundance of fibrotic woundmatrix, as is seen with hypertropic scars and keloids. Finally, there is theoverriding issue that all injured tissue heals with a scar.

In some eukaryotes, tissue can regenerate in response to injury sothat it approaches its original phenotype and function. Human skin hasthis capacity during prenatal development, where it has been observedto heal without scar. However, through an unknown mechanism, thiscapacity is lost in adult life.8 Instead, most tissues respond to a woundby creating a patch of cells (predominantly fibroblasts), deposit disor-ganized extracellular matrix (predominantly collagen), finally resultingin scar formation.

Wound healing occurs classically in three overlapping phases —inflammation, tissue formation, and tissue remodeling (Fig. 1). With tis-sue injury, the vascular network is disrupted, and platelets are attracted tothe wound to initiate hemostasis by formation of a platelet plug. At thesame time, platelets secrete various mediators of wound healing, includ-ing platelet-derived growth factor. These mediators activate macrophagesand fibroblasts to migrate to the wound.9

Neutrophils are the first responders during the inflammatory stage.These leukocytes migrate from local blood vessels into the wound, cleans-ing the area. Macrophages then engulf debris. The proliferation processthen ensues, during which fibroblasts, macrophages, and vascular tissuesenter the wound to begin formation of granulation tissue. Fibroblastsand myofibroblasts lay down connective tissue rich in collagen, and thewound contracts through the action of myofibroblasts and matrixremodeling.

Re-epithelialization begins once granulation tissue is created, duringwhich keratinocytes migrate over the granulation tissue to create a newlayer of epidermis. In a final remodeling stage, the granulation tissuematures, during which time the synthesis and turnover of structural

Stem Cells and Wound Repair 161

162 S. H. Ko et al.

Fig. 1. Classic stages of wound repair. Inflammation (A), new tissue formation (B), andremodeling (C). Originally published by Gurtner et al.67

proteins such as collagen continues over a period of six to 24 months. Atthe end of this process, the wound is healed with a scar, which approachesapproximately 70% of the tensile strength of the original tissue.10

Overhealing of tissues has been described for over 200 years. Keloidswere first described by Alibert in 1806 as tumorous growths or “chan-croids,” a name he later amended to “cheloid” as he developed betterunderstanding of these growths.11 Keloids and hypertrophic scars arebenign dermal growths of fibrous tissue that occur in those with a predis-position, usually in those with dark pigmented skin. These lesions oftencause disfigurement and symptoms such as pain, burning, and itching.Keloids and hypertrophic scars can result from any mechanism of injury,such as trauma, inflammation, surgery, or burns, although they have alsobeen described as occurring spontaneously.12 Histology shows over-deposition of collagen and glycoprotein in the scar tissue. Therefore,keloids and hypertrophic scars represent a derailment of the mechanism ofprotective wound-healing, and difficulty in delineating at which pointderailment occurs has made these lesions notoriously challenging to treat.13

In mammals, scars result from rapid deposition of fibrotic tissue,which is likely a protective mechanism developed to prevent infection andmechanical deformation. Though this rapid deposition clearly has its ben-efits, scar formation can be detrimental, as it prevents the regenerativeprocess that is seen in other organisms. In order to shift the balance fromscar formation to regeneration, it is thought that therapies must focus onlimiting the rapid fibrotic response of tissues to injury to allow multipo-tent cells, such as stem or progenitor cells, promote regeneration.14 Theearly inflammatory phase has been identified as a target to slow the dep-osition of fibrotic tissue and formation of scar.

After hemostasis is achieved with clot formation, stages of woundhealing begin with the inflammatory cascade and proceed to proliferation,matrix remodeling, and scar formation. The early inflammatory phase thatsets in within minutes of injury largely determines areas where scar willform, as it creates signals for cytokines and growth factors dictatingmovement and organization of cells. As a result of stress, during the earlyinflammatory phase, neutrophils infiltrate the injured area, elaboratingneutrophil-specific enzymes such as metalloproteinases and collagenases.These substances, along with macrophages that invade the area, break


down large amounts of tissue with free radicals and leave the injured areadevoid of matrix. Ultimately, through migration and proliferation offibroblasts, collagen production, collagen deposition, and angiogenesis,this area is filled with scar tissue.9

As a result of the inflammatory cascade’s role in scar formation, thenecessity of inflammation to the proper healing of wounds has beendebated. Recent knockout and knockdown studies suggest that regulationand limitation of inflammatory infiltrates to the wound bed may, in fact,enhance wound healing. To illustrate this point, in 2003, Martin et al.reported data on PU.1 knockout mice devoid of macrophages and function-ing neutrophils. These mice were shown to heal at a similar rate to wild typemice but without the formation of scar. The authors found that the cytokineand growth factor profiles in these wounds differed from those in the wildtype. As a result, cell death was reduced and scar formation was mitigated.15

Additional studies have been performed on other knockout mice, con-firming the suspicion that inflammatory cells may not be essential towound healing. Szpaderska et al. studied mice with induced thrombocy-topenia, showing that, as long as hemostasis is achieved, platelets are notnecessary for the normal healing process.16 Egozi et al. experimented withmast cell deficient mice. The data from this group shows that the absenceof mast cells did not affect overall wound healing compared to the wildtype. The authors noted, however, that these mice produced decreasednumbers of neutrophils early in the inflammatory phase, suggesting thatthe few mast cells present at the beginning of wound healing are involvedin creating early activating signals.17 Athymic mice and antisense geneknockdown experiments have further confirmed the inflammatoryresponse as an appropriate target for scar treatment and prevention.18–20

Proliferation, matrix remodeling, and scar formation involve complexinteraction between cells, cytokines, growth factors, and extracellularmatrix components. This process can last up to two years after injury.Many targets in the process of inflammation and collagen synthesis andlysis have been identified to modify wound healing and promote regener-ation, including tumor necrosis factor (TNF-α), platelet-derived growthfactor (PDGF), transforming growth factor (TGF-β), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor (ILGF), and epidermal growth factor (EGF).21 These targets

164 S. H. Ko et al.

are under continual investigation for improving the regenerative capacityof tissue.

3. Stem Cells and Wound Repair

3.1 Overview of stem cell biology

During cellular development, two distinct lineages emerge as a morulabecomes a blastocyst: the trophoectoderm and the inner cell mass.Embryonic stem cells, undifferentiated cells with renewal capacity, areproduced from the inner cell mass and are able to divide multiple timeswhile still remaining in an undifferentiated state. These cells arepluripotent and can, accordingly, differentiate to any of the three primarygerm layers — endoderm, mesoderm, or ectoderm22 (Fig. 2).

In culture, embryonic stem cells can be maintained as undifferentiatedcells or induced to differentiate into specific cell lineages. Developmentof embryonic stem cell research has allowed for genetic modification, as


Fig. 2. Stem cell generation and differentiation. Embryonic stem (ES) cells are derivedfrom the inner cell mass of the blastocyst. These ES cells can be induced to differentiateinto cells of all three primary germ layers: endoderm, mesoderm, and ectoderm.

well as the study of signals and differentiation steps involved in tissuedevelopment. This research has allowed for the creation of knockout miceand the study of specific genes involved in biological processes. However,the use of embryonic stem cells remains controversial, as ethical concernsarise regarding human cloning and the industrial production of embryosto obtain cells for study, as well as the potential of these cells to becomeneoplastic. As a result, attention has somewhat been redirected to the adultstem cell population as alternative source of cells with potential toremodel diverse tissues and organs.23

Adult stem cells retain many of the same characteristics of embryonicstem cells in that they have self-renewal capabilities, long life, high poten-tial for proliferation, and multipotency. However, these cells are typicallymore restricted in their ability to differentiate, and they often are lineagespecific.24

Adult stem cells are necessary for regeneration of tissue. In manytissues, stem cells are in a quiescent state until stimulated to revert backto the cell cycle to divide. Once stimulated by extracellular cues, thesecells produce undifferentiated progenitor cells without self-renewal capa-bilities. These cells then differentiate into effector cells. The ability forstem cells to remain dormant for long periods of time allows these cells tolargely avoid the risks of DNA replication and mitosis, such as mutationand exposure to harmful metabolites.25

Adult stem cells can be found in the bone marrow or in the tissuesthemselves. Tissue stem cells have been found to have a large role inmaintaining homeostasis in areas such as the skin’s epidermis, which dis-plays a fast rate of cell turnover and high regenerative potential. When celldivision and regeneration go awry in such tissues, malignancy can result.Tissue stem cells exist in functional niches, which are tissue microenvi-ronments that shelter stem cells from stimuli for replication and apoptosis,maintaining a balance between activity and quiescence.26

3.2 Epidermal stem cell biology

The cutaneous epithelium is constituted by epidermis, dermis, hairfollicles, sebaceous glands, and sweat glands. The outer epidermis isseparated from the inner dermis by a basement membrane. Human

166 S. H. Ko et al.

interfollicular epidermis is a constantly renewed, multilayered stratifiedsquamous epithelium made up of keratinocytes.27

The proliferation of keratinocytes in the basal layer of the interfollic-ular epidermis maintains tissue homeostasis. Basal cells in the epidermisare mitotically active but initiate terminal differentiation by detachingfrom the basement membrane, withdrawing from the cell cycle, andmigrating upward to the skin surface in a columnar fashion. When thesecells divide to produce committed progenitor cells, they eventuallybecome terminally differentiated and move to the skin’s surface. Throughterminal differentiation, these cells progress through three distinct stagesin which they become the three layers of the epidermis: the spinous layer,followed by the granular layer, and finally, the keratinocytes lose theirnuclei to become the stratus corneum. These cells must be continuouslygenerated throughout life to maintain a protective barrier, as stratumcorneum cells are constantly shed. According to Weinstein et al., theprocess of epidermal regeneration occurs at an average of 39 day cyclesthroughout life. This group averaged that the mean transit times throughthe three layers of the epidermis are as follows: 13 days, 12 days, and 14days for the spinous, granular, and stratus corneum layers, respectively.27

Several molecular signaling pathways necessary for epidermal differen-tiation, stratification, and acquisition of barrier function have been identified,including Notch, mitogen-activated protein kinase, nuclear factor-κB, p63,the AP2 family, CCAAT/enhancer-binding protein transcriptional regulators,interferon regulatory factor 6, grainyhead-like 3, and Kruppel-like factor 4.28

Notch and p63 signaling pathways have been found to be critical inepithelial terminal differentiation. These pathways dictate the switch frombasal cells to spinous layer cells, as has been demonstrated in gain andloss of function in vertebrates. Senoo et al. studied p63 null mice duringembryogenesis to determine whether p63 was necessary for epidermalformation from primitive ectoderm. This group noted that mice lackingp63 had sparse areas of epidermis in comparison to wild type embryos,which showed a continuous and well-formed epidermis.29 Likewise,through conditional ablation of RBPJ (a DNA-binding protein essential tothe Notch pathway)30 and knockout studies on Hes1 (one of the mainepidermal Notch target genes),31 Notch has been determined to be essen-tial to the conversion of basal cells to spinous cells.


The hair follicle is an appendage of the mammalian epidermis that iscomposed of an external outer root sheath attached to the basal lamina andcontiguous with epidermis, a channel, and a hair shaft. The hair follicle andits sebaceous gland together are called the pilosebaceous unit. The base, orbulb, of the hair follicle contains committed but proliferating progenitor cellsor matrix encasing the dermal papilla, which are specialized mesenchymalcells. The hair and its channel grow from this region (Fig. 3).32

Two thirds of the pilosebaceous unit contributes to the hair cycle,which is a process with three stages: catagen (or degeneration), telogen(the rest phase), and anagen (growth). In conditions supporting apoptosis,

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Fig. 3. Epithelial stem cells. Epithelial stem cells are located in the bulge area of the hairfollicle, in sebaceous glands, and in the lower layer of the epidermis. This figure shows thethree layers of the epidermis: the spinous layer, the granular layer, and the stratumcorneum. During regeneration, cells migrate vertically upward to the stratum corneum,where they flatten and act briefly as a physical barrier. These cells are then sloughed off.


the hair follicle proceeds through catagen and telogen, during which timehair growth ceases, the lower portion of each follicle degenerates, and arest period ensues. After the rest period, the dermal papilla initiates regen-eration (anagen onset) and the production of a new hair from follicleepithelial cells at the follicle’s base. This new follicle develops immedi-ately adjacent to the original follicle, and a bulge is created in the externalouter root sheath as regenerating cell progenitors reorganize. This bulge,which develops early on in skin development, is now a well-known andideal epithelial stem cell niche, existing in a protective, well-vascularized,innervated area.32

Ito et al. used fate mapping experiments to study the contribution ofbulge stem cells to epidermal repair after injury. The data from theseexperiments showed that, after the epidermis was injured, cells from thebulge region of the hair follicle initially migrated to the wound andacquired an epidermal phenotype, thereby assisting in wound healing.However, these cells are thought to be transient amplifying cells, cells thatare short-lived, initially responding to injury but sloughed within twoweeks. The data are notable in that they distinguish epidermal and bulgecells from each other but also show that bulge cells are able to repopulatethe interfollicular epidermis in response to stress.33 Experiments per-formed by Levy et al. confirm these findings.34,35 Furthermore, Oshimaet al. showed, through transplantation of the bulge region, that bulge cellswere capable of repopulating the epidermis, sebaceous gland, and allepithelial layers of the hair follicle.36

There are a number of stem cell niches in the epidermis, and it is nowthought that two described above — the interfollicular epidermis and thebulge region of the hair follicle — can supply each other when damaged.This idea is clinically important in that the skin, the largest organ of thebody, may be similar to bone marrow as a stem cell reservoir.

Adult stem cells in the human epithelium are responsible for main-taining tissue homeostasis and responding to injury. As previouslymentioned, early gestation fetal skin wounds have the ability to heal with-out scarring, while adult wounds heal with a scar.8 As humans age, theirwound healing ability diminishes. If stem cells are presumed to be respon-sible for cutaneous wound healing, these observations would suggest thatadult stem cells are affected by aging.

The effects of aging could negatively affect the quality of stem cellmachinery or deplete the number of stem cells available to respond to pro-liferation signals.32 In support of this hypothesis, studies of keratinocytesisolated from older human donors display a lower proportion of holo-clones in clonogenicity assay in comparison to those from youngerdonors.37 Another study showed that epidermal cells from elderly individ-uals show increased positivity for p16INK4A, a molecule essential for G1arrest.38

3.3 Mesenchymal stem cells

Mesenchymal stem cells (MSC), initially isolated on the basis of their plas-tic adherence, are non-hematopoietic stromal cells that are capable ofdifferentiating into mesenchymal lineages such as bone, cartilage, muscle,and fat.39 Although they are present as a rare population of cells in bonemarrow, representing 0.001% of the nucleated cells, they are expandable inculture while retaining their growth potential and multipotency.39 MSCshave been isolated from various sites other than the bone marrow, includingadipose tissue and amniotic fluid, and show phenotypic heterogeneity.40,41

Because MSCs can be expanded in culture with relative ease and differen-tiated into several tissue types in vitro, MSCs represent a promising sourceof stem cells for wound repair. Moreover, since MSCs are isolated fromadult tissues, the use of these cells could avoid some of the obstacles asso-ciated with the use of embryonic stem cells. For example, if adult stem cellsare transplanted back into the patient from whom they are harvested, thesecells should avoid rejection by the immune system.

Several studies show that MSCs have the ability to migrate acrossendothelial cell layers to reach injured tissue. One group harvested bonemarrow cells from enhanced green fluorescent protein (EGFP) transgenicmice and transplanted the cells into lethally irradiated mice via tailvein injection. These mice were then wounded. Many EGFP+ cells werefound in both normal cells and at the wound edges.42 Site-directed deliv-ery of MSCs has shown that these cells can home to a variety of tissues,particularly after injury. Many studies have used injection of allogeneicMSCs into the vascular system to demonstrate that these cells hometo various epithelium-lined organs including the lung, gut, skin, and

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cornea.43–47 Several groups have also reported that MSCs can migrate intoinfarcted myocardium.48–50 These experiments demonstrate the multipo-tency of MSCs and their potential utility in complex tissue repair andregeneration. However, mechanisms by which MSCs home to tissues andmigrate across endothelium are not well understood. It is likely thatinjured tissue expresses specific ligands or receptors to recruit MSC intothe site of injury, similar to leukocyte trafficking, adhesion, and infiltra-tion to site of inflammation.39

Increasing evidence has shown that MSCs participate in cutaneouswound healing. During injury repair, MSCs can differentiate and thendirectly participate in the structural repair of a wound, or use paracrine sig-naling with secreted factors to support wound healing and modulate theimmune system. For example, when MSCs were transplanted on the sur-face of deep burn wounds in rats, there was an acceleration of theformation of new vessels and granulation tissue along with a decrease ininflammatory cell infiltration.51 Also, application of MSC to cutaneouswounds led to accelerated closure in an excisional wound splinting modelin both normal and diabetic mice.52 MSCs were found to expresskeratinocyte-specific markers and high levels of vascular endothelialgrowth factor (VEGF) and angiopoietin-1, suggesting that MSCs promotedwound healing by differentiation and release of pro-angiogenic factors.52

Another study demonstrated that MSCs transdifferentiated into keratin-ocytes, endothelial cells, and pericytes in the wounded skin of mice thatreceived intravenous injection of MSCs.46 When human bone marrow-derived MSCs were applied to full-thickness skin defects in mice, all skinwounds were reported to heal without a scar or retraction.53 All thesefindings from animal transplantation studies demonstrate that MSCs cancontribute to the repair of injured skin and can serve as the cell source forregenerative therapy.

There are already some encouraging results from human studies uti-lizing MSC as a therapeutic agent for tissue repair. In a study of chronicnon-healing wounds that failed conventional therapy, application ofautologous bone marrow cells led to complete closure with evidence ofdermal rebuilding with less scar.54 More recently, the application of fibrinspray containing autologous bone marrow-derived MSCs produced accel-erated wound closure in both excisional and chronic wounds.55


Despite rapid progress in evaluating the efficacy of MSC transplanta-tion on wound healing, many questions still need to be addressed. Becauseof the crude method of isolating MSCs, uncertainties remain with respectto the defining characteristics of these cells, such as specific markers toidentify and trace the lineage of resident MSCs. A recent hypothesis is thatMSCs are pericytes, a supporting cell for blood vessels.56 This hypothesisraises an interesting connection between these cells and angiogenesis, akey component in wound repair. More research is needed to extensivelystudy not only MSCs, but also other cells and factors that make up themicroenvironment or niche that supports the survival and differentiation ofthe cells. Further studies characterizing the MSC and its niche would helpto determine the extent to which MSCs act as stem cells versus as sourcesof secreted factors. This research would also allow dissection of this het-erogenous cell type into more distinct and functional subpopulations.

3.4 Induced pluripotent stem cells

The reprogramming of adult cells to a pluripotent state is one of the mostexciting recent advances in stem cell biology. Takahashi and Yamanakapublished a landmark paper in 2006 that defined a specific set oftranscription factors capable of reverting differentiated cells back into apluripotent state, thus creating “induced” pluripotent stem (iPS) cells.57

Through screening of 24 pre-selected ES-cell specific factors in the murinesystem, four transcription factors — Oct4, Sox2, Klf4, and c-Myc — werefound to be sufficient to reprogram adult mouse fibroblasts into ES-likeiPS cells.57 The same combination of transcription factors has beendemonstrated to be sufficient for pluripotent induction of human cells aswell.58 Mouse and human iPS cells closely resemble molecular and devel-opmental features of bastocyst-derived ES cells.57,59,60 Different researchgroups have shown that iPS cells injected into immunodeficient mice giverise to teratomas comprising all three embryonic germ layers, similar toES cells. In addition, when injected into blastocysts, iPS cells generatedviable high-contribution chimeras (mice that show major tissuecontribution of the injected iPS cells in the host mouse) and contributedto the germline.57,59,60 Furthermore, various studies have shown that iPScells express key ES cell markers.57,59,60

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The reprogramming of adult cells to ES cell-like pluripotent statesprovides new exciting possibilities. iPS technology can be used to gen-erate patient-specific cell lines for therapeutic approaches, eliminatingthe concern for immune rejection or bioethical concerns associated withuse of ES cells. However, multiple issues need to be addressed beforethis technology can be used in patients. iPS cells are generated with theuse of retroviral and lentiviral vectors to activate the necessary repro-gramming transcription factors. Because of viral integration, the risk ofinsertion mutagenesis could lead to uncontrolled modification of thegenome.61 Much progress has been made recently in generating integra-tion-free murine iPS cells, and various studies using adenoviral,plasmid-based, and recombinant protein-based strategies have reportedthat viral integration is not required for the reprogramming process.62–64

The issue of viral integration aside, the safety of iPS cells needs to bevigorously tested because all essential reprogramming factors are onco-genes that, if overexpressed, can lead to cancer.65 Chimeras and progenymice derived from iPS cells had higher than normal rates of tumor for-mation than those derived from ES cells, which in some cases may bedue to reactivation of the transfected c-Myc oncogene.66 These key issuesneed to be further elucidated to define the safety of iPS cell use in regen-erative medicine.

4. Conclusion

The process of wound healing is one of the most complex biologicalprocesses, influenced by numerous secreted factors including growth fac-tors, cytokines, and chemokines. Complications in wound healing such asinadequate healing or hypertrophic and keloid scars can arise from abnor-malities in the repair process. Systemic and local influences such asmalnutrition and infection can impair the normal wound repair process.Even without such harmful factors, most injuries in adult human result indeposition of non-functioning fibrotic tissue.

Although tremendous progress has been made in our understanding ofnumerous factors involved in wound healing process, these findings havenot yet led to substantial advances in patient care. The cellular organizerof the wound repair process is still not well understood. One method to


address both underhealing and overhealing would be to manipulate thekey cell types that modulate this overall process. Stem cells have enor-mous potential to fulfill this role, as the cells could both coordinate theiractions with the immune system and mediate regeneration of lost tissue topromote an ideal wound repair outcome.

Recent advances in stem cell research offer promising opportunitiesfor regenerative medicine. Diverse cell types including ES cells, MSC,resident tissue stem cells (such as epithelial stem cells), and iPS cells arecurrently under intense investigation. Novel cell-based therapies usingstem cells to induce tissue regeneration hold great promise for woundrepair and modern medicine.

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10

Engineering Cartilage: From Materialsto Small Molecules

Jeannine M. Coburn and Jennifer H. Elisseeff

1. Introduction

This chapter begins by reviewing the structural and compositional makeup of articular cartilage followed by a brief discussion of osteoarthritis.Defects of cartilage that may lead to osteoarthritis are discussed alongwith associated surgical strategies for repair. The authors will alsodescribe numerous tissue engineering strategies aimed to repair cartilagedefects including materials and small molecules that direct stem celldifferentiation towards a chondrogenic lineage. The goal of this chapter isto not be all encompassing on each of these areas but to give an overviewto the reads of the exciting research being performed in the area ofcartilage tissue engineering and the current limitations.

2. Structure of Articular Cartilage of the Knee

Articular cartilage lines the surface of all diarthroidal joints including thehips, knees and shoulders. The diarthrodial joints are enclosed in a fibrouscapsule. Lining the inner surface of the capsule is the synovium which

181

contains synoviocytes. The synoviocytes secrete the synovial fluid thatprovides the nutrients to the articular cartilage along with lubrication. Dueto the smooth surface of the articular cartilage and the synovial fluid,minimal friction is exerted in the joint space during normal motion.1

Articular cartilage is predominantly composed of water and extracel-lular matrix (ECM). Additionally, a single cell population resides withinthe articular cartilage, known as chondrocytes, which emerge frommesenchymal precursor cells during limb development.2 The ECM isdivided into two major categories, collagen and proteoglycans (PGs). Themain type of collagen in articular cartilage is type II and to a lesser extenttype IX and XI.3 Collagen fibers are found throughout the cartilage andcontribute to the tensile strength of the tissue.1 Proteoglycans arecomposed of a core protein, typically aggrecan, with glycosaminoglycans(GAGs) bound to a serine residue via a trisaccharide linker. The predom-inant GAG molecules of articular cartilage are chondroitin sulfate andkeratin sulfate. Glycosaminoglycans are polysaccharides of which mostsugar moiety contains one or multiple negative charge(s) resulting in elon-gation of the PGs. Aggregates of PGs are formed when multiple PGs bindto hyaluronic acid, a non-sulfated GAG molecule, via link proteins.Moreover, due to the highly negative charge the molecules are extremelyhydrophilic and can trap in large amounts of water resulting in a highlyelastic tissue.1 The collagen network, with its high tensile strength, inter-acts with the PGs resulting in a fiber reinforced composite with highcompressive strength.

Similar to other connective tissue, articular cartilage functions via itsECM, offering its biological function and mechanical integrity. The resi-dent chondrocytes play a key role in ECM production and turnover. Theyserve to produce the collagen and PGs of cartilage along with the mole-cules that remodel the matrix, specifically matrix metalloproteinases(MMPs) and a disintegrin and metalloproteinase with thrombosondinmotifs (ADAMTS) which predominantly degrade collagens and aggre-cans, respectively. The anabolic and catabolic nature of chondrocytesresults in natural cartilage turnover. An imbalance in the natural cartilagehomoeostasis leads to arthritis.

There are four zones in articular cartilage, with the superficial tangentialzone containing flattened chondrocytes. Collagen and cells of this layer

182 J. M. Coburn and J. H. Elisseeff

are aligned tangential to the surface. This layer has been suggested to con-tain progenitor cells.4 Next is the transition layer, where the chondrocytesand collagen are randomly oriented followed by the deep zone. Collagenand cells of the deep zone are aligned perpendicular to the surface. Asmooth transition from articular cartilage to subchondral bone is markedby calcified cartilage starting at the tidemark. The tidemark is thebasophilic line that separates the calcified and uncalcified cartilage. Dueto the spatial orientation of the ECM and cells along with specializedmechanical properties, it is particularly difficult to engineer the full thick-ness of articular cartilage.

Articular cartilage is avascular, acquiring most of its nutrients from thesynovial fluid. Because of the dense ECM which limits chondrocytemobility, and the avascular nature, cartilage has a limited ability to self-repair. This is problematic when articular cartilage is damaged due totrauma or osteoarthritis making it an excellent candidate for tissue engi-neering strategies. The exact mechanism that triggers OA is not wellunderstood, however, when patients have cartilage lesions caused bysome form of trauma they are at higher risk of developing OA. In theUnited States alone, 27 million people live with OA.5

3. Osteoarthritis of the Knee

Osteoarthritis (OA) is a disease of the whole organ system, including thesynovium, subchondral bone and articular cartilage.6 It is unknown whichpathological changes initiate OA. However, in OA there is an imbalancein the rebuilding and degradation of the ECM resulting in a net loss of car-tilage. Radiographic changes of the articular cartilage include thinning ofthe joint space and sclerosis of the subchondral bone. Histologically, aloss of proteoglycans, or fixed charges (via safranin-O or toluidine bluestaining) is observed.6 A loss in total collagen occurs at later stages of OA,however, it is preceded by increased swelling properties of the cartilagedue to loosening of the collagen fibers7 seen at initial stages of OA. Thefirst stages of OA are marked by fibrillation of the cartilage surface; pro-ceeding to complete loss of articular cartilage such that the subchondralbone is exposed.6 The tidemark is also seen to thicken which correlates tocalcification of the deep zone, known as hypertrophy, and marked with an

Engineering Cartilage: From Materials to Small Molecules 183

increase in type X collagen.6 Articular chondrocytes are seen to beclustered, as opposed to single cells, due to initiation of proliferation.Bony growths, known as osteophytes,6 can also be found.

The two major classes of degradation molecules occurring naturallyin articular cartilage are increased in OA. A disintegrin and metallopro-teinase with thrombospondin motifs, specifically ADAMTS-4 and -5, playa crucial role in the degradation of aggrecan.8 Due to degradation ofaggrecan, the PGs disassemble and diffuse out of the articular cartilage,resulting in a decrease in compressive strength due to reduction of nega-tive charge. Matrix metalloproteinases play a key role in the degradationof collagen and gelatin. The three main MMPs prevalent in OA are MMP-2,-9 and -13.9 MMP-13 is a collagenase efficient at degrading type II colla-gen fibrils. Gelatinases, MMP-2 and -9, follow up by degrading the newlyformed gelatin. Both classes of enzymes play a pivotal role in matrixdegradation that causes a reduction in the mechanical integrity of articularcartilage. They also pose a particular challenge when attempting to engi-neer the tissue as they may serve to inhibit the fill tissue formation — anaspect not typically modeled during in vitro testing of tissue engineeringstrategies.

Inflammation also plays an important role in cartilage degradation andthe occurrence of OA.6 Specifically, interleukin-1 (IL-1) and tumor necro-sis factor (TNF) are inflammatory cytokines found to be increased inprimary cultures of OA chondrocytes compared to healthy chondrocytes.IL-1β and TNF-α have been found to be localized to the superficial zoneof articular cartilage.10 These cytokines induce the production of the pro-tease along with proinflammatory cytokines acting as a positive feedbackloop.11 Additionally, these cytokines are known to be associated with adecrease in chondrocytic gene.12 Both the increase in proteases anddecrease in terminally differentiated chondrocytic gene expressioncontribute to the reduction in ECM.

These cytokines are also known to activate many intracellular signalingpathways including JNK, p38 MAPK, ERK and NF-κB.13,14 The abundantform of NF-κB is a heterodimer of p65 and p50. It is sequestered in thecytoplasm via interacting with inhibitor of κB (IκB, either α or β ).13 TheNF-κB heterodimer is released from IκB after IκB is phosphorylated andtargeted for ubiquitin mediated degradation. This allows for NF-κB


translocation to the nucleus where it transcriptionally regulates manycytokines including proinflammatory cytokines IL-1 and TNF-α , andexpression of IκB-α 13 along with further downregulation of chondrocyte-specific genes.

Patient symptoms associated with OA include pain and instabilitywhich can result in immobility. Pain is felt in early OA coinciding withactivity level. However, as OA progresses, chronic pain may becomemore persistent, even in the absence of activity.15 The underlying cause ofpain with OA is not fully understood, as articular cartilage is aneural.However, some have theorized the pain resides from swelling of the syn-ovium, in turn affecting the peripheral nervous system.11 Also, MRIimaging has shown lesions in the bone marrow which is better associatedwith patients experiencing pain than those who do not.16 Non-surgicaltreatments for pain management include non-steroidal anti-inflammatorydrugs (NSAIDS) and intra-articular injections of hyaluronic acid orcorticosteroids.

4. Surgical Strategies for Repairing Focal Cartilage Defects

Focal cartilage defects can occur due to trauma, repetitive impact or pro-gressive mechanical degeneration. These defects, if left untreated, mayresult in further knee degeneration leading the osteoarthritis. There arethree types of cartilage defects: (1) partial thickness, (2) full thickness and(3) osteochondral which penetrate into the subchondral bone.17

Occurrence of OA appears on average ten years sooner in patients withcartilage defects. Because cartilage has a limited ability to repair itselfsurgical techniques are employed to repair the defect site by filling withbiological material to reduce or eliminate the progression to OA.

Surgical treatments for OA are only employed when non-surgicaltreatments have failed. There are three primary techniques utilizing torepair cartilage defects: (1) bone marrow stimulation, (2) mosaicplasty orosteochondral autograft transfer system (OATS) and (3) autologous chon-drocyte implantation (ACI). All of these procedures result in inferiorfibrocartilage formation and mechanical characteristics. This is due to thedisorganization of the fill tissue, predominantly composed of type I colla-gen, whereas articular cartilage has organized cellularity and ECM.


4.1 Bone marrow stimulation

Bone marrow stimulation utilizes natural repair strategies to fill cartilagelesions. The microfracture technique is the most widely used.18 This tech-nique involves debridement of the defect area down to the subchondralbone followed by creating multiple punctures into the subchondral boneto allow bone marrow to come into the defect site.18 A fibrin clot will formwithin the defect which contains mesenchymal stem cells and acts to facil-itate migration of additional mesenchymal stem cells within days.19 Thesecells will then go on to form new fibrocartilage tissue. Positive clinicaloutcomes include reduction in pain. However, results are found to deteri-orate within 18 to 24 months post-surgery.20,21

4.2 Mosaicplasty and osteochondral autograph transfersystem (OATS )

Mosaicplasty and OATS procedures employ osteochondral plugs trans-planted into the defect site. They are suitable for smaller size defects (lessthan 4 cm2) due to surgical challenges and donor site morbidity.22,23 Onelarge graft, or in the case of mosaicplasty multiple small grafts about1 mm in diameter, is isolated from a non-load bearing area of the knee andtransplanted to the defect site. The implanted graft maintains its articularcartilage structure. However, the bonding tissue between the implants isfibrocartilage in nature.

4.3 Autologous chondrocyte implantation

The final surgical procedure for cartilage repair is autologous chondrocyteimplantation (ACI).24 This is a two-step process. In the first stage cartilagefrom a non-load bearing area is removed. The chondrocytes are then iso-lated from the ECM and expanded in vitro. Expanded cells are thenreimplanted during the second surgery into the focal defect and held intoplace with a periosteal flap. The periosteal flap is believed to play a rolein defect repair due to a paracrine effect or may serve as a source for auto-genous cells.25 This technique has been found to have comparable resultsto microfracture after two years.26 In addition, histological evaluation of


the fill tissue has shown some hyaline-like cartilage with the rest beingfibrocartilage.24 However, many limitations to this procedure exist,including donor site morbidity, limited cell supply and dedifferentiation ofthe isolated cells. Dedifferentiation occurs due to in vitro expansion inmonolayer culture resulting in loss of cartilage specific gene expressionand ECM production. Redifferentiation of the expanded chondrocytes isnecessary in order to obtain hyaline-like cartilage.

5. Scaffolds for Assisting Operative Techniques

Multiple scaffolds have been generated to attempt to enhance the regen-erating cartilage. Materials have been utilized to anchor the cells in placeand augment the operative procedures. Using tissue engineering strategiesto augment operation procedures is of interest to the research communitybecause it offers many opportunities to direct the fill tissue. Specifically,materials have been utilized to direct redifferentiation of chondrocytes,differentiation of stem cells and fill tissue architecture to match that ofnative tissue. Cell-free and cell-laden scaffolds have been used in the clin-ical setting. Cell-laden scaffolds are homogenous in cell distribution,better recapitulate the native three-dimensional environment and help tomaintain or induce the chondrocytic phenotype.

5.1 Collagen scaffolds for augmenting ACI

Multiple collagen scaffolds have been developed and used clinically foraugmenting the ACI procedure. A type I/III collagen flap has beendesigned to eliminate the need for periosteal flaps.27 It had significantadvantages compared to the original technique, including less invasivesurgery, reduced surgery time and decreased postoperative pain. Thistechnique was limited in that suturing of the collagen membrane to thearticular surface is required and tedious.

Due to enhanced histological findings with the collagen flap a newtechnology was developed to eliminate the limitation of the collagenflap, a procedure known as matrix-induced autologous chondrocyteimplantation (MACI®).28,29 As opposed to injecting cells underneath theflap, cells are seeded directly onto a type I/III collagen membrane


isolated from porcine peritoneal cavity. The cell-laden collagen mem-brane is then placed into the prepared defect using a thin layer of fibringlue to secure it to the defect. The chondrocytes can penetrate into boththe collagen membrane and the fibrin glue and retain their chondrocyticphenotype. The resulting tissue is hyaline-like and positive for type IIcollagen.29 Similar results are seen with a porcine type I/III bilayermatrix, Chondro-Gide®.30

Another ACI augmenting collagen scaffold, NeoCart®, is a three-dimensional type I collagen scaffold seeded with expandedchondrocytes.31 The seeded scaffold is then cultured in a bioreactor. Thetotal time for implant development is 67 ± 18 days. A proprietary tissueadhesive, CT3 (Histogenics), composed of collagen and polyethylene gly-col is used to secure the implant in place. Since this implant is fairly new,minimal clinical outcomes have been observed. However, the treatedpatient’s pain score was lower than at baseline. Also, range of motion andknee function was seen to be improved.

5.2 Collagen scaffolds with autologous mesenchymalstem cells

Mesenchymal stem cells are a promising cell source for tissue engineer-ing because they have the ability to differentiate into multiple lineagesincluding bone, cartilage, fat and astrocytes.32 They can be isolated fromindividual patients in a minimally invasive manner and expandedin vitro without losing their ability to differentiate. Mesenchymal stemcells have been evaluated clinically for their ability to augment bonemarrow stimulation. In this work, 2 mm of the subchondral bone wasremoved until bleeding was seen.33,34 Perforation was then performedusing 1.2 mm Kirshner wire to facilitate further bleeding. MSCs werepreviously isolated using standard procedures and expanded throughone passage in vitro. The day before surgery, MSCs were lifted from thetissue culture plates (average 13 million) and embedding into 1.2 ml of0.25% type I collagen from porcine tendon. The cell suspension wasseeded onto collagen sheets (derived from bovine source) and allowedto gel. The collagen/MSC scaffold was cultured overnight in DMEMsupplemented with autologous serum and antibiotics. The composite


was placed into the defect with the collagen sheet covering the upperside. A perestium flap was used to secure the material in place. Controlpatients received the collagen scaffold with no cells and others did notreceive the perforation technique. The resulting fill tissue was found tobe mechanically weaker in all groups compared to the surround carti-lage. However, histologically (using toluidine blue) the cell-laden groupwas superior to the cell-free group, exhibiting a metachromatic stainingand some hyaline-like tissue.33,34 Further work is necessary to under-stand the origin of the cell population residing within the scaffolds afterimplantation.

5.3 Hyaluronic acid (HA) scaffolds

Hyaluronan is found in all soft tissues. Hyaluronan is an unsulfatedglycosaminoglycan of disaccharide repeat units, glucuronic acid andN-acetylglucosamine. Its molecular weight ranges from 4000 to 8 × 106

Da. As mentioned previously, it interacts with proteoglycans along withother proteins and molecules. In addition, cells expressing CD-44 canbind to and migrate on HA.35 Degradation products of HA contribute tomany biological activities including size-dependent affect on chondro-genic differentiation,36 vascularization37 and angiogenesis.38

Modified HA has been used as a scaffold for articular cartilage defectrepair, specifically Hyaff®. Hyaff® hyaluronic acid starts as a molecularweight of 180–200 kDa. Esterification of the glucoronic acid groups isperformed. By varying the alcohol used in the esterification reaction andthe degree of substitution bioresorbabilty, water solubility and residencetime can be varied.

HYAFF® 11 is biocompatible and resorbable without the presence ofan inflammatory response.39 It has been used to augment the ACI proce-dure.40 Isolated chondrocytes were expanded in vitro followed by seedingand culturing on the scaffold for an additional two weeks. Used in thisway, it is marketed as Hyalograft® C. In animal models, these scaffoldshave been shown to develop hyaline-like cartilage and integrate with thesurround tissue. Clinical trials have shown that patients treated withHyalograft® C had significant improvements in pain, physical activity andknee function.40


5.4 Fibrin scaffolds

Fibrin glue is a material that synthetically mimics fibrin clots, part ofnatural tissue repair after injury. It is a two-component system composedof fibrinogen and thrombin. Once mixed together, the thrombin degradesfibrinogen to fibrin which acts as a tissue adhesive and three-dimensionalscaffold. Fibrin glue is used as an adhesive to secure various scaffolds intocartilage defects due to its chondroinductive property.41

Scaffolds of fibrin can also be made and have been studied for carti-lage tissue engineering.42 Fibrin scaffolds have been used clinically, again,to augment the ACI procedure. In this treatment, the expanded chondro-cytes are mixed with fibrinogen and thrombin to form a cell-seeded fibrinscaffold. The defect site is then debrided to the subchondral bone and thesubchondral bone penetrated. A thin layer of fibrin glue is appliedfollowed by molding and implantation of the cell-seeded fibrin scaffold.Another layer of fibrin glue is then applied. This technique is superior toACI alone because it eliminates the need for a periosteal flap, reducingpatient recovery time.43

Zimmer Inc. has also developed a product using fibrin glue.44 Theproduct, known as DeNovo® NT Graft, utilizes mince cartilage tissuefrom allogous juvenile cartilage. Viable cartilage pieces are mixed intra-operatively with fibrin and placed into the defect site. A thin layer of fibrinis then applied over the implant to maintain placement. Utilizing viabletissue results in a cell source that can migrate out of the minced pieces andfill the defect area. Advantages over the ACI procedure include onlyrequiring a single operation and eliminating the need for costly in vitroexpansion.

5.5 Chitosan scaffold

Chitosan is a polysaccharide derived from deacetylating chitin isolatedfrom the exoskeleton of crustaceans. It is composed of D-glucosamineand N-acetyl-D-glucosamine linked via a β [1–4] linkage. Due to its pos-itive charge it behaves as a bioadhesive and can adhere to negativelycharged tissue.45 BST-CarGel® is composed of a mixture of chitosan anduncoagulated whole blood which gels within ten minutes. BST-CarGel®


has been evaluated for its ability to augment microfracture technique.46

First, the microfracture technique is performed and a “dry field” is cre-ated. At least 5 ml of peripheral blood is obtained, 4.5 ml of which ismixed with the supplied BST-CarGel®. This mixture is then placed withinthe prepared cartilage defect and allowed to gel for 15 minutes. Multipleanimal studies have been performed and have shown that using BST-CarGel® resulted in increased hyaline-like cartilage formation, GAGcontent, collagen content and repair tissue volume when compared tomicrofracture alone.46 Clinical studies have shown that BST-CarGel®

decreased pain and stiffness along with increased joint function.

5.6 Polyester-based scaffolds

Polyesters are synthetic polymers of repeating degradable ester groupswith various amounts of carbons separating the esters and side chains. Forexample, poly(lactic acid) and poly(glyocolic acid) contain one carbonbetween the ester bonds with poly(lactic acid) containing a methyl groupon the α-carbon. Another common polyester is poly(ε-caprolactone) withcontains six carbons between the ester groups. Polydioxanone is apolyester-ether containing three carbons along with oxygen (making theether) between each ester bond. The esters can be hydrolytically degradedto carboxylic acids and alcohols. Degradation rate of polyesters can becontrolled by the specific polyester used, copolymerization or mixingmultiple types of polyesters together. These variations change the degra-dation rate due to altering the degree of crystallinity. The range ofdegradation that can be achieved is a couple of weeks up to years.

One example clinically evaluated is a polymer-based scaffold ofpolylactic/polyglycolic acid (polyglactin, vicryl) and polydioxanonecollagen fleece coined Bioseed®-C.47,48 Chondrocytes isolated and expandedfrom the patient are seeded within the scaffold and secured using fibrin. Thescaffold is cut to the shape of the defect, armed with vicryl sutures at eachcorning and secured using K-wires passed through the femur. A press fittingtechnique is used to securely hold the Bioseed®-C scaffold in place. Thistechnique is more stable than ACI and eliminates donor site morbidity asso-ciated with periosteal flaps. The procedure is also performed completelyarthroscopically so it reduces adhesions associated with open surgeries.


However, in a comparative clinical study between ACI and Bioseed®-C nodifferences were seen in the clinical outcomes between the treatmentgroups.47

Another polyester system undergoing clinical evaluation utilizesmince cartilage pieces collected from the patient seeded onto a foam scaf-fold coined cartilage autograft implantation system (CAIS).44 The mincepieces are held into place with the use of fibrin adhesive. The scaffold isthen stapled into place. Evaluation of a similar scaffold in goats aftersix months of implantation resulted in completely filled defects withhyaline-like cartilage that had more type II collagen and less type Icollagen than scaffolds without minced cartilage.49

6. Mesenchymal Stem Cells for Cartilage TissueEngineering

Limitations exist in using articular chondrocytes. First, cells must beisolated from the donor site during the initial surgery which results intwo invasive procedures in order to repair the defected cartilage. Second,the number of chondrocytes that can be isolated is limited and thereforethe cells must be expanded in vitro. This results in a costly procedure anddedifferentiation of the chondrocytes due to cell expansion.50 Finally,donor site morbidity is of concern and limited research has been done toinvestigate these effects.

Alternative cell sources exist for cartilage tissue engineering. Theseinclude embryonic stem cells and mesenchymal stem cells. Embryonicstem cells are derived from the inner cell mass of an embryo. Due to theisolation technique, ethical issues surrounding embryonic stem cells havelimited the research and extent of use of these cells. Alternatively, mes-enchymal stem cells can be isolated from bone marrow, umbilical cordblood, adipose tissue, synovial cells, and peripheral blood.51 The firstthree are the most common sources under investigation. Bone marrow-derived MSCs have been studied more extensively than any other source.Umbilical cord blood-derived MSCs are limited by availability becausethey can only be isolated at birth. Adipose-derived MSCs can easily beisolated from lipoaspirate after cosmetic liposuction. This derivationtechnique has proven to result in higher cell yields than that of bone


marrow and umbilical cord-derived MSCs. However, this cell source islimited to individuals with excess fat to isolate cells from. Humanadipose-derived MSCs have also been shown to have an inferior ability todifferentiation towards cartilage and bone when compared to human bonemarrow-derived MSCs.

Differentiation of MSCs towards a chondrogenic lineage can beinduced by a variety of physical and chemical factors. A three-dimensional culturing system is required in order for adequatechondrogenesis to occur. Examples of three-dimensional environmentsused are pellet culture, high density culture, hydrogels and sponge-likescaffolds. Chemical factors are included into the medium to elicitmore specific differentiation. Dexamethasone is supplemented into themedium though the exact mechanism of how it induces chondrogenesisis not well understood.52 It is believed to increase Sox-9 geneexpression, a transcription factor that activates type II procollagen geneexpression.53 Various growth factors and proteins are also supple-mented into the medium to induce differentiation including transforminggrowth factor (TGF)-β1 and -β 3, IGF-1, bone morphogenic proteins(BMPs) and fibroblast growth factor (FGF)-2.54 These factors arealso known to be key regulators of chondrogenesis during skeletaldevelopment.2

7. Hydrogels for Directed Differentiation of MesenchymalStem Cells

Hydrogels have been used as a three-dimensional scaffold for studyingcellular function and differentiation. As mentioned previously, bioactivematerials have been used as scaffolds. Many bioinert materials have beenstudied as a means to encapsulate cells in a three-dimensional configura-tion. Examples of bioinert hydrogels include poly(ethyleneglycol)-diacrylate (PEGDA), poly(vinyl alcohol) (PVA) and alginate. Asa result the soluble components contained in the medium are the primarycontributors to directing cell fate. In the case of bioactive hydrogels, thecells can interact with the substrate resulting in materials that can be usedto direct cellular activity. When encapsulating MSCs into hydrogels it isof particular interest to engineer materials that direct differentiation. In


this way, scaffold assisted differentiation occurs as a result of the material-cell interaction; resulting in a scaffold with potential to enhance in vivodifferentiation, where factors contained in medium are not available.

7.1 Functionalized Poly(ethylene Glycol ) Hydrogels

Poly(ethylene glycol) hydrogels have been investigated extensively asbioinert scaffolds for three-dimensional cell culture. Poly(ethylene glycol)has been functionalized with acrylate groups to facility crosslinking vianumerous methods including ultraviolet light exposure and a free radicalinitiator, reduction-oxidation reaction, and Michael addition.55 They havebeen shown to support differentiation of MSCs towards a chondrogeniclineage via the factors present within the medium.

Numerous research groups have functionalized PEG hydrogels withRGD (arginine-glycine-aspartic acid), a cell-binding peptide found inmany proteins. When anchorage-dependent cells are encapsulated intoinert three-dimensional hydrogels adhesion is not possible resulting in alarge percentage of apoptotic cells. Therefore, incorporating adhesionpeptides, such as RGD, facilitates cell survival within the hydrogels.Nuttelman and colleagues56 demonstrated that human MSCs survival inPEG hydrogels increases from 15% to 75% when RGD was covalentlyincorporated into the network. Enhancing cellular survival is of particularinterest because tissue formation will be facilitated within the scaffold dueto an increase in total extracellular matrix deposition.

When evaluating human embryoid body-derived MSCs (MSCsderived from embryonic stem cells) Hwang and colleagues57 found a sig-nificant increase in chondrogenesis in the presence of covalently linkedRGD in PEG hydrogels. Specifically, increase in GAG and total collagenproduction was seen on a per cell basis when compared to PEG onlyhydrogels. This was confirmed by increased safranin-O and type I and IIcollagen staining. Though favorable to have an increase in type II colla-gen, type I collagen is less favorable. However, when gene expression wasevaluated, type II collagen, aggrecan and link protein increased in theRGD group; whereas type I collagen gene expression was similar to thatof PEG hydrogels alone.


Interestingly, when alginate hydrogels were functionalized withRGD chondrogenesis of bovine MSCs was inhibited in a concentrationdependent manner.58 Similar inhibition of sulfated GAG production wasobserved in bovine chondrocytes encapsulated in alginate hydrogelswith covalently link RGD.59 Silk scaffolds covalently linked with RGDalso exhibited similar inhibitor effects on chondrogenesis of humanMSC.60

Still RGD peptide does have a positive effect on cell viability. To com-bat the negative effect on chondrogenesis, Salinas and colleaguesdeveloped an RGD containing peptide with an MMP-13 cleavage site.61

Matrix metalloproteinase-13 activity was observed to peak at an interme-diate culturing time when MSCs were undergoing chondrogenesis whichresulted in cleavage of the RGD sequence. In this way, the increase in via-bility can be harnessed earlier followed by removal of the RGD toeliminate the inhibitor effect seen with RGD and chondrogenesis. Thoughcell viability was seen to decrease at the time at which cleavage wasbelieved to occur, GAG production relative to DNA increased in a timedependent manner even after the RGD sequence was cleaved. In a controlgroup, where the RGD peptide was not cleavable, GAG productiondecreased supporting the hypothesis that RGD is important for cell via-bility but necessary to remove to enhance chondrogenesis.

Salinas and colleagues also evaluated a decorin binding peptide,KLER (lysine-leucine-glutamic acid-arginine) in combination withRGD on the chondrogenesis of human MSCs.62 Decorin is known toinfluence fibrillogenesis of collagen at two major sites, RELH (arginine-glutamic acid-leucine-histidine) and KLER. When both RGD and KLERwere covalently linked to PEG hydrogels, collagen content increasedover the time course of the experiment when compared to RGD andRGD plus scrambled peptide. Gene expression for aggrecan was highestin the RGD/KLER peptide group compared to the controls at early timepoints, whereas type II collagen expression was highest at later timepoints.

Another peptide has been evaluated for its effects on chondroge-nesis. This peptide, known as collagen-mimetic peptide (CMP),immobilizes collagen via binding through a strand invasion route.63

Using a synthetic peptide to bind endogenous collagen is favorable


over collagen scaffolds because it eliminates possible immuneresponse. The material in which the peptide is covalently linked canalso be mechanically more robust than collagen gels making themfavorable from a practical aspect, also. When compared to PEG hydro-gels, CMP containing hydrogels had an increase in cartilage matrixproduction (GAG and type II collagen) and cartilage-specific geneexpression of differentiating goat MSCs. Whereas, type I collagenproduction, marker for bone formation, and type X collagen geneexpression was decreased.

In addition to peptides, GAG molecules can be covalently linked toPEG hydrogels to facilitate enhancement of chondrogenesis. One suchGAG molecule, chondroitin sulfate, has been shown to enhance the chon-drogenesis of goat MSCs compared to PEG hydrogels.64 Visualization ofthe hydrogels after chondrogenesis showed a nodule-like appearance.When investigated further, the MSCs were found to be undergoing a mes-enchymal condensation-like state based on gene expression for versicanand cadherin 11 (markers found naturally in mesenchymal condensation).CS-based hydrogels also enhanced total collagen production and type IIcollagen deposition. Also, hypertrophic markers were lower in the CS-based hydrogels. Overall, CS-based hydrogels have the ability to enhancechondrogenesis while reducing hypertrophy and may recapitulate thenatural process of mesenchymal condensation.

7.2 Naturally derived materials

Natural derived biomaterials, such as collagen and hyaluronic acid (HA),have been utilized as 3D scaffolds for MSC differentiation.65,66 Thesematerials mimic the natural ECM of cartilage and better recapitulate thein vivo environment. Limitations to these types of materials include min-imal control over spatial location and orientation of the biomolecule andmechanical weakness. Also, because they are derived from naturalsources (i.e. animal tissue) concerns of immune response exist.

Hyaluronic acid hydrogels have been developed via functionalizingHA with methacrylate groups to facilate free-radical crosslinking.66 TheseHA hydrogels have been compared in parallel to PEG hydrogels (withsimilar mechanical properties). In vitro chondrogenesis of human MSCs


was enhanced in the HA hydrogels based an increase in type II collagenand proteoglycans deposition along with chondrocytic gene expression.In vivo, cartilage specific gene expression was also seen to be higher inHA hydrogels. Three different groups were evaluated in vivo. The groupnot administered exogenous TGF-β3 or predifferentiated for two weeksin vitro before implantation demonstrated the optimal gene expressionprofile. This shows that HA hydrogels may be capable to differentiationMSCs towards a chondrocytic lineage without external stimulation.

As mentioned previously, collagen scaffolds have been used in vivo.They have been able to elicit a more hyaline-like cartilage tissue fill favor-able for cartilage tissue engineering. A study comparing type I collagen,type II collagen and a bioinert hydrogel, alginate, showed that type II col-lagen hydrogels enhanced chondrogenesis of bovine MSCs based on geneexpression and ECM deposition.67 Similarly, studies have shown enhance-ment of osteogenesis in type I collagen scaffolds (type I collagen beingthe predominant collagen type in bone).68,69 These findings support thehypothesis that ECM component found in the native tissue of interest canfacilitate enhancement of differentiation towards that tissue lineage.

8. Fiber-Hydrogel Composites

Hydrogels have been the predominant focus for researchers in developingtissue engineering strategies for articular cartilage. Mechanically, theybetter resemble of the developing limb in comparison to the more stiffmature cartilage. However, they are primitive mimics of articular cartilageas they lack the fibrous nature of native cartilage. Many research groupshave investigated methods of producing fibrous scaffolds to act as a phys-ical stimulus similar to that of the fibrous phase of articular cartilage.70,71

However, these systems then lack the hydrogel phase. Fewer researchershave investigated fibrous-hydrogel composites for cartilage tissue engi-neering.72–75 These composites employ the use of a hydrogel phase as adelivery vehicle for cells infiltration into the fibrous phase. The hydrogelphase also helps to maintain the round cellular morphology native tocartilage. Fiber-reinforced hydrogels have an added benefit of enhancingthe mechanical integrity of the scaffold; this may help in addressing largersize cartilage defects than currently feasible clinically.


Polyesters have been studied extensively as fibrous meshes for carti-lage tissue engineering. Favorable results have been obtained for bothchondrocytes and chondrogenic differentiation of MSCs seeded directlyonto the meshes. Chondrocytes have been seeded onto electrospunpoly(ε-caprolactone) meshes and were found to maintain their chondro-cytic phenotype.76 Chondrogenic differentiation of MSCs in the presenceof TGF-β1 resulted in rounded cell morphology similar to that of nativechondrocytes.77 In addition, favorable gene expression was found withchondrogenic MSCs, including increased type II collagen and decreasedtype X collagen. When compared to pellet culture, chondrogenic MSCson nanofiber scaffolds were found to have a two-fold increase in GAGproduction. These findings imply that fibrous polyester scaffolds are goodcandidates for cartilage tissue engineering. However, limitations exist inhigh-throughput development of fibrous meshes large enough to fill carti-lage defects. Also, cell infiltration into electrospun mats is limited due tothe small pore size.

A non-woven fleece of polygalactin, E210, has been implanted subcu-taneously in athymic nude mice after infiltrating with alginate containingbovine chondrocytes.72 Crosslinking of the alginate was facilitated byimmersing the composites in a calcium chloride solution prior to implan-tation. After explantation, E210 fleeces without cells and alginate weretoo weak to evaluate and broke when cut. E210 scaffolds seeded withcells, with and without aliginate, had similar levels of GAG and type IIcollagen production. However, the scaffolds with alginate had a moreeven distribution of cells and did not exhibit any shrinking.

A similar composite scaffold has been evaluated using a PGA meshand either alginate or type I collagen hydrogels.73 Rabbit MSCs wereseeded in a similar manner. At early time significant differences were seenwith the alginate-fiber compared to collagen-fiber and fiber-only scaf-folds. Cell morphology in the alginate scaffolds was maintained asrounded whereas in the collagen scaffolds a fan-shape was seen. However,the fan-shaped cells became more polygonal over time indicating chon-drogenic differentiation. Cell proliferation and differentiation was seen tobe delayed in the alginate scaffolds at three weeks. However, after sixweeks of culture the GAG content normalized to DNA was significantlyhigher in the alginate scaffolds compared to collagen scaffolds.


Finally, a woven scaffold composed of PGA yarn has been utilized asa reinforcement material for cartilage tissue engineering.74 Fibers wereoriented in the x, y and z directions resulting in a highly interconnectedfibrous network where the pore size could be controlled. The hydrogelphase was composed of either agarose or fibrin. The authors were able toshow that fibers enhance the mechanical properties of the hydrogels inde-pendent of the type of hydrogel used. From a biological perspective, theauthors were able to incorporate porcine articular chondrocytes homoge-nously throughout the fibrous scaffold with the use of vacuum-assistedinfusion. In a later study, chondrogenesis of human adipose-derivedMSCs in woven PCL scaffolds with fibrin and without exogenous growthfactors was investigated.75 Collagen-rich ECM was found to completelyencapsulate the fiber/hydrogel scaffolds and fill the inner pores. Whereasthe fiber alone scaffolds were only encapsulated by the collagen-richECM after 28 days of culture.

9. Small Molecules for Directing Chondrogenesis

Current system for directing stem cell differentiation towards a chon-drogenic lineage have been lacking in their ability to produce tissuecomparable to articular cartilage. This tissue does contain cartilage-likeECM however lacks in the quantity produced. Therefore, other methodsof directing differentiation are necessary in order to obtain comparable


Fig. 1. Electrospun fibers used to enhance hydrogel properties developed in ourlaboratory. (A) SEM of PVA-methacrylate fibers electrospun onto aluminum collector.(B) Electrospun PVA-methacrylate fibers that have been collected on a grounded ethanolbath. (C) PEGDA hydrogel having a transparent appearance. (D) PEGDA hydrogel withincorporated fibers from (B).

tissue to the native environment. Small molecules are commonly sup-plemented into the culture medium for directing differentiation in vitro,including ascorbic acid and dexamethasone. However, other moleculeshave been explored, typically in the presence of other chondrogenicinducing molecules. Small molecules are extremely useful for in vivoapplications as they can be administered locally to the site of tissuerepair. Controlled release systems can also be developed to allow thesmall molecule delivery over time allowing for longer residence time.

9.1 Glucosamine and its analogs

Glucosamine has been studied extensively for its effects on chondro-cytes.78–81 Many studies have shown that glucosamine enhancescartilage-matrix deposition along with cartilage-specific gene expressionof chondrocytes. A limited number of studies have been performed toevaluate the effects of glucosamine on chondrogenesis. Glucosamine andits analogs (N-acetyl glucosamine, glucosamine oligomers from chi-tosan, lactose, lactosamine and N-acetyl lactosamine) have beenevaluated on a chondrogenic cell line, ATDC5 cells.82 By evaluatingalkaline phosphatase (ALP) activity, a marker for mineralization, glu-cosamine was found to be the only molecule capable of decreasing boneformation. Therefore, it was the only sugar evaluated in subsequentexperiments. Glucosamine was shown to increase alcian blue staining forsulfate GAG and decrease gene expression for matrix gla protein (MGP),SMAD 2 and SMAD 4. MGP is a matrix protein that plays a role in min-eralization of cartilage and bone. SMAD 2 is believed to regulatehypertrophy. SMAD 2 and SMAD 4 both play a role in chondrocytedifferentiation. Therefore, glucosamine may have an effect on mineral-ization by affecting genes associated with the transition from cartilageto bone.

Hwang and colleagues83 looked at the effect of glucosamine on chon-drogenesis of mouse embryoid bodies (derived from mouse embryonicstem cells). Glucosamine at concentrations of 2 mM and 10 mM (in thepresence of TGF-β1) were shown to decrease metabolic activity and DNAcontent of EBs encapsulated in PEGDA hydrogels. However, at 2 mM theGAG production was significantly higher than control. Aggrecan gene


expression was also found to be enhanced at this concentration. In addition,when TGF-β1 was removed from the culture medium staining for sulfatedGAG was only positive in the cells conditioned with 2 mM GlcN and thestaining was comparable to groups treated with TGF-β1 alone. This isindicative that glucosamine has chondroinductive properties at a concen-tration of 2 mM.

9.2 NINDS library screening

The National Institute of Neurological Disorders (NINDS) has puttogether a library of small molecules to aid in the discovery of new ther-apies for neurological disorders. In it is a collection of 1040 smallmolecules. Huang and colleagues84 developed a system to perform high-throughput screening of chondrogenesis of bovine bone marrow-derivedMSCs. Pellet culture was used as a 3D model to minimize the number ofcells required to analyze a single substance. Inducers of chondrogenesiswere defined as those molecules capable of increasing GAG productionby a factor of 1.5 or more compared to chondrogenic medium (in theabsence of exogenous growth factors). Five small molecules were foundto induce chondrogenesis at concentrations of 10 μM: doxylamine succi-nate, pergolide mesylate, perphazine, eszopiclone and colforsin. Furtherinvestigation into each individual molecule is necessary in order to fullycharacterize the chondrogenic state of the cells and to optimize theconcentration for induction.

10. Conclusion

Tissue engineering strategies have been developed to repair cartilagedefects to ultimately reduce the prevalence of osteoarthritis. These strate-gies include scaffolds for directing tissue formation and stem celldifferentiation in addition to small molecules for directing stem cell dif-ferentiation. This chapter has been a brief summary of articular cartilagearchitecture, osteoarthritis, and surgical techniques for repairing cartilagedefects along with the current research areas designed to enhance thesurgical techniques available.


Acknowledgments

This work was supported by NIH R01EB05517-01 and NIHF31AG033999.

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57. N. S. Hwang, S. Varghese, Z. Zhang and J. Elisseeff, Chondrogenic differ-

entiation of human embryonic stem cell-derived cells in arginine-

glycine-aspartate-modified hydrogels, Tissue Eng 12: 2695–2706 (2006).


58. J. T. Connelly, A. J. Garcia and M. E. Levenston, Inhibition of in vitro chon-

drogenesis in RGD-modified three-dimensional alginate gels, Biomaterials

28: 1071–1083 (2007).

59. N. Genes, J. Rowley, D. Mooney and L. Bonassar. (2000) Culture of chon-

drocytes in RGD-alginate: effects on mechanical and biosynthetic properties.

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2000, Orlando, Florida, Vol. 25 (2000).

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and G. Vunjak-Novakovic, Engineering cartilage-like tissue using human

mesenchymal stem cells and silk protein scaffolds, Biotechnol Bioeng 88:

379–391 (2004).

61. C. N. Salinas and K. S. Anseth, The enhancement of chondrogenic differen-

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62. C. N. Salinas and K. S. Anseth, Decorin moieties tethered into PEG networks

induce chondrogenesis of human mesenchymal stem cells, J Biomed Mater

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woven composite scaffold for functional tissue engineering of cartilage,

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dimensionally woven poly(ε-caprolactone) scaffolds for cartilage tissue

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77. W. J. Li, R. Tuli, C. Okafor, A. Derfoul, K. G. Danielson, D. J. Hall and R. S.

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78. S. Varghese, P. Theprungsirikul, S. Sahani, N. Hwang, K. J. Yarema and J. H.

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sis, and gene expression, Osteoarthr Cartil 15: 59–68 (2007).


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Ann Biomed Eng 36: 1909–1921 (2008).


11

Adult Stem Cells for Articular CartilageTissue Engineering

Sushmita Saha, Jennifer Kirkham, David Wood,Stephen Curran and Xuebin B. Yang

1. Introduction

Healthy articular cartilage is vital for proper functioning of our joints.However, the absence of neural tissue and blood vessels coupled with asparse cell population and slow metabolic activity greatly limits the repar-ative process of this connective tissue.1 Articular cartilage can be damagedin numerous ways, including congenital pathologies such as paediatricgrowth plate disorders, trauma induced injuries and age related degenera-tive joint disorders such as osteoarthritis.2 Current treatment for cartilagedamage has been primarily symptomatic ranging from the use of anal-gesics and anti-inflammatory drugs to surgical interventions that includedebridement, cell therapy/grafting, joint replacement and joint fusion.3

These methods have had varying success with either partial healing or theformation of fibrocartilage which is mechanically inferior to nativehyaline cartilage.

211

Functional tissue engineering offers hope to the field of cartilageregeneration/repair by combining three basic elements: (1) a suitablesource of cells that have a high chondrogenic potential, are easily expand-able and can be maintained for long periods of time in culture; (2) abiocompatible and biodegradable porous scaffold to act as a carrier todeliver and/or support cell growth; and (3) bioactive regulators whichcould be either biomolecules such as transforming growth factor-β3(TGF-β 3) or bone morphogenic proteins (BMPs) and/or mechanicalfactors such as hydrostatic pressure, stress or strain which can stimulatecell proliferation and differentiation.

Successful repair of cartilage defects by autologous chondrocyte trans-plantation was first reported in 1994 by Brittberg et al.4 A year laterGenzyme employed this technique and produced “Carticel”, an FDAapproved autologous chondrocyte implantation (ACI) treatment forpatients with knee cartilage injuries.5 Autologous grafts or chondrocytetransplantation have been shown to be efficacious, however, they involvean invasive procedure with limited cell availability, loss of differentiationcapability in vitro and a risk of donor site morbidity.6 Owing to theirintrinsic properties, stem cells are now emerging as a favourable cellsource for use in cartilage tissue regeneration. However, the application ofembryonic stem cells (ESCs) is not ideal due to ethical issues and prob-lems on the regulation of cell differentiation in vivo. For these reasons,adult mesenchymal stem cells are preferred for cell based therapies.7

A stem cell is defined as a cell which possesses the ability to self-renewand differentiate into multiple mature cell lineages.8 Adult stem cells/mul-tipotent cells possess limited self-renewal capabilities unlike embryonicstem cells which are considered to be pluripotent. Many researchers areinvestigating the trans-differentiation capacity/plasticity of adult stem cellswhich remains a highly controversial subject.9 In spite of the numerousquestions regarding their trans-differentiation capabilities, mesenchymalstem cells (MSCs) have emerged as a popular cell source in cartilage tis-sue engineering. MSCs cultured in vitro have been documented to lackmajor histocompatibility complex (MHC) class II cell surface markers.These cells possess only class I MHC surface markers without any co-stimulator molecules making them an ideal source in auto/allo/xenogenictherapeutic applications.10 It is also well understood that MSCs have a

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trophic effect, i.e. depending on their local environment and activity statusthey secrete large quantities of bioactive molecules which help bring abouta therapeutic response.11 The presence of MSC immunomodulatory andtrophic functions in vitro has opened up a new era of cell mediated thera-pies. In the last five to ten years a plethora of sources of stem cells withchondrogenic potential have been reported. However, there is no uniformlyaccepted definitive phenotype and/or cell surface markers to assist withMSC isolation. Thus, in order to be classified as an MSC by theInternational Society for Cytotherapy, the cell population must: (1) adhereto plastic under standard culture conditions, (2) express cluster of differen-tiation (CD) markers — CD105, CD73 and CD90, and (3) be able todifferentiate down chondro/osteo and adipo-genic lineages.12 This chapterwill present a concise synopsis of adult stem cell sources, functions andidentities (e.g. bone marrow, fat pad, synovial fluid, dental pulp, perios-teum) with potential for use in cartilage tissue engineering along with adiscussion on the more recent discoveries of stem cell sources.

2. Human Bone Marrow Mesenchymal Stem Cells(hBMMSCs)

An estimated one in every 10,000 nucleated cells in the bone marrow ofnewborns is a stem cell.13 hBMMSCs form the non-hematopoietic com-ponent of the stem cells found in the bone marrow. Friedenstein et al.(1960s) provided the earliest evidence of the existence of stem cells in thebone marrow when they generated a haematopoietic ossicle by trans-planting a whole bone marrow under the kidney capsule in mice.14 Sincethen, the field of mesenchymal stem cell research has gained immensepopularity, with scientists regularly reporting new sources for MSCs.Bone marrow mesenchymal stem cells (BMMSCs) still remain the moststudied and best understood stem cell source used in cartilage tissue engi-neering. Bone marrow aspirates (containing the BMMSCs) are easilyaccessed by introducing a needle directly into the bone marrow, mostly atthe iliac crest.15,16 The ability of BMMSCs to undergo successful chon-drogenesis has been studied in a variety of models. The most popular arehigh density cell pellets/micromasses or cell-3D scaffold constructsin vitro and in vivo under various mechanical conditions with/without the

Stem Cells for Cartilage Tissue Engineering 213

addition of bioactive regulators.17 A typical chondroinductive cocktailcontains dexamethasone (Dex) and ascorbic acid with/without a growthfactor in a serum free culture medium. Positive identification of an appro-priate cartilaginous phenotype is based mainly upon observed expressionof the transcription factor Sox9 and the extracellular matrix proteinscollagen type II, aggrecan and cartilage oligomeric matrix proteinamongst others.17

Numerous studies have looked into the factors that contribute to thechondrogenic differentiation of hBMMSCs. For example, chondrogenicpreconditioning of hBMMSCs in aggregate cultures with fibroblastgrowth factor-2 (FGF-2) was observed to enhance chondrogenesis intissue engineered constructs while withdrawal of TGF-β3 was observedto differentiate the hBMMSCs to a hypertrophic state.18,19 Enhancedglycosaminoglycans (GAG) synthesis was observed when hBMMSCswere cultured in a hyaluronan-alginate layer culture system.20

Evaluation of the efficacy of hBMMSCs to undergo chondrogenesisin vivo has yielded mixed results in animal models. BMMSCs seeded onvarious scaffolds and implanted into rabbit cartilage defects resulted inhypertrophy of the tissue with large areas of bone replacement visible.21,22

BMMSCs seeded on a poly(lactic acid) (PLA)-alginate amalgam in acanine model led to the formation of fibrous tissue.23 Chen and colleagues(2005) showed the formation of cartilage-like tissue in a sheep defectmodel when they seeded autologous BMMSCs on a poly(lactic acid-co-glycolic acid) (PLGA) scaffold. Cartilage-like tissue formation wasobserved from four to eight weeks without any visible signs of bone for-mation.24 Autologous hBMMSCs seeded on a collagen gel and implantedin the patello-femoral joint covered with autologous periosteum or syn-ovium led to an improvement of clinical symptoms with the defect beingfully repaired after 12 months with formation of fibrocartilage tissue inone of the patients.25 Yang et al. have shown the potential of usinghBMMSCs in combination with biomimetic biomaterial scaffolds to formcartilage-like tissues in different in vivo models (Fig. 1).26

One of the major drawbacks observed when working with hBMMSCsis the difficulty in maintaining their plasticity in vitro. The “Hayflicklimit” in hBMMSCs has been observed where successive passagingin vitro leads to a reduced proliferation and differentiation capacity.27

214 S. Saha et al.

Another drawback in the lack of a definitive in vivo model usinghBMMSCs to generate high quality hyaline cartilage, which requiresfurther research for the understanding of the basic biology of hBMMSCsand their behavior in a physiological condition.


(a) (b)

(c) (d)

Fig. 1. Cartilage tissue formation by hBMMSCs in vivo: (a) after seven days in achorioallantoic membrane assay model; ( b) after ten weeks in a diffusion chamber modelintraperitoneal implantation in Nu/Nu mice (Yang X.B. et al., 2004); (c) in a diffusionchamber model by hBMMSCs after adenoviral BMP-2 gene transfer (reprinted fromBBRC, 292: 144–152, Partridge et al., copyright (2002), with permission from Elsevier);(d) in a diffusion chamber model enhanced by pleiotrophin (reproduced from J Bone MinerRes 2003; 18: 45–57, Yang et al., copyright (2003) with permission from the AmericanSociety for Bone and Mineral Research).

3. Adipose Derived Mesenchymal Stem Cells (ASCs)

Adipose tissue (mesodermal origin) has been a recent hot bed for thediscovery of stem cells. ASCs were first formally documented by Zuk andcolleagues in 2001 in human lipoaspirates.28 ASCs are isolated from fattissues via collagenase digestion and differential centrifugation followedby plastic adherence.29 The white adipose tissue (WAT) present mainly inthe intra-abdominal viscera and subcutaneous tissues contains highernumbers of ASCs with enhanced proliferation and plasticity properties incomparison to brown adipose tissue (BAT) which is generally present inabundance in newborns.30,31

Similar to hBMMSCs, ASCs have been found to be immunosuppres-sive due to the absence of MHC class II antigens on their surface. Inaddition, ASCs have been observed to exert an inhibitory effect over allo-genic lymphocytes in vitro. However, freshly isolated ASCs are capableof eliciting a T-cell proliferative response at passages 0 and 1 whichdisappears in latter passages.32,33

2D culture of ASCs is unfavourable for maintaining chondrogenicphenotype but culturing these cells on an elastin like polypeptide wasobserved to significantly enhance chondrogenesis.34 Using a chondroin-ductive cocktail including TGF-β, expression of collagen type II,aggrecan and sulphated proteoglycans was observed. Enhanced chondro-genesis of ASCs was observed when the cells were cultured with BMP-6.In contrast, hBMMSCs underwent osetogenesis upon BMP-6 exposure.35

Henning et al. (2007) further showed that a combination of TGF-β withBMP-6 was the ideal cocktail for chondroinduction of ASCs.36

In vivo studies using ASCs have shown that these cells are capable ofretaining their chondrogenic phenotype for up to 12 weeks.37 Fibrin gluescaffolds seeded with ASCs and implanted in vivo for eight weeksexpressed aggrecan and type II collagen.38

Much controversy lies in determining whether ASCs have higherchondroinductive abilities in comparison to hBMMSCs. hBMMSCs arereported to possess a higher chondrogenic potential than that of adiposetissue derived MSCs in the pellet culture system in the presence of TGF-β1/β2.39,40 However, Lee et al. (2004) reported ASCs were superior tobone marrow stromal cells in respect to maintenance of proliferating

216 S. Saha et al.

ability. Microarray analysis of gene expression revealed differentiallyexpressed genes between ASC and bone marrow stromal cell. But theirphenotypes and the gene expression profiles are similar.41

A drawback with the use of ASCs is their expression of embryonicmarkers such as Oct-4, UTF-1 and Nodal in short term culture. This hasthe potential to trigger the transformation of these cells into cancer-likecells over extended periods of culture.42

4. Periosteum Derived Stem/ Progenitor Cells(PDSCs/PDPCs)

The outer surface of bones at the non-articulating junctions are covered bya thin layer of connective tissue known as the periosteum. The outerfibrous layer of the periosteum is made up of fibroblasts while the innercambial layer contains progenitor cells responsible for increasing bonewidth.43 The presence of progenitor cells in the periosteum that are capableof differentiating into the three mesodermal lineages (oseto/adipo/chondrogenic) has been demonstrated since the early 1990s.44 de Bariet al. (2006) isolated and characterized PDPCs with MSC surface markers.These PDPCs were observed to possess MSC-like multipotentiality whenanalysed via single cell lineage analysis and they underwent chondrogen-esis when cultured as micromasses.45

A major plus point to using PDPCs is their ability to proliferate at ahigher rate compared to BMMSCs.46 PDPCs are routinely used in theclinic to regenerate bone, however their clinical use in repairing cartilageis undocumented so far.47,48 The ability of PDPCs to undergo chondroge-nesis in vivo was demonstrated in a rabbit model where the formation ofectopic cartilaginous tissue was observed 20 days following dissection ofthe periosteum (7 × 15 mm2 periosteum defect). However, the newlyformed cartilaginous tissue turned to bone by day 40. The advantage ofsuch a technique is the elimination of the need for cell culture.49 In anotherreport, a similar approach was used to create an artificial space (“biore-actor”) between the tibia and periosteum in which angiogenesis wasinhibited (via local administration of the anti-angiogenic factor suramin ina hyaluronic acid (HA)-based gel matrix) to promote a more hypoxic


environment within the “in vivo bioreactor” space. Cartilage formationwas observed within the in vivo bioreactor after ten days.50

While these cells possess MSC-like multipotentiality and higherproliferation rates than hBMMSCs, a major disadvantage of using PDSCsis the need to carry out a surgical procedure for their isolation, limitingtheir application as a potential autograft transplant. The probability of cellloss in the cambial layer when harvesting the periosteal cells coupled withthe variation in MSC numbers in the cambial layer depending on donorage yields inconsistent results.51,52

5. Synovium Derived Mesenchymal Stem Cells (SMSCs)

The non-articular surfaces of diarthrodial joints are lined by a thinmembranous tissue known as the synovium. The main function of thistissue is to maintain the synovial fluid cavity which nourishes the articu-lating cartilage.53 De Bari and colleagues (2001) successfully extractedMSCs from the synovial membrane for the first time.54 The immuno-genicity of these cells has been found to be similar to that of hBMMSCs.SMSCs are negative for the expression of class II MHC molecules andsuppress T-cell proliferation in a mixed lymphocyte reaction.55

Numerous evidences point to the chondrogenic potential of these mul-tipotent cells. Crawford et al. (2006) reported the ability of cell culturesisolated from synovial nodules from a patient suffering from primary syn-ovial chromatosis (PSC) to undergo chondrogenic differentiation. Thebenign synovial metaplasia in PSC is thought to arise due to the prolifer-ation of mesenchymal progenitor cells.56 The expression of chondrogenicmarkers such Sox9, aggrecan and cartilage oligomeric matrix protein(COMP) has been documented to be 2%–25% higher in synovial calftissue when compared to committed articular cartilage.57

SMSCs have the highest proliferation ability in the presence of TGF-β1,IGF-I and FGF-2, while synergistic action of TGF-β1 and IGF-I enhancedSMSC chondrogenesis.58 Shirasawa et al. (2006) documented enhancedhuman SMSCs chondrogenesis in pellet culture as a result of the syner-gistic effects of TGF-β3, Dex and BMP-2.59 However, further studiesreported the inhibitory effect of Dex on BMP-2 chondroinduced synovialcells.17,60

218 S. Saha et al.

In vivo, like PDSCs, SMSCs migrate from the synovium to partialthickness articular cartilage defects in adult rabbits and yucatan pigs butthe cells differentiated into fibroblasts and filled the cavities with afibrous connective tissue.61 Pei et al. (2009) seeded allogenic SMSCs ona polyglycolic acid (PGA) mesh, cultured the resulting constructsin vitro for one month and then implanted the neo-tissue into full thick-ness femoral condyle cartilage defects in rabbits. They observed theformation of hyaline-like cartilage tissue with no detectable levels ofcollagen type I.62

Although the main advantage of using SMCs is their high proliferativecapacity54 and enhanced chondrogenic differentiation capabilities com-pared to other MSCs,59,63 however, the invasive surgery needed to accessthese stem cells coupled with an unclear understanding of theirdifferentiation mechanism may hinder their popular use.

6. Human Dental Pulp Stem Cells (HDPSCs)

HDPSCs have been isolated mainly from the pulp tissues of third perma-nent molar teeth and can be maintained for up to 25 passages. A numberof studies have reported that HDPSCs could be a suitable cell source forbone tissue engineering64 but there are few references reporting theirpotential in cartilage regeneration. A STRO-1(+) DPSC populationhas been thought to possess a higher multilineage potential compared tonon-sorted cells, probably because of their homogeneous nature.65 TheseSTRO-1(+) cells can be differentiated down neurogenic, osteogenic/odon-togenic, adipogenic and myogenic lineages. However, chondrogenicdifferentiation of these primary cells was found to be inferior to myogenicdifferentiation by the same cells.64

Wei and colleagues (2008) demonstrated multilineage differentiationin all HDPSC samples with collagen type II expression levels beingsignificantly upregulated after chondro-induction.66 The main advantage ofusing HDPSCs lies in the ease of obtaining these cells without any inva-sive surgery by banking milk teeth. However, their superiority in terms ofchondrogenic differentiation over the other stem cell sources available stillneeds to be determined by a set of robust comparative studies.


7. Umbilical Cord/Cord Blood Derived Stem Cells

Wharton’s Jelly in the human umbilical cord has recently been reportedto possess MSC-like cells (human umbilical cord perivascular cells:HUCPVCs) and depending upon how these cells are harvested, they candifferentiate down the neuronal or cardiac pathways.67,68 These HUCPVCsare located close to the vasculature of the cord and have a colony formingfibroblast frequency of about 1:3000, expressing an immuno-privilegedand immuno-modulatory phenotype.69 A comparison of the chondrogenicpotential between these cells and hBMMSCs revealed that when culturedas pellets for 21 days along with TGF-β3, HUCPVCs formed larger sizedpellets than HBMMSC pellets. The sGAG content and alcian blue-picrosirius staining intensities were comparable between the two celltypes indicating similar matrix formation. Unlike hBMMSCs, these cellsdemonstrate the potential to grow in multi-layers, overlying cellularaggregates. It is however important that independent studies ascertain thepossibility of these cells transforming to a cancerous phenotype and toidentify the molecular mechanism involved in such multi-layered cellulargrowth.

Unrestricted somatic stem cells (USSCs) derived from cord blood arebelieved to be a promising source of therapeutic stem cells and can bedifferentiated down all three germ layers (e.g. ectoderm, mesoderm andendoderm).70 However, when compared to hBMMSCs cultured as pelletsfor 21 days, there was a significant decrease in sGAG content of theUSSC compared to hBMMSCs pellets suggesting reduced matrixformation.71 Further studies of chondro-induced 3D constructs of cordblood stem cells also showed lower levels of sGAG as compared to nativecartilage.72

8. Other Potential Cell Sources with a ChondrogenicPotential

As an alternative to MSCs, mesenchymal progenitor cell populations(MPCs) were recently isolated from traumatised muscle tissue that hadbeen surgically debrided due to an orthopaedic wound. The MPCs hadsimilar morphological, proliferation and differentiation capacities as

220 S. Saha et al.

hBMMSCs.73 However, before research using these cells can progress, itis important to demonstrate that they can undergo robust in vitro expan-sion and maintain their properties and differentiation capabilities in anin vivo environment.

The anterior cruciate ligament (ACL) has been recently identified asanother source for MSCs with chondrogenic potential. Stem/stromal cellsfrom ACL can be enzymatically released and cultured as monolayers onplastic dishes.74 These cells were observed to have similar phenotypiccharacteristics as hBMMSCs. FGF-2 and TGF-β 1 can enhance theproliferation of these cells along with extracellular matrix proteinproduction.75,76 However, in other studies, only one cell line out of sixdonors was found to be capable of tripotent differentiation. ACL derivedcells were observed to be more beneficial in the formation of ligamentfibroblasts than cartilage tissue.77

The dogma of articular cartilage being a non-regenerative tissue isnow increasingly being challenged with the hypothesis of the presence ofa progenitor/stem cell population in the superficial layer of articular car-tilage.78 Morphological variations are observed in the different layers ofarticular cartilage: cells of the flattened superficial zone secrete lubricin;the rounded and columnar arranged middle zone cells produce cartilageintermediate layer protein (CILP); while the considerably larger deepzone cells express type X collagen and alkaline phosphatase.79,80 Hayeset al. (2001) demonstrated the presence of slow-cycling cells, akin toprogenitor cells, in the superficial zone of articular cartilage through theuse of BrdU (bromo-deoxyuridine) injections. These cells expressedNotch-1 and possessed a high colony-forming efficiency.81

Grogan and colleagues (2009) demonstrated the localization of pro-genitor cells in healthy cartilage using Notch-1, Stro-1, and vascular celladhesion molecule-1 (VCAM-1) as stem cell markers with highestfrequencies of labelled cells being observed in the superficial zone.82

Progenitor cells were also isolated from osetoarthritic (OA) cartilage. Thefrequency of Notch-1, Stro-1 and VCAM-1 positive cells was found to behigher in the middle and not the superficial zone for OA tissue. However,a similar frequency (0.14 ± 0.05%) of progenitor cells in healthy and OAcartilage was observed. These progenitor cells possessed chondrogenicand osteogenic capabilities but not adipogenic differentiation potential.


However, the authors suggested that Notch-1, Stro-1 or VCAM-1 may notbe useful in identifying progenitors in cartilage and that their increasedexpression in OA cartilage perhaps indicates their involvement in OA.82

While the presence of these cell populations highlights the possibilityof cartilage possessing an intrinsic regenerative capacity, further researchis required to elucidate their role before they can be used in transplantstudies.

9. Conclusion and Future Directions

A myriad of sources of MSCs with chondrogenic potential have beenidentified. Under appropriate conditions, these cells can be maintainedand differentiated down the chondrogenic lineage. Some of the MSC pop-ulations are more easily isolated and differentiated than others, yet they alloffer promising solutions to the field of cartilage tissue engineering.Before selecting an appropriate MSC source for cell therapy, it is impor-tant to consider whether the cell of choice can be expanded/maintained/differentiated in vitro, cultured on a scaffold and easily assessed via con-ventional methods of histology. At present there is a need for more robusttests to characterise cells classified as stem cells by using clongenic-lineage-specific gene marking with multipotentiality visible under in vivoconditions. Upon fully understanding the biology of the stem cellsisolated we can go on to design methodologies to stimulate these cells torepair the damaged articular cartilage tissue in the body. However, thetranslation of research from lab to bedside could be another challenge forfunctional articular cartilage tissue engineering.

Acknowledgments

SS was supported by a PhD studentship from Smith & Nephew Ltd.

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12

Stem Cells for Disc Repair

Aliza A. Allon, Zorica Buser, Sigurd Berven and Jeffrey C. Lotz

1. Introduction

The intervertebral disc forms an avascular, fibrocartilaginous jointbetween adjacent vertebral bodies and provides flexibility while routinelysupporting several multiples of body weight. The disc is composed ofthree major sub-tissues, the gelatinous nucleus pulposus (NP), the fibrousannulus fibrous, and cartilaginous endplates (Fig. 1). The NP is centrallylocated and composed primarily of sulfated-glycosaminoglycan (GAG),type II collagen, and water. The NP serves as the osmotic mechanism thatgenerates volume and hydrostatic pressure because the high GAG contentmakes the tissue very hydrophilic. The annulus fibrosus is firmly attachedto the vertebral edges to serve both as a ligament to guide intervertebralmovement, and as a barrier to contain nuclear swelling and thereby allowdisc pressurization. The endplate is a thin (0.1 to 1.6 mm) hyaline carti-lage layer that separates the NP from the adjacent vertebra. The endplatefunctions as a semi-permeable membrane to allow diffusive communica-tion between disc nuclear cells and vertebral vasculature, as well as toprevent large molecular weight GAG from leaving the nuclear space.

231

Pain of spinal origin afflicts most adults at some point in their lives:the annual US incidence of acute and/or chronic back pain is approxi-mately 100 million.1 Intervertebral disc degeneration underlies severalpainful low back disorders including intervertebral disc herniation(IVDH), degenerative spondylolisthesis (DS), spinal stenosis (SS), anddegenerative disc disease (DDD). For the first three (IVDH, DS and SS),recent randomized prospective clinical studies have demonstrated advan-tages of surgical care compared with non-operative care.1,2 However,DDD management remains the most difficult challenge because theunderlying source of pain is unclear, causing uncertainty when develop-ing guidelines for operative and non-operative care and therapieswith improved efficacy.3 As a result, estimates suggest there are between1.5 and 4 million adults in the US with DDD-related chronic low backpain (CLBP) that have failed conservative management and await thera-peutic intervention, of which there are few options beyond spinal fusion.2,3

CLBP is a common indication for spine fusion surgery, and morerecently total disc replacement. Although success rates from spinal fusionare in the range of 70%, has several disadvantages including a decrease inspinal range of motion and acceleration of degeneration at adjacentlevels.4,5 Disc replacement (where a prosthetic disc is implanted between

232 A. A. Allon et al.

Fig. 1. Mid-sagittal sections through a health (left) and moderately-degenerate (right)human disc. In the healthy state, the nucleus possesses a significant capacity to swell andsupport spinal forces. The early stages of degeneration are characterized nuclear fibrosisand annular fissuring.

vertebrae) is an attractive alternative to fusion since it maintains near-phys-iologic movement, but there are currently barriers to widespread use, suchas limited insurance reimbursement, and potential surgical and implant-related complications.6,7 Spine fusion will likely remain the standard ofcare for advanced disease, where arthritic changes prevent return of normalfunction even with newer motion-sparing technologies. The concept ofminimally-invasive biologic disc repair for less advanced cases of degen-eration has grown in recent years. These include gene therapy, injection ofvarious growth factors, and cell implantation (with or without scaffold).

2. The Demanding Intervertebral Disc Environment

The process of age-related disc degeneration can be considered chronicdysfunctional matrix remodeling in response to physical inputs such asimpaired transport and/or abnormal mechanical loading, with the responseto both likely modulated by yet undefined familial risk factors.8 At an earlyage (before ten years) there is a marked decrease in endplate vascularityand beginnings of structural disorganization. After age 20, the discbecomes sealed-off from the vertebral blood supply by the cartilage end-plates and subchondral bone.9,10 Thereafter, disc cell survival is dependenton diffusion from capillaries in the adjacent vertebra (for nuclear cells) andsurrounding vascularized tissues (for annular cells).11 The capillarieswithin the vertebra terminate just above the hyaline cartilage endplate, pro-viding a continuous capillary bed across the bone disc interface.6 Oncenutrients reach the endplate, movement of small solutes (e.g. glucose andoxygen) pass through disc matrix primarily by diffusion.11–15 (larger solutesmay also be influenced by convective fluid flow created by mechanicaldisc compression and recovery). Cells compete for nutrition, making it dif-ficult to sustain high cell densities at the long distances from the nutritionsource typical of human lumbar discs (approximately 8 mm).12–15

This disc transport limitation has several negative consequences.Tissue oxygen concentrations are low, in the range of 0.5% to 5%.16 Thesehypoxic conditions inhibit matrix synthesis: sulfate incorporation at 1%oxygen is one-fifth that at 5%.11,16 Because of limited oxygen, the nucleuspulposus cells produce energy through anaerobic glycolysis, which utilizesglucose and generates lactic acid as a by-product.11,14 Accumulation of

Stem Cells for Disc Repair 233

lactic acid decreases disc pH (to near pH 6.3) and is detrimental to thematrix as it decreases glycosaminoglycan production, tissue inhibitor ofmetalloproteinases (TIMP) production, and cell viability.17 The depend-ence on anaerobic glycolysis for cell production of ATP makes glucose acritical nutrient. Disc glucose concentrations are typically considered tobe in the range of 0.5 to 5 mM, where disc cells die within 24 hours atconcentrations below 0.2 mM.14 Other factors in serum are also importantas serum deprivation results in decreased cell proliferation and increasedcell senescence.18 Moreover, the influence of these factors is not neces-sarily independent, since some research indicates hypoxia supportsnucleus cell survival during serum withdrawal.18

Another main feature of the disc nucleus is osmolality. The disc func-tions biomechanically by using a high osmotic pressure (generated byproteoglycan fixed charge) to attract water and produce physical pressure tosupport spinal compression. This osmotic stress (in the range of 250 to450 mOsm) causes changes in cell volume and stimulates cell behavior viacytoskeleton rearrangement.14 Under hypo-osmotic conditions (250 mOsm)disc cells increase gene expression for aggrecan and type II collagen, whileat hyperosmotic conditions (450 mOsm) there is a downregulation ofbiglycan, decorin and lumican in nucleus pulposus cells.15

3. Evaluating a Stem Cell-Based Therapy

3.1 In vitro outcome measures and assessments

Since disc degeneration is considered to initiate in the nucleus, regenera-tive strategies target nucleus regeneration. As a result, stem cell-basedtherapies focus their efforts on replicating the characteristics of the nativenucleus pulposus cells (NPCs). Stem cell performance is typically evalu-ated by characterizing gene expression and the matrix synthesis. In orderto assess differentiation stage and the cell fate, the gene expression levelsare measured for the positive chondrogenic marker Sox9, as well as severalnegative markers including the fibroblastic marker Collagen 1, the hyper-trophic marker Collagen X, and the osteogenic marker Runx2. Majormatrix proteins aggrecan and Collagen 2 are often evaluated to assess thecells’ level of protein synthesis. In addition, matrix metalloproteinase (MMP)genes that are responsible for degrading tissue are generally measured, in


particular MMP-2, MMP-9 and MMP-13 are commonly measured indegenerate discs.19 For quantitative analysis at the protein level, proteogly-cans are typically measured using a dimethylmethylene blue assay andCollagen 2 can be quantified using an enzyme-linked-immunosorbent-assay.20,21 Both proteins can also be assessed qualitatively using histologicstaining with Safranin-O or immunohistochemistry techniques.20

Biological efficacy should be demonstrated in models of increasingcomplexity. Two- and three-dimensional cell culture systems can be use-ful for initially demonstrating cellular effects, dosing, and toxicity.Three-dimensional systems are preferable to maintain cell phenotype, andaugmenting stimuli to include other disc mimetic conditions such as pres-sure, hypoxia, and inflammation are important as these factors cansignificantly influence cell function.22 The degenerative disc conditionswill vary with the degree of degeneration, however, the cells implanted ina human degenerative disc will generally experience hypoxia (about 4%O2), high pressure (350 KPa at rest), inflammatory cytokines (particularlyTNF-alpha and Il-2b), and a drop in pH (as low as pH 6.7). To simulatethose conditions, a number of different methods can be used. The oxygencontent can be controlled with a hypoxic incubator and a pressurized envi-ronment can be stimulated with the aid of a bioreactor. In addition, theinflammatory cytokines and pH levels can be replicated in the culturemedia. Therefore, the in vivo degenerative disc environment can be mim-icked in vitro and should be considered during testing and optimization.

3.2 Models for efficacy and safety: In vivo preclinical models

Ultimately small animal studies are a critical next step because of theimportant in situ interactions between disc cells and spatially-varying hostfeatures unique to the healing disc environment: pressure, hypoxia,degraded matrix, cytokines, other stromal and inflammatory cells, plussystemic factors. Along with an increasingly complex model comechallenges in response interpretation. Outcome measures should be cou-pled to the designed treatment mechanisms, but should also includeoverall indices of disc quality such as histology and biomechanics. Timedependence of the outcomes is critical to establish whether the therapeu-tic response is persistent above the background degenerative responsetypically triggered by the therapy delivery. Design of experiments (DOE)


techniques for study design and statistical analyses can help establishsample size and efficiently optimize treatment parameters.23

Safety also needs to be established in preclinical models. The avascu-lar nature of the disc environment can lead to persistence of activetherapeutic agents, secreted cytokines, as well as carrier degradation prod-ucts. Consequently, even though a growth factor or carrier has anestablished use track record in other tissues, they need to be evaluated inthe unique NP environment. Adverse reactions can manifest through inter-discal toxicity, inflammatory cell recruitment, and matrix erosion. Forexample, cytokines and scaffold degradation products can diffuse from thedisc and incite a sclerotic reaction in the adjacent vertebral endplates, alongthe delivery wound site, or outside the annulus fibrosus.24 Equally impor-tant is to establish the reaction to extradiscal placement of the therapeuticmaterials and delivery vehicles. It is likely that these can escape from thedisc during surgery or early in the post-surgical period. Inflammation andthe mass effect induced by these materials can adversely affect adjacentnerve roots and other paraspinal tissues.25 The safety of injected materialsor cells should also be confirmed under the worst case scenario to avoid acatastrophic event, such as the one seen for chemonucleolysis.26

After efficacy and safety are established in vitro in small animals, largeanimal studies are required to motivate clinical use, principally because ofsize effects on disc transport13 and biomechanics. Typical animals used forthis purpose include goats, sheep and mini-pigs. As with other preclinicalmodels, efficacy may be difficult to establish due to a lack of relevant start-ing points and clinical metrics that match the intended patient population.Yet, biologic plausibility should be supported as well as safety through his-tological, biochemical, and biomechanical assays. Comparisons to negativecontrols (surgical procedure without treatment delivery) and untreatedlevels can help judge effect size and potential clinical relevance.

4. Non-Stem Cell-Based Regeneration Strategies

4.1 Gene therapy and growth factors

Several groups have published promising results using either in vivo orin vitro gene therapy.27 Growth factors such as TGF-beta, BMP2;


transcription factor Sox9; inhibitors IL1Ra (interleukin-1 receptor anatag-onist) and TIMPs have been successfully delivered to NPCs. Anotherapproach that modulates cells at the gene level is RNA interference whichis designed to silence genes that are the potential cause of disc degenerationwithout needing a viral delivery vehicle.28

The most straightforward acellular biologic strategy is to inject growthfactors into the disc. Several studies have reported in vivo effects of OP-1(osteogenic protein-1), TGF-β (transforming growth factor-beta), GDF-5(growth differentiation factor-5) that have led to an increase in disc heightand GAG content.29 Although promising results have been reported usingthese techniques, the relative acellularity of human degenerated discsraises the concern that the patient’s own disc cells may be insufficient tomount a therapeutic response. Other concerns regarding these strategiesinclude the activation of the host immune response due to the presence ofviral vectors during gene therapy and the short half-life of bioactive mol-ecules used in growth factor therapy.

4.2 Autologous NPCs

The introduction of cells capable of surviving within the intervertebraldisc and producing appropriate amounts of matrix is an important com-ponent of disc tissue engineering. One type of cell considered is anautologous (derived from the patient) NPC cell-line.30 Even with somepromising data, there is legitimate clinical concern over donor-site mor-bidity, since harvesting the patient’s own cells requires damage to anadjacent disc, which will likely induce degeneration in that level. Also,disc acellularity will require a slow in vitro cell-culture expansion step toobtain sufficient cell numbers. Furthermore, autologous cells will be sim-ilarly aged to the diseased level and potentially limited in their ability tomount therapeutic repair response.

5. Stem Cells for Disc Repair

5.1 Cell carriers

Carriers for cell-based disc regeneration strategies fulfill multiple roles. Theyserve as delivery vehicles to attain acute cell retention in the high-pressure


disc nucleus. They preserve nucleus volume and defend against scar tis-sue encroachment by adjacent annular tissue during early healing. Byaugmenting nuclear volume, carriers also serve to enhance acute biome-chanical stability. In addition to these biomechanical functions, carriersneed to provide an environment that supports the desired biological activ-ity of the delivered cells. Unfortunately, biomechanical and biologicalroles may create conflicting design constraints, with stiffer materialsbeing more suitable for biomechanical retention and stability, and porous,pliant materials being more appropriate for nutrient transport and a 3Dmilieu conducive to a disc cell phenotype. Importantly, these materials,which may or may not degrade over time, have to be synergistic with cellfunction over the long term. If non-degrading, they need to be biocom-patible and non-migratory under complex loading/pressures. If degrading,the degradation kinetics should ideally be timed with cell matrix synthe-sis. Also, because of disc size and avascularity, degradation products mayhave a longer persistence and achieve higher concentrations thanobserved in other applications.

Many types of carriers have been used that include gels with or withoutporous solid scaffolds. Examples include synthetic polymers such aspoly(lactide-co-glycolide) (PLGA),31 polyglycolide (PGA),32 and polylactide(PLA).33 Many forms of natural scaffolds are available, such as hyaluronan,34

collagen/atelocollagen,35 chitosan,36 alginate,37 agarose,38 calcium polyphos-phate,39 demineralized bone particles,40 fibrin sealant,31,41 and small intestinesubmucosa (SIS)42 — a natural extracellular matrix. So-called smartscaffolds can contain bioactive agents such as growth factors, cytokineinhibitors, or antibiotics.

5.2 Autologous versus allogenic

Adult mesenchymal stem cells (MSCs) are attractive for disc tissue engi-neering since they can differentiate into a variety of cell types, includingNPC-like cells. Depending on the therapy, the MSCs can be autologous orallogenic (derived from a donor). In the case of autologous transplanta-tion, the patient would have a preliminary outpatient procedure whereMSCs would be harvested from bone marrow or adipose tissue. SinceMSCs represent only a small percentage of the cells in either of these


donor tissues, the MSCs would need to be separated and expanded in vitroto have sufficient numbers desired for the therapy. The implantation pro-cedure would be performed several weeks later. In the case of an allogenictransplant, the patient would be treated with MSCs from an organ donorin a one-procedure cost-effective approach. While host rejection of allo-genic cells is a concern, several studies have indicated that MSCs areimmunoprivileged and do not elicit a rejection response.

5.3 Differentiation of stem cells before implantation

A primary concern regarding the use of MSCs for disc repair is whetherthey survive the harsh in vivo conditions and appropriately differentiatein situ. Although the NPC lineage is not fully characterized, it is generallyagreed to closely match that of chondrocytes.43,44 While MSCs are knownto readily differentiate into this cell type under controlled conditionsin vitro, it must still be established whether they will spontaneously dif-ferentiate and thrive in situ, or alternatively require augmentation withsupplemental, differentiation factors.

Environmental cues can provide MSCs with important differentiationsignals. Several studies have traced labeled MSCs implanted within discsand observed that they persist, integrate with host tissue, and differentiateover time.45,46 Similarly, in vitro studies have shown that MSCs culturedin 3D scaffolds also exhibit some levels of differentiation.47,48 Yet, severalstudies have shown greater persistence and matrix deposition with MSCsthat are first predifferentiated or implanted along with stimulatory fac-tors.49 Consequently, the introduction of key bioactive molecules iscommonly used to enhance desired MSC differentiation.

The use of adenoviral vectors encoding chondrogenic growth factorsin MSCs is one possible strategy to ensure the differentiation and sus-tained performance of implanted MSCs. Gene transfer therapy enables thesustained synthesis of the encoded bioactive transgene products that maybe effective since these factors would not likely be present in a degenera-tive in vivo environment. The transfer of key genes including TGF-β1 andBMP-2, to MSCs in vitro have lead to the sustained upregulation of keymatrix production and differentiation genes.50,51 Transfected cells can beimplanted alone or in combination with untransfected cells.52 However,


the use of viral vectors and genetically modified cells represent importantsafety hurdles when considered for clinical application.53 These safetyconcerns will likely slow clinical adoption.

Exposing MSCs to key growth factors either before or during implan-tation is a more popular strategy. The most common approach is to cultureMSCs in 3D alginate bead culture with TGF-β 1 supplemented media,where MSCs have robust differentiation and matrix synthesis.54 Thismethod is also convenient because the cells can easily be released fromalginate using sodium citrate washes and reimplanted without compro-mising cell viability. Controlled release of growth factors such as TGF-β1and other such molecules can also be incorporated into scaffolds that areseeded with cells and implanted.55,56 The main concern regarding TGF-β-induced differentiation of MSCs is the progression of MSCs towardshypertrophy whereby the MSCs begin secreting collagen X and MMP-13.57 This is a concern since the mechanical properties of matrix secretedby hypertrophic cells do not match those desired for disc regeneration.58

5.4 Co-culture techniques

A newly emerging technique is to co-culture MSCs with mature instruc-tive cells. This approach was originally explored to identify interactionsbetween implanted stem cells and host cells. However, when synergisticeffects were observed, co-culture was investigated for potential therapeu-tic benefits. In this context, co-culture involves creating 3D cell pelletsthat allow for contact between the two cell types. This synergy has beenshown to increase overall matrix production and promote differentiationof MSC without leading to hypertrophy.20 The NPCs are thought to pro-vide sustainable signaling cues to the MSCs and the MSCs are alsothought to be providing the NPCs with stimulatory signals. The combina-tion of these two effects is very attractive therapeutically in creating aself-sustaining implant that does not require external cues.

Yamamoto et al. report that cell-cell contact between MSCs and NPCshad synergistic effects in monolayer, however, it remained unclearwhether the MSCs were differentiating or acting as feeder cells to reacti-vate the NPC.59 Ultimately, Richardson et al. employed a similartwo-dimensional co-culture system and demonstrated that NPCs cause


MSCs to differentiate into an NP-like phenotype as assessed by geneexpression after FACS sorting.60 They observed that a 75% NPC/25%MSC ratio was optimum for MSC differentiation, as indicated by SOX9,collagen 2, and aggrecan gene expression. Another study has since madesimilar observations in 3D culture using a randomized mixture of MSCsand degenerative NPCs.61 These studies highlight the beneficial effects ofrecreating a condensation shape and the unique signaling arising from co-culturing. However, none have reproduced the key induction processwhere two layers of different cell types communicate both with heteroge-neous signaling across the interface and homogeneous signaling withinthe layer of the same cell type.

Our current strategy seeks to regenerate the disc using a novel bilaminarcell pellet (BCP) (Fig. 2). The BCP is a co-culture pellet composed of aninner sphere of MSC enclosed in an outer shell of NPC. The cell composi-tion ratio is 75% MSC and 25% NPC with a total of 500,000 cells androughly a 1mm diameter. The bilaminar structure allows for homotypicinteractions between cells of the same type within the layer and for het-erotypic interactions between different cell types across a defined interface.This organization mimics the processes of condensation, where cell aggre-gates form, and induction, where a mature layer of tissue directs thedifferentiation of a naïve one. The two cell types provide one another withstimulatory signaling which eliminates the need for growth factors orgenetic manipulation since the BCP provides self-sustaining cues.

Synergistic interactions are apparent within the BCP suggesting NPCsdirect MSC differentiation. After three weeks in culture, MSCs in the BCP


Fig. 2. (A) Co-culture in monolayer by Yamamoto58 and Richardson.59 (B) Random 3Dco-culture pellet.41,60 (C) Bilaminar co-culture pellet (BCP).20,41,61 (D) Frozen sectionhistology of BCP with MSCs dyed with Dil (red).20,61

exhibit significantly higher gene expression of aggrecan (two-fold), colla-gen II (675-fold), and SOX9 (175-fold) and a significant downregulationof MMP13 (three-fold) and ColX (eight-fold) over MSC controls.20 Inconjunction, NPCs in the BCP exhibit significantly lower levels of expres-sion of aggrecan and collagen II (both two-fold) but a similar level ofSOX9. Spatial and temporal gene expression patterns also provide cluesto the nature of cellular interactions within the BCP. At early culture times(one week), the expression of both the aggrecan and collagen II is prima-rily on the BCP periphery, where the NPCs are located. As timeprogresses, there is increased aggrecan gene expression by MSCs at theBCP center.20 Taken together, these results indicate that MSCs are differ-entiating due to their interaction with the NPCs within the BCP.

BCP culture results in a 30% increase in proteoglycan production afterthree weeks as compared to single cell type controls.20 Consistent withgene expression patterns, early aggrecan production is primarily restrictedto the outside layer presumably by the NPCs. By three weeks of culture,the aggrecan staining is widespread, indicating that the MSCs have begunsynthesizing the protein (Fig. 3).

Importantly, BCPs demonstrate superior performance when culturedunder conditions that mimic those anticipated for the degenerate disc envi-ronment: hypoxia (4% O2), pressure (350 KPa), and inflammation (10 ng/ml


Fig. 3. Immunohistochemistry staining for aggrecan (dark gray) on paraffin sections ofBCP. (A) BCP at one-week time point. The aggrecan staining is localized to the outer layerwhere the NPCs are present. (B) BCP at three-week time point. The aggrecan staining isthroughout the pellet including the center of the pellet where the MSCs are located. Thisindicates a progression in MSC differentiation towards an NPC phenotype between oneand three weeks.

of TNF-α and interleukin 1-β ). As expected, single-cell type pellets consist-ing of NPCs produce more matrix than MSCs alone or BCPs underphysiologic disc conditions of hypoxia and pressure. However, when cul-tured in the presence of cytokines, BCPs and MSCs produce significantlymore proteoglycan than NPCs alone. In this setting, the NPC performancewas dramatically reduced indicating their high sensitivity to the inflamma-tory environment. When hypoxia, pressure, and inflammation are combinedto simulate the pathologic disc environment, BCPs produce significantlymore proteoglycan than MSCs and NPCs.21 These results demonstrate thesensitivity of cell performance to culture conditions, highlighting the impor-tance of mimicking the anticipated in situ environment during in vitrooptimization of cell-based tissue engineering strategies.

The resilience of BCPs to the simulated pathologic disc environmentin vitro suggests advantages for in vivo application. This is borne out bypreliminary studies in rat caudal discs. Two weeks after implantationusing a fibrin carrier into denucleated discs, cell retention and survivalwas increased by 50% with BCPs versus MSCs or NPCs alone.62 At fiveweeks, the BCP-treated discs demonstrated significantly better disc-morphology (assessed histologically by a blinded-scoring-scheme) thaneither untreated or fibrin-only groups, and tended toward better scoresthan MSC- and NPC-only conditions. The BCP-treated discs uniquelyexhibit histologic evidence of proteoglycan synthesis, and tended to bettermaintain disc height than the other groups (Fig. 4).


Fig. 4. Rat disc paraffin section histology with Safranin-O staining five weeks aftersurgery. (A) This disc was a control disc with no treatment. (B) This disc was injected withthe fibrin carrier alone. In images (A) and (B), the disc has collapsed and there is no pro-teoglycan in the disc space. The end plate and growth plate are severely disrupted. (C) Thisdisc was treated with a BCP and fibrin carrier. The disc height is maintained with someproteoglycan staining in the disc space. The end plate and growth plate are both continuous.

These BCP studies indicate the value of adapting inductive strategiesutilized during normal joint development to guide appropriate MSC dif-ferentiation in the challenging wound healing environment. As opposed tousing a single growth factor supplement or genetic manipulation, thismethod leverages the totality of NPC signaling to program appropriateresponse in MSCs. This programming inhibits hypertrophy and promotesresistance to inflammation. BCPs synthesize substantially more disc-likematrix than either NPCs or MSCs alone. Thus, BCPs, are a promisingstem cell-based approach for disc repair.

6. Conclusion

The successful design of cell-based treatments for low back pain is con-founded by ambiguities of disease and pain mechanisms in patients, andlack of consensus regarding ideal preclinical models. In particular, the pri-mary clinical endpoint — pain relief — is currently not directly testablein animals. Yet, these therapies can be advanced by establishing biologicplausibility of efficacy and safety using models of increasing complexity,starting with cell culture, small animals (rats and rabbits), then largeanimals (goat and mini-pig) that more closely mimic nutritional, biome-chanical, and surgical realities of human application. Ultimately, successwill hinge on carefully designed clinical trials with well-defined patientselection criteria and objective outcome metrics that demonstrate signifi-cant benefits relative to gold-standard control treatments, such as spinalfusion.

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62. A. A. Allon, N. Aurouer, B. B. Yoo, E. C. Liebenberg, Z. Buser and J. C. Lotz,

Bilaminar co-culture of stem cells and disc cells prevent disc degeneration in

a rat model, in submission (2010).


13

Skeletal Tissue Engineering:Progress and Prospects

Nicholas J. Panetta, Deepak M. Gupta and Michael T. Longaker

1. Introduction

The breadth of conditions afflicting the skeletal system, as well asvariety of patients beset with defects and deficits of the skeletalsystem, is expansive. To this end, the extent of this clinical need hasgarnered a significant amount of effort from surgeons and researchersto develop novel therapeutic interventions aimed at achieving the endgoal of improved patient outcomes. However, despite progress insurgical techniques and our understanding of skeletal biology thatguides the endogenous regenerative process, reconstructive modalitiescurrently at the disposal of surgeons continue to frequently produceless than ideal results. This largely stems from the inability of modernreconstructive techniques to simultaneously restore both the form andfunction of native bone. As such, the field has been witness to a shiftin investigative focus in recent years. Skeletal regenerative medicine,and more specifically progenitor cell-based tissue engineering, offer anew paradigm in the treatment of disease processes involving the

251

skeletal system currently intervened upon utilizing standard therapies.Where current reconstructive modalities fail, ideal cell-based skeletaltissue engineering strategies are focusing on the utilization of autoge-nous osteoprogenitor cells paired with novel biomimetic scaffoldmaterials capable of delivering targeted pro-osteogenic molecular sig-nals in a spatiotemporally controlled fashion in a single operativeprocedure (Fig. 1). Substantial progress has been appreciated fromefforts to achieve this overarching goal. However, with every noveldiscovery, additional light is shed not only on the critical importanceof each of these components comprising a cell-based skeletal recon-structive strategy, but the need to understand the harmonious interplaythat must exist between each of these components in order to result ina functionally competent skeletal regenerate.

What are the ideal osteoprogenitor cells, scaffold materials, and pro-osteogenic molecular signaling pathways that will be critical to thetranslation of tissue engineered bone to the clinical arena? For investiga-tors focusing their efforts in the field of skeletal tissue engineering,answering these questions, as well as defining the interactions thattranspire between each of these components during the formation of

252 N. J. Panetta, D. M. Gupta and M. T. Longaker

Fig. 1. Bedside tissue engineering.

de novo tissue engineered bone, has taken center stage. Throughmultidisciplinary collaboration between surgeons and scientists in thefields of stem cell biology, developmental biology, and bioengineering,significant advances have been appreciated. Of note, recent advances inthe study of candidate osteoprogenitor cells have moved us closer to iden-tifying a multipotent cell population possessing the necessarycharacteristics for successful application in skeletal tissue engineeringstrategies. Additionally, work in our laboratory and others continues toadvance our understanding of molecular signaling pathways critical toosteogenesis and amenable to targeted manipulations, with the goal ofenhancing the osteogenic differentiation of progenitor cells. Indeed,encouraging results from in vitro and in vivo studies, as well as earlyhuman applications, have been appreciated to date.

As alluded to above, improved patient outcomes resulting fromsurgical intervention have been observed as a direct result of increasingknowledge regarding the molecular underpinnings guiding skeletaldevelopment and regeneration, in addition to technological advan-cements in skeletal reconstruction. Largely, this knowledge has beenderived from the study of endogenous bone tissue engineering in theform of distraction osteogenesis. By refining our understanding of howbiomechanical forces alter skeletal remodeling and regeneration,surgeons have developed and optimized techniques to harness theendogenous regenerative capacity of bone.1 Furthermore, the study ofcongenital skeletal anomalies resulting from miscues in genetic andmolecular biology during the developmental process, including cran-iosynostosis, have yielded a wealth of information regarding signalingpathways critical to the process of osteogenesis. It is such knowledgeupon which the foundation of our understanding of skeletal regenerativebiology has been built and allowed the pursuit of cell-based skeletaltissue engineering strategies to evolve.

The extent of diseases affecting the skeletal system that potentiallyrequire surgical intervention impart an underappreciated yet substantialburden on the US healthcare system. Upon examination of data collectedin conjunction with the US Health Care Utilizations Project, one canbegin to grasp the significance of this trend. Data from 2006 regarding the

Skeletal Tissue Engineering 253

cumulative cost of intervention to treat fractures of the hip, extremities,and craniofacial skeleton surpassed US$9 billion dollars in the US.2 Thisdid not include the treatment of pathologic fractures, upon which an addi-tional US$1 billion dollars in expense was incurred.2 Furthermore, recenttrends suggest that this burden can be expected to substantially expand inthe not-so-distant future. A greater than two-fold increase in the annualnumber of inpatients discharged with the diagnosis of osteoporosis andother chronic diseases afflicting the skeletal system has been observedover the course of the past decade.2 Together, these data allude to the pres-ence of a substantial and growing clinical need for novel skeletalreconstructive strategies.

In addition to escalating demand, the shortcomings of modern recon-structive interventions to treat skeletal defects and deficits, whetherarising from congenital malformations, oncologic resection, or trau-matic tissue damage/loss, has further heightened awareness regardingthe need for disruptive innovations in the field. Despite substantialincremental improvements in modern complex microsurgical techniquesand reconstructive material, current reconstructive efforts centered onautogenous tissue transfer, allogeneic tissue implantation, and theimplementation of improved alloplastic materials all have shortcomings.The limited amount of autogenous donor bone that can be harvestedfrom any of a variety of locations (calvarium, ribs, iliac crest, etc.) priorto imparting clinically significant donor site morbidity related to theharvest remains an inherent limitation of the technique.3 In addition,complications associated with allogeneic tissue and alloplastic materialreconstruction include infection, immunologic rejection, and structuralfailure. Such deficiencies point to a clear need to improve upon our cur-rent capacity to treat skeletal disease requiring surgical intervention. Inthe following discussion, we will probe the historical work in whichmuch of our current understanding of skeletal biology is founded.Subsequently, a review of the current state of studies investigating eachof the critical components of cell-based skeletal tissue engineeringstrategies (progenitor cells, molecular biology of skeletal regeneration,scaffolds) will be presented. Finally, effort will be made to present whatcritical steps remain in order for the successful translation of such strate-gies to the clinical arena to be appreciated.


2. Lessons Learned from Endogenous Skeletal TissueDevelopment, Healing and Regeneration

2.1 Distraction osteogenesis: endogenous skeletal tissueengineering

At its essence, distraction osteogenesis utilizes controlled mechanicalforce to stimulate and guide osseous regeneration. As such, it can beviewed as skeletal tissue engineering in its purest form. The characteris-tics of bone formation observed following successful distraction arerepresentative of those desired from a cell-based skeletal regenerate; bonyhealing over the course of a relatively short period of time that results ina regenerate of similar in size, shape and quality as the native bone. Firstdescribed by Codivilla in 1905 using a limb model, several decadespassed prior to Gavril Ilizarov describing the physiologic and mechanicalfactors necessary in governing successful regenerate formation using along bone fracture model.4,5 Subsequently, similar principles were appliedto the craniofacial skeleton. First studied using animal models in the1970s, it was not until 1989 when McCarthy and colleagues utilized dis-traction in the human craniofacial skeleton to lengthen the mandible.6

Subsequently, distraction osteogenesis has grown to be the cornerstone ofsurgical intervention to treat a multiplicity of mandibular and midfacedeficiencies.7

Over the course of distraction osteogenesis, deficient bone is ini-tially osteotomized. Subsequently, following a period of latency, theosteogenic fronts are gradually separated from one another.7 A periodof consolidation follows, during which callus formation and de novobone formation are observed. Additionally, during the course ofosteogenic distraction, surrounding soft tissues are also observed toexpand and adapt to increased regional needs.7 Thus, resulting from acomplex interaction between externally applied mechanical forces andmesenchymal tissues, progenitor cells and osteoblasts are stimulatedand guided to endogenously produce a skeletal regenerate with struc-tural form and biologic function that mirrors that of native tissues. Theimportance of understanding the molecular mechanisms underlyingthis impressive endogenous skeletal regenerative capacity has notbeen underappreciated. Indeed, a great deal of knowledge that is


currently guiding cell-based regenerative efforts stems from lessonslearned here.

For tissue engineers, harnessing an understanding of the events transpir-ing on a molecular level to guide the differentiation of progenitor cellstoward the osteogenic lineage in the formation of mature skeletal tissue hasbeen of utmost importance. Studies have revealed a multitude of small mol-ecules that are elaborated during distraction osteogenesis. Of these, TGF-β,BMP, and FGF family members have emerged as important mediators ofthe osseous regenerative process.8–11 Furthermore, additional work has high-lighted the importance of signaling mediated by FAK, MAPK/ERK, VEGF,HIF-1, and SDF-1 in guiding the processes of osteogenesis andangiogenesis critical to skeletal regeneration resulting from distraction.12–16

To elaborate, the role of TGF-β1 promoting collagen production dur-ing bone healing, as well as the inhibition of matrix metalloproteinasesand osteoclasts, has been well described.17,18 Additionally, TGF-β1 mayplay an important role in the neovascularization of new tissue through theup regulation of the pro-angiogenic factors VEGF and bFGF.19–22 Finally,of potential interest to skeletal tissue engineers are the temporal differ-ences in expression of TGF-β1 during distraction osteogenesis comparedto fracture repair.23,24 Distraction osteogenesis is marked by an early riseand sustained expression of TGF-β1, whereas an early downregulationand subsequent late increase in expression are observed during the processof fracture healing.

Effort has also been invested in defining the molecular mechanisms thatregulate the transduction of externally applied forces to stimulate de novo osseous regeneration. Bradley and colleagues probed this questionby developing a system that allowed for the application of both lineardistraction and compression to MC3T3 preosteoblasts suspended in colla-gen type I gel.25 Here, it was observed that constant distraction augmentedthe proliferative capacity treated cells, while cyclic distraction and com-pression lead to increased expression of markers of osteogenicdifferentiation. In vivo work by Tong and colleagues made effort to clarifywhat molecular signaling pathways may be contributing to these observa-tions. Here, focal adhesion kinase (FAK) was immunolocalized to tissuescomprised of regenerated bone following distraction, speaking to theimportant role this signaling pathway may be contributing to the process.13


2.2 Craniosynostosis

Where the study of distraction osteogenesis has lead to important observa-tions regarding the molecular underpinnings of endogenous skeletal tissueregeneration, so to has the study of congenital anomalies of the craniofacialskeleton. Indeed, work aimed at understanding the genetic and molecularsignals involved when developmental cues fail, resulting in craniosynostosis,have further matured our understanding of important biologic principlesthat are today important to efforts in skeletal tissue engineering.

The identification of molecular miscues resulting in craniosynostosishave contributed to our current understanding of the molecular underpin-nings of osteogenic biology. Numerous signaling pathways have beenimplicated in the evolution of craniosynostosis, including TGF-β and FGF.26

Of particular interest is the role that bone morphogenetic protein (BMP)family members, as well as their antagonists, have been observed to play inthe development of aberrant cranial suture phenotypes.27–29 To elaboratehere, work by Warren and colleagues made interesting observations regard-ing the contribution of the BMP antagonist Noggin in normal mouse cranialsuture fate.27 In their study, they were able to demonstrate that Nogginexpression was observed in patent (sagittal and coronal), but not fusing(posterofrontal), sutures. However, when misexpressed via viral-delivery tothe posterofrontal suture, pathologic patency was observed. Furthermore,Noggin was shown to be suppressed by FGF-2 and syndromic gain of func-tion FGFR signaling. Thus, it can be postulated that syndromicFGFR-mediated craniosynostoses may result from inappropriate downreg-ulation of Noggin expression. Of interest to current skeletal tissueengineering efforts, conceptually such observations speak to the potentialbenefits from simultaneous stimulation of pro-osteogenic agonists and inhi-bition of antagonists in an effort to maximize the osteogenic potential ofmultipotent progenitor cells.

3. Progenitor Cell-Based Skeletal Tissue Engineering

Indeed, it is the unique endogenous capacity of skeletal tissue to self-renew and regenerate subsequent to injury that makes it such an attractivearea of focus for cell-based tissue engineering efforts. However, despite


this profound endogenous regenerative capacity, skeletal defects anddeficits resulting from congenital anomalies, chronic disease (osteoporo-sis), surgical resection (oncologic), and traumatic injury frequently exceedthis capacity and necessitate surgical intervention. It is here that skeletaltissue engineering and regenerative medicine aim to improve on the inad-equacies of current reconstructive modalities. Despite advances insurgical technique and biomaterial technologies, less than desirablepatient outcomes following skeletal reconstructive efforts are far too oftenobserved. Thus, a shift in focus has transpired toward investigating thecapacity of tissue engineering, and specifically progenitor cell-basedtissue engineering, to meet this need.

What are the critical characteristics of a multipotent osteoprogenitorcell that can potentially fulfill this need? First, it is important that theydemonstrate a potential to undergo efficient and predictable osteogenicdifferentiation. Additionally, the cells must display a robust proliferativecapacity, be accessible in sufficient numbers (ideally in quantities thatavoid the need for ex vivo expansion prior to utilization), and be acquiredby means that do not impart undo donor morbidity.

One needs to search no further than human embryonic stem cells(hESC) to identify a cell population that clearly fulfills all of these criteria.Furthermore, utilizing a true pluripotent cell population, capable of regen-erating multiple tissue types arising from all three germ layers, opens doorsto the possibility of addressing increasingly complex reconstructive sce-narios. However, enthusiasm surrounding the application of hESC inregenerative medicine strategies has largely been squelched by politicaland ethical concerns regarding their clinical application.30 It is thesehurdles that have lead scientists to escalate investigations in pursuit ofidentifying a postnatal progenitor cell populations capable of displayingthe aforementioned biologic characteristics necessary for application intissue engineering strategies. Encouragingly, coming forth from workstudying mesenchymal stem cells (MSC), a body of evidence has accumu-lated that supports their capacity to potentially meet this need. Nowisolated from a multitude of adult tissues, and observed to have a multipo-tent capacity to differentiate down osteogenic, cartilaginous, adipogenicand myogenic lineages, efforts to expand our understanding of this cellpopulation to meet the needs of skeletal tissue engineers has expanded.


3.1 Bone marrow-derived mesenchymal stromal cells

Earliest investigations on MSC focused on those derived from bonemarrow, coined bone marrow mesenchymal stem cells (BMSC). Withregard to the multipotent potential of this progenitor population, early in vitro work by Pittenger and colleagues described their ability toundergo multilineage differentiation, including osteogenic, when culturedin the appropriate milieu of small molecules.31 Since that time, a wealth ofinformation has accumulated displaying the capacity of both BMSCharvested from small animals, as well as bone marrow aspirates derivedfrom humans, to augment osseous regeneration in vivo.32,33

Despite these encouraging results, multiple hurdles have been putforth impeding the widespread clinical application of these cells in skele-tal tissue engineering strategies. Factors including donor morbidityresulting from bone marrow aspiration, as well as potential age-dependentdecline in proliferative and osteogenic capacity that may exist, hinderpotential for clinical application.34 Furthermore, the paucity of BMSCfound in bone marrow aspirates (one in 27,000 cells) would likely neces-sitate their ex vivo expansion, on the order of weeks, to obtain adequatecell numbers to produce a single reconstructive construct.35 As such, sub-sequent efforts on the part of investigators have focused on a more readilyobtainable and abundant source of MSC to mitigate the aforementionedfactors that have impeded the use of BMSC in the past.

3.2 Adipose-derived mesenchymal stromal cells

To this end, an abundance of evidence now exists that suggests thatadipose-derived stromal cells (ASC) may be up to the task. Early on, Zuket al. observed ASC to have a similar potential for mesenchymal lineagespecific differentiation as BMSC (bone, muscle, adipose, cartilage), aswell as display a surface antigen profile that equated that found in BMSCwith little exception.36,37 Furthermore, it appears as though ASC do notdisplay a decline in proliferative capacity with age as was previouslyobserved in BMSC.38 Given these early observations, paired with the ben-eficial characteristics of ASC compared BMSC (relative abundance andpotential for acquisition with relatively little donor morbidity), our


laboratory and others have invested significant effort in pursuing thetranslational potential of this mesenchymal progenitor population in thesetting of skeletal tissue engineering.

In vivo studies have put forth promising results regarding the potentialto utilize ASC in the development of cell-based skeletal tissue engineer-ing strategies. In this context, the bone forming capacity of ASC was firstdescribed by Lee and colleagues, when they observed ossification of ASCseeded PLGA scaffolds implanted subcutaneously.39 Subsequently,Cowan et al. demonstrated the ability ASC to regenerate bone in critical-sized calvarial defects.40 In their study, murine ASC were seeded onapatite-coated, PLGA scaffolds. After implantation into critical size pari-etal bone calvarial defects, comparable bony healing was noted in defectstreated with ASC relative to bone formation in study groups treated withBMSC and osteoblasts.40 Most recently, Yoon et al. reported a significantincrease in the healing of calvarial defects created in nude mice, andrepaired with human ASC-seeded PLGA scaffolds, compared to micetreated with scaffolds alone.41

3.3 Induced pluripotent stem cells

Most recently, pluripotent cells originating from adult tissues, now coinedinduced pluripotent stem cells (iPS), were described by Takahashi et al. in2006.42 Observed to have similar pluripotent capacity and cellular charac-teristics as hESC, iPS cells have been until recently derived fromembryonic and adult postnatal fibroblasts reprogrammed to express Oct4,Sox2, c-Myc, and Klf4. Regarding their translational potential, advocatesof iPS cells believe that these cells hold the potential to harness all of thepositive attributes of hESC, while largely circumventing the aforemen-tioned political and ethical concerns surrounding their study and clinicaluse. However, despite the substantial exuberance that has surrounded theirdevelopment, iPS cells are not without their critics. The need for retrovi-ral or lentiviral transfection in order to induce transcript expression, andthe associated risk of viral integration into the recipient genome, posesignificant concerns to application in humans. As such, efforts haveensued to derive iPS cells utilizing non-viral means, and moderate successhas been appreciated. Okita et al. observed the successful production of


virus-free iPS cells from embryonic fibroblasts through repeated transfec-tion of two independent plasmids containing complementary DNAsequences of Oct3/4/Sox2/Klf4 and c-Myc.43

Additional concerns have been raised regarding the extended period ofex vivo culture required for both adequate numbers of fibroblasts har-vested from skin to be obtained for iPS derivation, as well as the timerequired in culture for the formation of iPS colonies arising from fibro-blasts to be observed. The period of culture that has been observed to datefor the iPS colonies to be generated post-transfection is currently on theorder of weeks. Furthermore, the relatively low efficiency of transforma-tion of fibroblasts to iPS cells, as well as the need to be cultured on afeeder layer of mouse cells, have presented additional concerns regardingthe realistic feasibility of clinical translation. Thus, our group and othershave begun to probe the potential of alternate adult cell populations toachieve this goal more expeditiously and efficiently. To this end, collabo-rative efforts between the Wu and Longaker Laboratories at StanfordUniversity have put forth exciting progress on this front. Sun et al. havenow reported that deriving iPS cells from human ASC (hASC-iPS) can beachieved in significantly less time (16 days vs. 28 days) and with sub-stantially greater efficiency (0.2% vs. 0.01%) relative to their fibroblastcounterparts.44 Furthermore, although with reduced efficiency, hASC-iPScan be derived and maintained in culture in the absence of a feeder layerof cells; additional properties not previously appreciated when derivingiPS cells from embryonic or adult fibroblasts.44 Such advances are poten-tially of paramount importance to the future of complex skeletalreconstructive scenarios. Through the application of truly pluripotent pro-genitor cells, the potential to address reconstructive needs involving notonly multiple mesenchymal-derived tissue types, but tissues derived fromall three germ layers, becomes increasingly feasible.

4. Pro-osteogenic Molecular Biology

Today, it is believed that successful transition of cell-based skeletal tissueengineering strategies to the bedside will necessitate the application of tar-geted molecular manipulations of osteoprogenitor cells in order tomaximize their osteogenic potential and skeletal regenerative capacity.


Furthermore, an additional goal of delivering pro-osteogenic small mole-cules and cytokines via reconstructive constructs will be aimed atenhancing the endogenous regenerative capacity of surrounding nativetissues, as well as incorporation of implanted reconstructive constructswith these tissues. Heretofore, presented is a current working knowledgeregarding the molecular signaling pathways they have been identified asplaying an important role in skeletal developmental biology, fracture heal-ing, and the osteogenic differentiation of osteoprogenitor cells.

4.1 Bone morphogenetic protein

Of the important pro-osteogenic signaling pathways that have been iden-tified, BMP has been most widely examined. To briefly provide somebackground, BMP is a member of the transforming growth factor-beta(TGF-β) superfamily. To date, more than 14 types of human BMP havenow been described.45 Of these, BMP-2, -6, -7, and -9 have been observedto promote robust osteogenesis in vitro and in vivo.45 BMPs function in adose dependent fashion, and demonstrate the greatest pro-osteogeniceffects when present as the heterodimers BMP-2/6, -2/7, and -4/7.46,47

Regarding the osteogenic differentiation of multipotent mesenchymal pre-cursor cells, BMP signaling has been observed to be mediated throughType I or II receptors, resulting in subsequent downstream activation ofSMAD transcriptions factors through phosphorylation, and modulation ofthe transcription factor Cbfa1/Runx2.45

Multiple investigations have highlighted the importance of BMP sig-naling to the osteogenic differentiation of both mouse BMSC andASC.48,49 Furthermore, investigations by Panetta and Gupta et al. in theLongaker laboratory have made strides in demonstrating the importanceof BMP signaling to the osteogenic differentiation of human ASC. Thiswork has been able to clearly define that human ASC respond to BMP ina dose dependent fashion. Furthermore, by means of qRT-PCR andELISA, it was elucidated that human ASC display increased BMP geneexpression and protein elaboration during the course of osteogenic differ-entiation. The specificity of this pro-osteogenic response to BMP wasclarified utilizing BMP-2 specific neutralizing antibodies. Here,osteogenic differentiation was impaired in the presence of neutralizing


antibody to BMP-2, and could be rescued through exposure to exogenousprotein. These observations are critical, as they increase the viability oftargeted molecular manipulations of BMP signaling, and potentially thatof its antagonists, to enhance the osteogenic capacity of human ASC foruse in cell-based skeletal tissue engineering strategies. In vivo, Cowanet al. observed augmented bony healing and bone turnover in critical sized4 mm murine calvarial defects reconstructed with osteoblasts, BMSC, andASC stimulated with rhBMP-2 ex vivo and seeded onto apatite coatedpoly-lactic-co-glycolic acid scaffolds.50 Encouraged by these in vitro andin vivo observations, considering their potential translational implications,ongoing studies are aiming to probe whether augmented BMP signalinghas the capacity to enhance in vivo healing mediated by human ASC.

Speaking to the potential translational importance of BMP to bonetissue engineering efforts, clinical applications of BMP to augmentendogenous skeletal healing have already achieved success in the clinicalarena. FDA approval was first granted in 2002 for the delivery of rhBMP-2 on a Type I collagen sponge to treat of degenerative lumbar diskdisease.38 Subsequently, preclinical trials by McKay and colleaguesdemonstrated that 94.5% of rhBMP-2 treated patients, compared to 88.7%of autograft treated patients, demonstrated disk fusion radiographicallytwo years post-intervention.51 Today, the application of rhBMP-2 in thetreatment of tibial fractures has also been approved for clinical use.38

Collectively, both these and the aforementioned observations stronglysupport the potential of augmented BMP signaling to enhance bothendogenous and cell-mediated healing of bony defects.

4.2 Wnt

Recently, evidence that supports the previously unappreciated role of Wntsignaling to the osteogenic differentiation of mesenchymal progenitorcells has been put forth.45 Briefly, Wnt signaling is mediated by the bind-ing of Wnt ligands, comprised of multiple secreted glycoproteins, to thetransmembrane receptors Frizzled/LRP5/6 co-receptors. Upon binding,phosphorylation of Disheveled protein occurs. Next, through a complexinteraction between Axin, Frat-1, and APC tumor suppressor, phosphory-lation of β-catenin by GSK-3β is inhibited. This leads to subsequent


cytoplasmic accumulation of β-catenin, nuclear translocation, and tran-scription of downstream target genes. Regarding the role of Wnt signalingin the osteogenic differentiation of MSC, Gaur et al. demonstrated thatWnt signaling lead to the activation of Runx2 in mesenchymal osteo-progenitors.52 Additionally, Day and colleagues described that β-cateninmediated Wnt signaling enhanced osteogenic, and inhibitedchondrogenic, differentiation of MSC.53

4.3 Fibroblast growth factor

The family of FGF ligands consists of 23 related proteins, and signaltransduction is mediated by four tyrosine kinase transmembrane recep-tors. Speaking to the important role FGF signaling plays in bonehomeostasis, FGF-2 null and haploinsufficient mice have been observedto express reduced levels of FGFR-2 and Runx2, with resultant decreasedbone density, relative to wild type mice.54 In an effort to clarify the speci-ficity of these observations as they related to a decreased presence ofFGF-2, subsequent studies observed that the osteogenic capacity ofosteoblasts harvested from null and haploinsufficient mice was rescued in vitro by the addition of exogenous FGF-2 ligand. The important role ofFGF signaling in the osteogenic differentiation of mesenchymal cells hasalso been described. Important to the identification of osteogenic signalingpathways in ASC that may potentially be amenable to targeted manipula-tions aimed at enhancing the osteogenic potential of these progenitorcells, Quarto et al. recently described the dynamic expression profile ofFGF-2, -4, -8, and -18 in vitro during the course of mouse ASC osteogenicdifferentiation.55

4.4 Hedgehog

The important role of the Hedgehog family of proteins in the process ofskeletal development and osteogenesis has been observed. Although con-tributing in distinct fashions, two of the three constituent family memberproteins (Sonic Hedgehog, Indian Hedgehog, and Desert Hedgehog) arenow known to contribute to the regulation of bone formation. SonicHedgehog (Shh) plays an integral role in the process of skeletal


patterning. Alternatively, Indian Hedgehog (Ihh) expression is critical toendochondral ossification.45 During the activation of these proteins,Hedgehog proteins are cleaved from their secreted form to a 19 kDa N-terminal fragment. Active fragments then function in a paracrine fash-ion through binding to the cell surface receptors Patched (Pct) andSmoothened (Smo). Upon binding, Smo is released from the complex,allowing for the activation of downstream signaling factors Gli1, 2, and 3and expression of Hedgehog target genes.45 Speaking to the importance ofthis cascade of events to the differentiation of osteoblasts, Hu et al.demonstrated that interruption of Ihh in primitive osteoblasts at a point indifferentiation prior to the expression of Colα1, alkaline phosphatase, andRunx2, resulted in the arrest of the differentiative process.56 Having madethis observation, ongoing studies are aiming to elucidate the role ofhedgehog signaling in multipotent progenitor populations with potentialfor application in tissue engineering strategies.

4.5 Hypoxia-inducible factor 1-alpha

A growing body of evidence continues to illuminate the importance ofangiogenesis in the process of skeletal regeneration and healing.Participating in this complex process, Hypoxia-inducible factor 1-alpha(HIF-1α) is now known to be critical to successful osseous healing. Wangand colleagues observed that HIF-1α over expression in osteoblasts ofdeveloping bone lead to increased levels of VEGF, resulting in highlyvascularized bones with high bone mineral density.15 Additionally, and ofsignificant importance to cell-based tissue engineering efforts, Cetrulo et al. demonstrated that systemically administered progenitor cells prefer-entially localized to ischemic regions of osseous healing, where HIF-1αupregulation was observed. This observation contributes additional sup-port to further investigate the potential of this signaling mechanism toaugment skeletal regeneration mediated by MSC.16

In the future, simulation of angiogenesis will likely play a critical rolein successful application of cell-based bone tissue engineering constructsin the operating suite. This is secondary to the fact that constructs studiedto date have been limited in size do to their avascular nature, making cellsurvival dependant on diffusion of nutrients from surrounding tissues.


Through the incorporation of pro-angiogenic signals, it is the hope of sur-geons that the viability of larger constructs will be appreciated, increasingthe scope of potential clinical applications.

5. Advances in Skeletal Tissue Engineering Scaffolds

Although progenitor cells hold substantial potential to produce functionalregenerated skeletal tissue on a scale not previously appreciated, they lackthe capacity to put forth the necessary mechanical stability important toskeletal reconstruction during the process of ossification and the matura-tion of regenerated bone. Providing such mechanical stability is critical,while at the same time possessing degradation kinetics that allow for thetimely replacement of the scaffold material by regenerated bone.Furthermore, as alluded to in our previous discussion, the utilization ofpro-osteogenic and angiogenic small molecules and cytokines to augmentthe regenerative process will likely be a critical component of futurereconstructive constructs. However, to appreciate optimal outcomes andavoid untoward effects of therapy, it will be imperative to deliver pro-osteogenic small molecules and cytokines in a very specificspatiotemporally controlled fashion to both progenitor cells and sur-rounding endogenous tissues. It is these factors that largely continue toguide the efforts of material scientists developing scaffold materials withan architecture and composition the successfully meets the needs of thereconstructive surgeons and patients requiring surgical intervention.

5.1 Scaffold composition

The composition of scaffolds is maybe the most important factor thataffects their osteoinductive potential. In the broadest sense, scaffold mate-rials can be divided into four categories: (1) natural scaffolds (i.e. collagen,hyaluronic acids, calcium alginate, chitosan, fibrin, thermoplastic starchbiodegradable plastics, etc.); (2) mineral-based scaffolds (i.e. calciumphosphate ceramics, bioactive glass, etc.); (3) polymer scaffolds (i.e. polylactic acid, polyglycolic acid, polylactic co-glycolic acid, poly-doxanonone, polycaprolactone, polyfumarate, etc.); and (4) hydrogels.57

As each of these scaffold materials bring to the table specific strengths


and weaknesses, the advent of composite scaffolds has ensued. Examples of such composites include hydroxyapatites/polymers, calciumphosphates/polymers, and bioglasses/polymers. Here, efforts have beenmade to draw upon the positive attributes of each component material,while mitigating insufficiencies appreciated individually. Investigationprobing the capacity of composite scaffolds to achieve this end have beenencouraging. Lastly, material properties specific to hydrogel scaffolds,including their capacity to undergo temperature dependent phase change,in addition to the potential to have specific control over their chemical,material, and three-dimensional characteristics, collectively make hydro-gels very attractive candidate scaffold materials for tissue engineers.58,59

With such malleability in composition as is seen in hydrogels, very specificcontrol over the incorporation of small molecules and cytokines, as well asof their release through controlled degradation, becomes increasinglyrealistic.

5.2 Scaffold structure

In so much as scaffold composition dictates the osteoinductive propertiesof a scaffold, scaffold structure largely defines its osteoconductive char-acteristics. Innate to defining the osteoconductive properties of a scaffoldare its capacity to allow for the integration of implanted osteoprogenitorcells with surrounding endogenous skeletal tissue to regenerate bone thatmirrors endogenous tissues in form and function. Several scaffold struc-tural characteristics have been identified that can significantly alter theircapacity to achieve this end. Degree of porosity, pore and/or tube size, andresultant surface to volume ratio all represent structural properties that canaffect the ability of a scaffold to direct neovascularization and nutrientdelivery to regenerating bone.57 If inadequate, these factors can have asubstantial negative impact on cell-mediated osseous healing.

5.3 Scaffold fabrication techniques

With a growing body of knowledge in hand informing tissue engineers ofscaffold compositions and structures that may best guide osseous regen-eration, the challenge becomes to produce scaffolds possessing these


properties in a reproducible and efficient manner. To this end, significantprogress has been appreciated secondary to the development of novel fab-rication technologies. Bioengineers and material scientists have set forthto develop techniques capable of generating scaffolds with a microarchi-tecture that closely resembles that of endogenous skeletal extracellularmatrix. It is believed that doing so will help to guide progenitor cells todeposit matrix in an arrangement that mimics that of endogenous tissues.Here, current efforts are being focused on refining nanofiber technologies(photolithography, electrospinning, laser patterning, self-assemblingpeptides).60 Most recently, self-assembling peptide nanofiber scaffoldshave garnered significant attention from scientists. Undoubtedly, clari-fying all of the compositional and structural scaffold characteristics thatwill optimize skeletal regeneration, as well as the development of tech-nologies that enable such scaffolds to be produced in a predictablefashion, will be critical for delivery of skeletal reconstructive modalitiesto the marketplace.

6. Summary and Future Directions

Undoubtedly, significant progress has been made in expanding our under-standing of all of the critical components of cell-based skeletal tissueengineering constructs. Yet, for these technologies to someday become areality and achieve translation to the bedside, as substantial amount ofwork to address unanswered questions remains. First, harnessing thecapacity to define MSC subpopulations that may exist within the popula-tion of cells isolated by current techniques has remained elusive. It iscertainly plausible, if not even probably, that the current population ofcells we define as MSC is in reality a “minestrone soup” of progenitorswith varying capacity for differentiation toward the various mesenchymallineages. Identifying the unique cell surface antigen profiles of progenitorsubpopulations may play a critical role in isolating osteoprogenitors withthe greatest osseous regenerative capacity.

Second, the need for continued evolution of current scaffold technolo-gies is unquestionable. We have now reached a point where futureprogress must focus on the development of increasingly biologicallyactive scaffold materials. A wealth of knowledge is now in hand regarding


molecular signaling pathways critical to the osteogenic differentiation ofmultipotent osteoprogenitors. As such, novel scaffold materials must havethe capacity to deliver small molecules and cytokines in a specific spa-tiotemporally controlled fashion, altering the agonist/antagonistrelationships involved in the regulation of these pathways in a pro-osteogenic fashion. Additionally, the goal of such interventions should notonly be to augment the osteogenic capacity of implanted progenitor cells,but simultaneously enhance the endogenous regenerative capacity of sur-rounding native bone. The envisioned end point of such manipulationswould be the rapid and efficient production of a robust skeletal regener-ate. With due diligence, success in addressing these needs, as well asneeds that remain unrecognized, will assuredly come to fruition. In turn,such advances will make possible the realization of improved patient out-comes through the translation of skeletal tissue engineering to the bedside.

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14

Clinical Applications of a Stem Cell BasedTherapy for Oral Bone Reconstruction

Bradley McAllister and Kamran Haghighat

1. Introduction

We have observed a rapid evolution in both surgical techniques andmaterials utilized in the promotion of regenerative therapy for oral recon-structive procedures. In particular, bone augmentation has been promotedthrough different methods which include the use of growth and differen-tiation factors, particulate and block grafting materials,1–4 distractionosteogenesis,5–7 as well as membrane-assisted guided bone regeneration(GBR).8 A central theme to all of these oral reconstructive technologies isthe need for rapid and complete revascularization.

Early research that introduced us to the concept of using barrier mem-branes in what is referred to as GBR outlined the need for exclusion ofundesirable soft tissue cellular contents and provision of a secluded spaceinto which osteogenic cells from various sources can migrate for successfulbone healing.9–11 The pattern of bone regeneration involves angiogenesisand ingress of osteogenic cells from the defect periphery towards thecenter to create a well-vascularized granulation tissue. This provides a

277

scaffold for woven bone proliferation and bone apposition within thedefect.12 The size of the defect influences the bone healing capacity. Incircumstances where the defect size is too large to generate a biomechan-ically stable central scaffold, bone formation will become limited to themarginal stable zone with a central zone of disorganized loose connectivetissue. Critical to the outcome of GBR is maintenance of primary woundclosure throughout the healing period.13,14

Perforation of the cortical bone layer has been advocated in GBR as ithas been postulated that this will increase the vascularity of the woundand release growth factors and cells with angiogenic and osteogenicpotential. Aggressive recipient bed preparation with decortication, intra-marrow penetration has also been supported due to increases in the rate ofrevascularization, the availability of osteoprogenitor cells and theincreased rate of remodeling.12,15 In addition to the surrounding bone, theperiosteum is also widely considered as an important source of cells withosteogenic capability. Despite the desirable graft containment and cellularexclusion characteristics of most barrier membranes, limiting the accessof periosteal-derived osteogenic cells during the early phases of woundhealing may not be of benefit to the healing of larger sized defects.

The autograft, allograft, alloplast and xenograft materials all havereported success either alone, or in combination, for particulate boneaugmentation.16 The particulate autograft is currently recognized as thegold standard for most bone grafting, including the treatment of dentalimplant related defects. Several studies have demonstrated the effectivenessof particulate autograft due the availability of cells with osteogenicpotential, as well as osteoinductive and osteoconductive properties.17,18

However, autografts have recognized limitations, such as donor sitemorbidity, increased cost, potential resorption, size mismatch, an inade-quate volume of graft material, as well as the unpredictability in thequantities of osteogenic precursor cells.

Allografts have the advantage of being available in higher quantitiesand eliminate the morbidity associated with a second surgical site.Biochemical extraction techniques have shown that growth and differen-tiation factors are present in demineralized freeze-dried bone allograft(DFDBA) preparations; however, quantities have been shown to be vari-able from lot to lot indicating a potential variation in performance.19–21

278 B. McAllister and K. Haghighat

Thus, allografts primarily act as a scaffold for the in-growth of capillar-ies, peri-vascular tissues, and osteoprogenitor cells from the adjacentrecipient bed.

The absence of differentiating precursor cells or osteoblasts in ade-quate quantities will ensue in limited bone formation. Osteoblasts containthe cellular machinery for production of bone matrix, but they are unableto undergo further division and have limited migratory capacity. Thislimits the expected benefits of cells contained in oral derived autografts,for example, which exhibit high variability in the numbers of cells withosteogenic potential.

Based on our current understanding of graft healing and the prerequi-sites for optimal bone regeneration, tissue-engineering research has beenfocused on providing the necessary cellular machinery, namely themesenchymal stem cells (MSCs) and osteoprogenitor cells, directly insites that require bone regeneration. It is this concept that has been utilizedin the processing of the commercially available graft material that will bediscussed here. Historically the majority of efforts for bone grafting withMSCs and osteoprogenitor cells have focused on the concept of harvestingthese cells followed by in vitro culture expansion for later implanta-tion.22,23 The following methodology section describes a novel approachthat leaves the MSCs and osteoprogenitor cells found within allogeneicbone and substantially depletes unwanted cells.

2. Procurement Methodology for Stem CellContaining Allograft

The allogeneic bone graft material described in this chapter (Osteocel®) iscommercially prepared for NuVasive, Inc.TM from cadavers recovered bylicensed tissue procurement agencies (AlloSource) and distributed intothe dental market by ACE Surgical Supply. Cadaver tissues are rushed tothe processing facility on wet ice and processing is begun within 24 hoursof the donors’ death. In parallel rigorous safety testing, donor screeningand evaluation for bacterial, fungal and spore contamination begins.Screening measures consist of physical examination and evaluation ofboth medical and social history, including a next of kin interview.Comprehensive serological and microbial testing are also performed

Oral Bone Reconstruction with Stem Cells 279

which includes nucleic acid testing (NAT) for Hepatitis-C and HIV. Donorassessment culminates with a complete medical record review by alicensed physician. Cortical bone is separated and processed into dem-ineralized bone particles for adding back to the cellular graft component.A process of selective immunodepletion, that involves several extensivewash steps, is initiated to remove undesirable cells, such as red blood cellsand lymphocytes that can provoke an immune response. These unwantedcells are substantially depleted leaving the remaining cell rich cancellousbone matrix. The cellular cancellous bone component then undergoes abroad-spectrum antimicrobial treatment (vancomycin, gentamicin sulfateand amphotericin) designed to eliminate potential contamination whilepreserving the viability of the cells. These remaining viable MSCs andosteoprogenitor cells remain attached to the cancellous bone matrix.Approximately 20% demineralized cortical bone particulate from thesame donor is then combined to the cell containing cancellous bonematrix component. A standard cryopreservation solution containing 10%DMSO with human serum albumen (HSA) is added and the product isstored at −80 ± 5 degrees Celsius (°C), permitting a five-year shelf life.

During the product processing validation, FACS (fluorescence acti-vated cell sorting) testing was performed to confirm the retention ofMSCs that are positive for cluster of differentiation 105 (CD105) andCD166 while being negative for CD45.24 Figure 1 depicts a representativeFACS Scatter Plot of the cellular allograft. While there is not a singleidentifiable surface marker for MSCs, this marker combination profile isindicative of MSCs and osteoprogenitor cells. Quality testing is performedon every lot of Osteocel® to validate a minimum cell count of 50,000cells/cc, and a minimum cellular viability of 70% of the enzymaticallyreleased cells. Another iteration of the cellular allograft product(Osteocel® Plus), which was not utilized in the clinical and histologicalaspect of this chapter, has quality testing for a minimum cell count of250,000 cells/cc. The cell count and viability are determined on releasedcells by a Trypan Blue dye exclusion test with a hemocytometer. Cellularosteogenic activity of each lot is also validated by performing in vitro celldifferentiation and alkaline phosphatase assays (Fig. 2).

The cellular bone graft material is stored at −80°C, shipped to the clinicon dry ice where it is prepared as per the manufacturer’s recommendation.



Fig. 1. Representative FACS Scatter Plot of Osteocel®. (A) Forward Scatter and SideScatter dot plot from the donor 2 population of Osteocel® Plus cells with [R1] 94.44%,[R2] 99.93% and [R1 + R2] 94.42%. (B) Dot plot showing positive expression of CD105and CD166 markers, after gating for CD45- from the donor 2 cells. Quadrant gating UL0.82%, UR 99.14%, LL 0.04% and LR 0.00%.

Fig. 2. Macroscopic aspect of the Osteocel® product. Following the appropriate thawingof the cellular allograft, there is a recommended four-hour window for its use. (Inset)Cellular image from the Osteocel® derived cells demonstrating positive osteogenic activity(staining positive for alkaline phosphatase activity).

Since the graft contains vital cells the maximum temperature of the waterbath used during the thawing process should not exceed 37°C. After thecryopreserved cells are thawed, the liquid is decanted and the cells arerinsed with sterile saline. The cell containing graft is then ready forimplantation, with a working window of four hours (Fig. 2). Dependingon the defect treated, the particle size (1–3 mm) is often found to be toolarge for oral reconstructive procedures. In such instances rongeurs can beused to carefully reduce the particle size.

Scanning Electron Microscopy (SEM) images from this particulategraft have consistently revealed the cellular component of the allografttogether with the extracellular matrix surrounding them. Figure 3 containsexample SEMs at different magnifications. The SEM preparation startswith a 0.1M cacodylate buffer containing 5% sucrose rinse. The cells arefixed with 2.5% gluteraldehyde for one hour. Following three rinses withcacodylate buffer, the samples are soaked in 1% osmium tetraoxide solu-tion in water for one hour at 4°C. The samples are subsequently dehydratedstep-wise in increasing concentrations of ethanol starting at 50% andcontinuing to 100% for approximately two minutes at each step. A criticalpoint dryer is used to dry the samples. The bone particles are attachedto the SEM plates with adhesive and silver paint followed by a gold/platinum sputter coating prior to imaging. Images were captured utilizinga Quanta Model 600 (FEI, Hillsboro, OR) with a Tungsten filament athigh vacuum mode and Soft Image Solutions (Olympus, Inc., Germany)software was employed for image collection.

3. Ridge Augmentation

With the greater acceptance and awareness of dental implant therapy as thestrongest method of tooth replacement, amongst practitioners and patientsalike, it is not uncommon to encounter reconstructive scenarios that requirebone augmentation in the overall treatment plan. This is particularly seen incases with long-standing edentulism, trauma and infection. Augmentationof the alveolar ridge for the ideal placement of an implant thus becomesnecessary for an optimal esthetic outcome.25 The various techniques andmaterials employed in ridge augmentation procedures have been discussedelsewhere and is beyond the focus of this chapter.4 Of relevance, Osteocel®,



Fig. 3. (A) SEM image showing the cancellous bone coated with native cells. The cellsare covered by extracellular matrix. (B) Higher magnification (2000×) of the box in(A). (C) Higher magnification (4000×).

as a cellular based grafting material, provides an attractive option for ridgeaugmentation, particularly in larger defects.

It is hypothesized that the cellular contents within Osteocel® wouldbenefit from a rapid revascularization. This revascularization process takesplace at a much faster rate from the periosteal source than bone. To exploitthis notion, use of a classic barrier membrane should ideally be avoided,when possible, to facilitate this process during the initial stages of healing.Other graft materials containing molecular enhancement products havelikewise been shown in certain studies to undergo a slower healing ratewhen used in conjunction with a barrier membrane.26,27 Figure 4 shows thesuccessful use of Osteocel® alone for a small defect. The use of barriermembranes, however, may be deemed necessary when treating larger nonspace-maintaining defects for graft containment. In larger sized defects, aswell as in cases where control of the location of the graft is critical, suchas grafting against dental implants, a space-maintaining device in the formof a titanium mesh has been successfully employed.4,28,29

Titanium mesh offers resistance to bone graft collapse without the com-promise in revascularization found with many products. However, whenfaced with a thinner tissue biotype caution is necessary when using anynon-resorbable device as ensuing soft tissue dehiscences can lead to a com-promised outcome.14 Recently Pieri and colleagues demonstrated titaniummesh use in combination with a mixture of intraoral autogenous bone andxenograft on 16 partially edentulous patients.30 They reported a mean hor-izontal augmentation gain of 4.2 mm. Only one of the cases showed earlyexposure of the mesh device. In cases where it was desired to regeneratemore than 3 mm of horizontal bone we have predictably used the graft inconjunction with a titanium mesh for both space maintenance and graftcontainment preventing the cellular allograft from collapsing (Fig. 5).

4. Sinus Augmentation

The posterior maxilla represents an area that has historically posed a chal-lenge for treatment with dental implants. These range from comprisingsites with poor bone quality to unfavorable bucco-lingual resorptionpatterns and inadequacy in the vertical dimension of available bonefollowing extraction of the teeth.31,32 In addition, bone regeneration within


the graft material is dependent upon it being populated by osteogenic cellsthat primarily originate from the osseous floors and walls, and to a smallerdegree from the Schneiderian membrane.33,34 Thus, cellular infiltration,vascularization, de-novo bone formation and graft replacement oftenrequire long healing times to produce bone of adequate quantity andquality for implant placement in the posterior maxilla.


Fig. 4. Horizontal bone augmentation with Osteocel®. (A) Clinical view showing thedental implant dehiscence at the time of placement. Note intra-marrow penetrations havebeen made. (B) Clinical view with the cellular allograft in place. No membrane was utilizedand the flap was sutured to tension-free primary closure with VicrylTM sutures. (C) Thefour-month post-operative clinical view showing 2–3 mm of lateral bone augmentation andcomplete coverage of the implant threads. (D) A four-month post-operative CT scanshowing the regeneration of a 2–3 mm thick buccal plate of bone over the implant.

Ongoing maxillary sinus pneumatization and normal post-extractionbone atrophy has been managed successfully by the sinus augmentationprocedure either before or simultaneously with implant placement. Theliterature is inundated with reports describing this procedure using avariety of grafting materials. The use of a variety of materials has showna varied bone quality and quantity with reported percentage bone areasthat range from as low as 5% to over 40%.35,36 In addition, studies havedemonstrated that it can take in excess of nine months to achieve optimalbone formation for implant stability.37,38

Dental implant technology has endeavored for a faster osseointegra-tion period to allow for more rapid restoration of the lost dentition. Theconcept of molecular enhancement of graft materials with either growthor differentiation factors for a more rapid regenerative outcome becomesdesirable.39,40 Cellular enhanced bone graft materials potentially offer thistiming advantage as well. Our experiences with Osteocel® as a sinus


Fig. 5. Horizontal bone augmentation with titanium mesh and Osteocel®. (A) Pre-operative clinical view. (B) Mucoperiosteal flap elevation revealing a deficient alveolarridge in the horizontal dimension. Perforation of the cortical layer with intra-marrowpenetration has been performed for revascularization of the Osteocel® graft. (C) A titaniummesh has been adapted and secured with screws for containment of the cellular allograftmaterial. (D) The four-month post-operative view following titanium mesh removalrevealing a 3–4 mm increase in horizontal ridge dimensions. (E) View of bone regenera-tion and Straumann implant placement after a partial reflection of the pseudoperiosteumthat is often found when using titanium mesh. (F) Closure of soft tissues after implantplacement, completely within the healed bone graft, showing a normal facial ridge profile.

augmentation bone graft material has consistently provided promisingoutcomes.24 This initial report based on histomorphometric analysis ofgrafted sinuses with Osteocel® showed an average vital bone content of33% (range 22%–40%) and an average residual graft content of 6% (range3%–7%) for cases that had an average healing period of 4.1 months (rangefrom three to 4.75 months). These results were confirmed in a recent


Fig. 6. Sinus augmentation with Osteocel®. (A) Sinus access after a classic lateralwindow approach with simultaneous implant placement. (B) Sinus after grafting with thecellular allograft. No membrane was used to cover the bone graft and lateral wall accesswindow. (C) A CT scan of the grafted sinus immediately after graft and implant placement.(D) A CT scan of the same grafted sinus after four months of healing. Note the radi-ographic evidence of increased bone density in the bone graft region surrounding thedental implant. The radiographic findings of significant bone formation are consistent withthe histologic data showing average percent bone areas in excess of 30%.


Fig. 7. Histological evaluation of the healed bone after sinus augmentation with cellularallograft residual. (A) Representative mineralized histologic core of a cellular allograftresidual grafted sinus. The portion of the core shown goes from the most superior aspect(left side) to the original sinus floor (right side). It was harvested four months followingthe cellular allograft sinus grafting procedure. The red stained tissue is either the residualmineralized cellular allograft material (lighter red, osteocyte nuclei not always visible) ornewly formed bone (darker red, osteocyte nuclei visible). The green stained tissue is theresidual demineralized allograft material (no cells visible, non-vital bone). New bone for-mation can be appreciated throughout the core. Bone has formed directly on the residualmineralized and demineralized allograft particulate as well as in areas without residualgraft material (original magnification of 20×). (B) New bone of varying levels of

larger multicenter study.41 A faster graft healing time with respect to newbone formation in adequate quantities has encouraged an earlier initiationof implant placement and restoration. A sinus augmentation case withOsteocel® is shown with the radiographic follow-up (Fig. 6) and histolog-ical evaluation after four months (Fig. 7).

5. Discussion

Stem cells can be derived from a variety of sources, including bone mar-row, and are used in a variety of medical therapies. In the future, medicalresearchers anticipate being able to use technologies derived from stemcell research to treat a wider variety of systemic diseases, in addition to sitespecific repair as was described in this chapter. To optimize these excitingapplications for stem cells a thorough characterization and understandingof stem cell biology will be required. Although stem cells can be isolatedbased on a distinctive set of cell surface markers, in vitro culture conditionscan alter the behavior of cells, making it unclear whether the cells willbehave in a similar manner in vivo. In fact, debate exists whether some pro-posed adult stem cell populations are truly stem cells. Because of theircombined abilities of unlimited expansion and pluripotency, embryonicstem cells remain a theoretically viable source for regenerative medicineand tissue replacement after injury or disease. Differentiating embryonicstem cells into usable cells and ultimately organs is a challenge that tissue


Fig. 7. (Continued ) maturation can be appreciated in this higher magnification view(original magnification of 100×). (C) Osteoblasts can be seen lining the newly formedbone (original magnification of 200×). (D) Several areas of bone graft resorption can beappreciated in this view. This osteoclastic activity along with the low original packingdensity is likely responsible for the small percentage area that is the original cellular graftmaterial. Multiple multinucleated cells can be seen (original magnification of 200×).(E) The difference in the level of bone maturity with areas of immature woven bone (rightside) and areas of mature lamellar bone (left side) can be appreciated (original magnifica-tion of 100×). (F) Previous field (E) seen under polarized light. The demineralized boneareas (green) did not refract the polarized light to the extent the mineralized bone areas did.The difference in the level of bone maturity with areas of immature woven bone (right side)and areas of mature lamellar bone (left side) can also be appreciated with respect to the levelof polarized light refraction (original magnification of 100×).

engineering researchers will face for years to come. Additionally, the useof embryonic stem cells is more controversial than adult stem cells.

Stem cells and progenitor cells act as a repair system for the body, notonly replenishing specialized cells, but also maintaining the normalturnover of regenerative organs and tissues. In spite of this importantfunction, pluripotent adult stem cells are rare and generally small in num-ber within the body, with most being lineage-restricted (multipotent).They have also been shown to decrease in number with age. Bone marrowcontains numerous cell types from both the hematopoietic stem celllineage (for example platelets, osteoclasts) and the non-hematopoieticstem cell lineage (for example MSCs, osteoblasts).42 During the Osteocel®

procurement procedures, immunogenic cells and tissues are substantiallydepleted. The potential for immune response is why typical fresh-frozenbone allografts are not as attractive of a grafting option, even though in afew reports success for oral reconstructive procedures has been demon-strated. One recent study on 21 patients reported high success with dentalimplant placement into fresh-frozen bone allograft regenerated bone.43

Since only a disinfection process is performed with typical fresh-frozenbone allografts and not a complete immunodepletion process, there arelikely large numbers of undesirable cells remaining in the graft. Thiscould potentially impact the graft performance and the host immuneresponse.

For multiple reasons MSCs enjoy a hypoimmunogenic host response.MSCs lack MHC-II and co-stimulatory molecule expression. MSCs arealso immunomodulatory in that they prevent T-cell responses indirectlythrough modulation of dendritic cells and directly by disrupting NK aswell as CD8+ and CD4+ cell function.44 MSCs also induce a suppressivelocal microenvironment through cytokine production (prostaglandins andinterleukin-10) and expression of indoleamine 2,3-dioxygenase whichdepletes the local milieu of tryptophan. One of the major proteins pro-duced by MSCs is transforming growth factor-beta (TGF-beta) thatregulates the host T-cells by promoting T-regulatory cells.

The cellular content of autogenous bone grafts varies based on the indi-vidual patient’s medical profile, the harvest technique (aspiration or openharvest), anatomic location of the harvest (intra-oral or extra-oral), type ofbone harvested (cortical or cancellous), age and gender. The cellular


content has also been shown to have an effect on the bone graft perform-ance.45 Therefore, the identification of MSCs and osteoprogenitor cellsand determination of their concentrations in different anatomical tissueshas been an area of recent investigation.46–48 Evaluation of bone marrowaspirates from the anterior iliac crest revealed a fairly small count ofMSCs, although a higher percentage of cells that tested positive forCD105 was found in the iliac crest aspirates when compared to peripheralblood.48 McLain and colleagues compared the osteoprogenitor cell con-centrations between iliac crest and vertebral body aspirates.46 Theirfindings show that vertebral aspirates (465 cells/cc marrow) have aslightly higher mean concentration than the iliac crest aspirates(356 cells/cc marrow). The process used to prepare the cellular allograftbone matrix described in this chapter involves the selective removal ofimmunogenic cells in hematopoietic lineage from cell-rich cancellousbone, while retaining the osteopotent cells in the mesenchymal lineage.The minimum number of MSCs and osteoprogenitor cells found in thecommercially available Osteocel® product is 50,000 cells/cc and forOsteocel® Plus product is 250,000 cells/cc. Clearly these cell counts are adramatic improvement over the cell counts for aspiration harvests andmay result in an enhanced clinical result. Recently Cuomo and colleagueshave conducted a preclinical trial evaluating MSCs from human bonemarrow aspirates in combination with demineralized bone matrix inathymic rat femur critical size defects.49 Unprocessed MSC concentra-tions were found to vary from 64 to 2933 cells/ml with an average of1010 +/− 960 cells/ml as determined from fibroblast colony forming unit(CFU-F) culture assays. MSC enriched bone marrow aspirate (centrifuga-tion concentration) improved the yield to an average of 6150 cells/ml.Interestingly, the bone forming capabilities were found to be only compa-rable to the demineralized carrier alone. The authors mention the cellnumber may be insufficient to stimulate a robust bone formation. Theclinical ramification of cell number has also been discussed by Hernigouand colleagues when treating tibia non-unions with marrow aspirates.They have identified 30,000 cells/ml as the minimum number of progen-itor cells necessary to induce healing in this indication.50 Anotherpossibility is that since the MSCs have not had a chance to adhere to thecarrier some may wash out of the wound further reducing the MSCs


influence on the healing. All of these considerations could have clinicalimplications when performing oral reconstructive surgery with bonemarrow aspirates as has been presented by some clinicians.48

This exciting new cellular allograft technology will further assist us inthe management of challenging oral regenerative procedures. Ongoingresearch is aimed at optimization of the clinical techniques and determin-ing the long-term success of their application.

Acknowledgments

The Osteocel® sinus augmentation research project was supported byAce Surgical, Brockton, MA. The authors gratefully acknowledge theassistance of Hari Prasad, Senior Research Scientist, Hard Tissue ResearchLaboratory, University of Minnesota School of Dentistry, and Dr. MichaelRohrer, Professor, Director of the Hard Tissue Research Laboratory andOral Pathology Laboratories, University of Minnesota, School ofDentistry, for the preparation of the specimens and the histological data.

The authors would also like to thank Dr. Tim Moseley, Chief ScientistNuVasive Inc.TM, for the technical assistance in preparing this chapter.

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15

Therapeutic Strategies for Repairingthe Injured Spinal Cord Using Stem Cells

Michael S. Beattie and Jacqueline C. Bresnahan

1. Introduction

Spinal cord injury (SCI) is a devastating condition that affects 10,000 to12,000 new individuals per year in the United States. In a recent surveyby the Reeve Foundation, chronic cases were estimated to be 1,275,000.The current standard of care includes the optional use of high dosemethylprednisolone,1 optional surgical stabilization and decompression,2

and rehabilitation.3 Recovery after SCI occurs, but is limited. Unlike themore progressive degenerative diseases, SCI affects mainly younger,healthy individuals, although this is changing, with increasingly moreelderly people being injured, especially from falls.4 Since SCI involvesboth acute and progressive secondary injury to the CNS that results inneuronal and glial cell loss, cellular replacement therapies have long beensought for this condition.5 With the advent of modern stem cell biology, itis obvious to ask whether stem cell technology can be brought to bear onthis problem, and many laboratories are pursuing this goal in experimen-tal models of SCI. However, it is not so obvious what the targets of such

297

therapies might be. While axonal regeneration has been the holy grail ofSCI research, strategies aimed at reducing secondary injury and inflam-mation and replacing myelinating oligodendrocytes are also clear targetsfor repair. Since there have been a number of recent attempts to use cell-based therapies in human SCI,3,6–9 it is important to have as clear a notionas possible as to the types of cells, and types of targets that might be usefuland, “to do no harm.” Here, we will briefly review the biological featuresof SCI that might be amenable to stem cell therapy, and consider some ofthe approaches to therapy based on current knowledge of stem cell biol-ogy. Rather than a comprehensive review,5,10,11 we will attempt to identifysome of the highlights of recent work in this field, and the problems andpromise that they present.

The use of the terms stem and progenitor cells in this article needsome definition, as they are often differently applied. Stem cell is meantto include cells from embryonic, fetal, or adult organisms, includinghumans, that are pluripotent and self-renewing. Progenitor cells repre-sent cells that have more limited potential, usually limited to cells of asingle germ layer. In the CNS, these are usually referred to as neuralprogenitors. Neural progenitors (or multipotential precursors) candivide and differentiate into lineage specific progenitors, includingneuronal and glial progenitor cells. Of course, CNS repair alsoinvolves cells originating from all germinal layers (e.g. endothelialcells, immune cells like microglia, etc.). The use of human embryonicstem cells per se has actually been somewhat limited in this field, butthere is much to be learned from the use of other cell-based therapeuticstrategies that have used fetal or adult stem and progenitor cells fromboth humans and animals. There may be diverse sources for stem cellsthat eventually will be applied to clinical use. Very recent advances inre-programming human somatic cells to produce the so-calledinducible pluripotent stem cells (hIPSCs)12 point to the rapid changes instem cell technology that are likely to affect clinical uses. Thus, we willconsider data from multiple experimental paradigms using a variety ofstem and progenitor cell types, since it is possible that the features ofeach of these phenotypes could be generated from multiple origins. Theideal cell, perhaps, would be an autologous, pluripotent stem cell

298 M. S. Beattie and J. C. Bresnahan

population that could be conditioned to form any of the many parts ofthe mature CNS without forming tumors.

2. Secondary Injury and Endogenous Repair After SCI

Experimental studies of spinal cord injury (SCI) have made significantadvances in recent years with the development of multiple approaches tomodeling the human condition, and real advances in the understanding ofsecondary injury and repair. It is now well established that the initial injuryis followed by a cascade of events that exacerbate the primary lesion viaexcitotoxicity, components of acute inflammation, and longer termprocesses that can result in apoptosis of myelinating oligodendrocytes thatmay contribute to demyelination and loss of function.13 It is also clear thatthe sequelae of SCI and other CNS injuries include regenerative and repar-ative processes that tend to restore or protect the remaining CNS, includingthe proliferation of endogenous progenitor cells, and the walling off of theinjured zone by astroglial hypertrophy.14 It is somewhat difficult to catego-rize many of these post-injury events as either positive or negative withrespect to the final functional outcome. Thus, while the astrocytic responseto injury may be protective, as is scar formation in peripheral tissues, theglial scar presents a serious impediment to axonal regeneration.15 Similarly,acute inflammation is thought to be destructive to oligodendrocytes,13 andyet aspects of the cytokine response appear to promote oligodendrocyteprecursor proliferation and differentiation. There appear to be many simi-larities between the cellular events leading to demyelination andconduction failure in multiple sclerosis (MS) and in SCI, and in both cases,a reparative response of the resident precursor population is mounted, butis insufficient to provide complete remyelination and recovery.16

Microglial activation is a prominent feature of the early injury response inrats and humans17 and, like the more general concept of inflammation,microglial activation has been associated with both cellular degenerationand repair.18 These mixed degenerative and regenerative events provide thebackground for targeting repair using exogenous stem or progenitor celltransplantation, that is, therapeutic targets for cellular transplantation canbe defined by the biology of injury and repair (Fig. 1).

Stem Cells and Spinal Cord Repair 299

3. Therapeutic Targets for Transplanted Stem andProgenitor Cells

Targets include protection of cells that survive the primary insult (bothneurons and glia), replacement of lost cells (neurons for re-establishingcircuitry and glia for remyelinating spared axons). Exogenous glial cellscan also provide a substrate for axonal growth and regeneration as well asproviding growth and trophic factors that reduce long term secondary cellloss and enhance axonal regeneration and sprouting, and have been shownin some cases to reduce the endogenous glial scar.


Fig. 1. Spinal cord trauma induces a cascade of responses in the injured nervous system,some of which are illustrated here. The initial insult causes frank damage to cells andblood vessels. Subsequent secondary events lead to further damage (so-called secondaryinjury) including excitotoxic necrotic cell death, inflammation and activation of microgliaand macrophages, and the initiation of active cell death (apoptosis). Especially importantin loss of function is the loss of oligodendrocytes inducing demyelination of spared axons,and changes in membrane properties of axons at the injury site. Not illustrated is theabortive endogenous repair process, including proliferation of progenitor cells, regenera-tion of axons, collateral sprouting in remaining axonal systems and reorganization ofuninjured but affected neural systems. Transplanted stem and progenitor cells can alter thebiology of the lesion via reduction of excitotoxic cell death (neuroprotection), reducing theinflammatory response (e.g. Ref. 54), and protecting oligodendrocytes.

4. Animal Models of Spinal Cord Injury

The usefulness of animal models of spinal cord injury for preclinicaltesting of repair strategies depends upon their reliability and validity.19,20

In recent years, most studies of experimental SCI have used rodents,mostly rats, but increasingly mice to take advantage of current technologyfor genetic manipulation. More recently, a return to larger animal modelsin SCI has been discussed and initiated.21 Rodent models are convenientand relatively inexpensive, and, many would argue, that the basic biologyof the lesion and the repair processes are similar in humans. However,there are many different ways to injure the spinal cord, and these may notalways be similar to the injuries incurred in clinical settings. Thus, manylaboratories use partial transection or complete transection injuries, eventhough most human injuries are more like the contusion and compressionmodels that have been characterized extensively in the past twodecades.22–24 The manner of injury will affect not only the normal patternsof recovery but also the strategies and utility of transplanting stem andprogenitor cells (see Fig. 2). Complete transections of the cord have beenused to test strategies aimed at true axonal regeneration, i.e. the regrowthof severed axons across a gap in the CNS (Fig. 2A). In this case, replace-ment cells must survive in the hostile lesion environment, integrate withthe host tissue at the cut ends of the cord, and provide a tissue bridge andsubstrate sufficient for axons to grow across and reconnect with the cau-dal spinal circuitry. In addition, the glial scar, consisting of proteoglycansand hypertrophied astrocytes, along with multiple chemical signals repul-sive to axonal elongation,15 extends across the entire access route to thegap. Despite this, axonal growth bridging a complete transection has beenaccomplished in a number of laboratories using either peripheral nervegrafts25 or using immature and proliferating Schwann cells in matrigelbridges,26 as well as grafts of fetal tissue containing stem and progenitorcells.5 And, there are reports of success with transplanted progenitor cellsincluding olfactory ensheathing glial cells and bone marrow stromalcells.27 However, these dramatic anatomical reconnections have resultedin only modest if any recovery of function. In addition, there are practicalissues with these experiments: the animals (usually rats) are severely dis-abled, lack bladder function for weeks, and require intensive nursing care.


Thus, these studies have been confined to a few laboratories. The fact thatonly a minority of human SCIs are in fact complete anatomical transec-tions has also contributed to the rationale for not pursuing these difficultstudies.

More laboratory studies have used partial transections, and as can beseen in Fig. 2, the lesion presents a less stringent situation for repair;


Fig. 2. Different experimental approaches to study spinal cord injury have differentfeatures, and test different targets for stem and progenitor cell therapies. SCI lesion modelscan affect transplantation results and interpretation. Targets for complete transectionstudies have included “true regeneration” cellular replacement, and scar reduction. Theyare not as well suited to studies of neuroprotection and remyelination. Targets for hemi-section studies include regrowth around a lesion, sprouting of spared axons and scarformation. The interpretation of results in these studies is complicated by the presence ofspared contralateral tissue. The contusion lesions may be more representative of mosthuman SCI, and present opportunities for targeting therapies aimed at reducing cell death,reducing scar formation, and are especially useful for studies of remyelination. The sparedrim of tissue provides another challenge in interpreting the results of efficacy of trans-plantation strategies.

damaged axons can grow around the lesion, the glial scar is limited toone side leaving “normal” tissue as a substrate for growth, spared axonscan sprout below the lesion, and the transplanted cells can providetrophic or growth factors to the adjacent tissue. Work from the laborato-ries of Mark Tuszynski at UC San Diego and the Drexel research groupin Philadelphia have attempted repair of partial cervical cord transectionsusing, fibroblasts engineered to produce neurotrophic factors,28–30 or bonemarrow stromal cells31 and report both sparing of intact axons and thestimulation of growth around, and sometimes into, the transplantedlesion site. These studies provide for more “high throughput” results thancomplete cord transections or large contusion lesions, but there is alwaysa question of whether functional effects are due to true regeneration,sprouting, or neuroprotection, and whether the results will translate to thecontusion lesions most often seen in human SCI.

The contusion lesion (Fig. 2C) presents additional issues; it is moreextensive than the cut lesions, affecting a larger area and potentiallyrequiring significantly larger numbers of cells for transplants, but it nearlyalways has a rim of spared tissue (Fig. 3) that contains surviving axonswhich subserve spared function and which might be susceptible todamage from the transplantation process.32 This rim also could provide aphysical substrate for regrowth and regeneration of cut axons. The sparedrim can contain demyelinated axons, which may be dysfunctional due toconduction loss. These may be a target for therapies that is not availablein the partial transection models.33 The glial scar in this case is also largerthan in the transection models. The center of the lesion is frequently occu-pied by cystic cavities which contain rafts of macrophages especiallyearly after the injury, but which also provide ideal receptacles for trans-planted tissue. Recently, much more attention has been given to attemptsto use cell-based therapies in this kind of model, with a variety of celltypes and successes (see below).

In all of the models used, important questions involving the survival ofthe transplanted cells, the identification of host versus donor cells, thelesion microenvironment, and the role of inflammation and immune rejec-tion need to be asked and answered. the answers may vary substantiallydepending upon the type of lesion used.



Fig. 3. Remyelination as a target for stem cell therapies. (A) Cross-section of the spinalcord at the lesion center three weeks after a 25 mm MASCIS injury in the rat. A spared rimof fibers can be observed to have survived the injury (arrow). This rim is separated from aforming cystic cavity by astrocytic processes that wall off the remaining tissue (doublearrows). The central region of the injury site is occupied primarily by large numbers ofphagocytic macrophages which have taken up the degenerating debris, and ingrowing cordsof Schwann and mesenchymal cells as well as regenerating axonal sprouts from the adja-cent dorsal roots (DR; see Ref. 32). (B–D) The spared rim of tissue (C, ventral funiculus)is also present after a contusion injury in the primate (500 gm-cm injury; ten-week survival).The central cavitated region contains macrophages (macs) and is walled off by astrocyticprocesses (double arrows in B). The remaining peripheral rim of white matter containsmany smaller myelinated axons which have survived the injury interspersed with degener-ating ones that have not yet been phagocytosed, and microcysts left by axonal and glial celldeath. In the more central regions (D) closer to the cavity, there are few surviving myeli-nated axons and many demyelinated ones (large arrows) interspersed among astrocyticprocesses, macrophages (mac), and microcysts (MC). (Toluidine blue stained 1 μm thicksections; dorsal is up in all sections; A — 4X, B and C — 20X, and D — 63X.)

5. Types of Stem and Progenitor Cells Usedfor Transplantation in SCI

As noted, there is a long history of transplantation of tissue into the spinalcord using peripheral nerve tissue, fetal grafts, and other approaches,34

and Schwann cells have been used for many years to “bridge the gap” instudies of complete transections of the cord.26 Recent work suggests thatthese cells may be useful in combination with other treatments incomplete cord transections35 (here using olfactory ensheathing glialprogenitors), and in contusion injuries as well. Pearse et al.36 reported thatSchwann cells in combination with cAMP treatments meant to stimulateaxonal growth could impact both axonal growth through a contusionlesion, and have some effect on neurological recovery using several meas-ures. While Schwann cells are not stem or progenitor cells, they canundergo proliferation in response to injury, and so constitute a cell thatcan be expanded and used for transplantation. Schwann cells can be takenfrom peripheral nerve biopsies and expanded, providing for a source ofautologous cells for transplantation into chronic SCIs. Schwann cells alsoproduce neurotrophic factors, and share many of the features of progeni-tor and stem cell populations considered elsewhere in this article.Schwann cells can also be derived from stem or progenitor cells. Forexample, mouse skin has been used as a source for neural crest-derivedneurospheres and stem cells that can be treated in culture to produce stem-like cells, and these have been transplanted into spinal cordinjuries.37 These cells, termed “SKPs”, differentiated into a Schwann cell-like phenotype (compared to neurospheres or “naïve” SKP cells) andtransplanted after a moderate thoracic contusion injury in rats have beenshown to (1) reduce lesion size (neuroprotection), (2) reduce the deposi-tion of chondroitin-sulfate proteoglycans (CSPGs) around the injurycavity, (3) support the regrowth of axons from the spinal cord and brain,(4) increase the migration and proliferation of endogenous peripheralSchwann cells into the cord, and (5) have some, although modest, effecton neurological outcome. Such studies emphasize the complexity ofinterpreting cell-based therapies, but also suggest that the right cell typecan act on several of the proposed targets.


Both mouse and human embryonic stem cells have been transplanteddirectly, or used as progenitor cells for deriving more restricted popula-tions of cells for transplantation into mouse and rat spinal cordinjuries.38,39 Cells derived from embryonic or fetal spinal cord and brainhave been used as well, especially a population of cells isolated fromthe embryonic day 13–14 rat spinal cord that includes glial restricted pre-cursor cells and neuronal restricted precursor cells (GRPs and NRPs).10,40

Human and rodent bone marrow-derived stromal cells (BMSCs) havebeen used extensively in spinal cord injury models11 and have also beenused in initial human clinical trials.41,42 BMSCs can be delivered directlyinto the cord parenchyma,43,44 but have also been delivered by intravenousand intrathecal injections.45 There are a number of reports of neuroprotec-tion and even transdifferentiation into neurons,46 but the generation ofneural cells from BMSCs has been questioned.31 Timing of delivery maybe particularly important when using BMSCs, with reports that acutedelivery provides more neuroprotection than when cells are transplantedat one week or later.47 This is in contrast to reports of neural progenitors,which seem to flourish best when transplanted a week after injury. The useof BMSCs for transplantation in the cord is especially attractive becauseof the potential for using autologous cells, although most of the experi-mental work has used human or rodent allogeneic sources. This has beenaccomplished in a limited clinical trial with additional treatment withgranulocyte colony stimulating factor (gcsf),48 and although the results ofthis trial are preliminary, this approach is likely to receive additionalattention in the future. In addition, human BMSCs can be engineered toexpress growth factors49 or transcription factors like Olig250 that mayenhance their usefulness in promoting recovery.

6. Evidence for Effects on Regeneration and Sprouting

Examples of reports of axonal regeneration across a complete transectioninjury after transplantation, with associated functional changes, include astudy of transplanted olfactory ensheathing glial cell progenitors27 and arecent study in which olfactory ensheathing glia were paired with cAMPtreatments and Schwann cell transplantions.35 In partial transectionmodels, Davies et al.,51 transplanted astrocytes derived from embryonic


glial-restricted precursors (GRP-derived astrocytes, GDAs) acutely intothe hemisected rat cervical spinal cord and compared the results to“naïve” GRPs. GRPs survived and filled the lesion, but failed to providesupport for growing transplanted dorsal root ganglion cell axons. GDAs,on the other hand, supported extensive growth of DRG axons across thelesion through an aligned network of astrocytic processes. Some of therecent studies using BMSCs have reported enhanced sprouting of axons,including the cortico-spinal tract.49

7. Evidence for Effects on Neuroprotection

Transplantation of stem or progenitor cells may affect lesion size and cellsurvival by reducing excitotoxicity and inflammation,13 or by providingtrophic factors that reduce longer term apoptotic cell death.52,53 Trans-plantation of glial restricted progenitor cells in our lab reduced the apparentinflammatory response, and in addition, reduced the reactive glial scar sur-rounding a contusion lesion.54 Others have shown similar effects.55

Transplanted Schwann cells along with cAMP treatments reduced the inflam-matory response to contusion lesion and appeared to result in enhancedneurological outcome.36

8. Evidence for Replacement of Neurons

McDonald et al.38 reported that transplantation of mouse embryonic stemcells nine days after contusion injuries resulted in the formation ofhuman cell-derived astrocytes, oligodendrocytes, and neurons, with anapparent effect on locomotor outcome. Lepore and Fischer56 transplantedrat E14 fetal spinal cord, and mixed neuronal and glial progenitor cellsisolated from fetal cord into partial hemisection injuries. These cells weretaken from the hPLAP transgenic rat (as in Hill et al.54) so they couldmonitor cell survival and integration into the adult host. Many of the cellsdied within four days of transplantation, but those that survived prolifer-ated to fill the hemisection cavity by three weeks, and included manydonor-derived neurons. This group went on to transplant GRP/NRP com-binations into the contused rat spinal cord nine days after injury,57 andreported not only reduction of lesion volume (i.e. neuroprotection), but


also the presence of mature glial and neuronal cells, and improved recov-ery of motor and bladder functions. Cummings et al.58 isolated humanadult neuronal stem cells from neurospheres and transplanted them intocontusion lesions in immune-compromised (scid) mice, and foundmature neurons and glial cells, along with improved behavioral recovery.Yan et al.59 recently reported that human fetal spinal cord-derived stemcells produced both neurons and oligodendrocytes when transplantedinto the uninjured or lesioned lumbar cord of nude rats. Many of thesestudies have used other cell types (e.g. fibroblasts) as negative controls,and the evidence for neural replacement using stem cells is mounting.Human spinal stem cells, produced from eight-week human fetal spinalcord tissue, were also used in a model of spinal cord ischemic injury,60

where they were found to replace lost GABAergic interneurons selec-tively lost to the ischemic insult. Thus, there is now ample evidence thatneuronal replacement may be a useful strategy in treating spinal cordinjury. Still needed are more clear demonstrations that these replacedcells can integrate into the host circuitry and affect useful functionalrecovery.

9. Evidence for Oligodendrocyte Replacementand Remyelination

Mouse embryonic stem cells, “neuralized” by treatment with retinoic acid(RA) were reported to produce neurons, astrocytes, and oligodendrocytesafter implantation into the contused rat spinal cord nine days after injury.38

Further, these animals exhibited better neurological recovery on the“BBB” locomotor scale, showing plantar stepping with weight support asopposed to controls that scored about two to three points lower (no weightsupport or stepping). In a later study, this same group used similar cells,but with factors added to the culture medium that induce oligodendrocytelineage differentiation (including tri-iodothryronine, T3) in transplantsinto the demyelinated dorsal columns (rats) or into the cord of shiverermice lacking normal myelin.61 These cells appeared to produce remyeli-nation. Several reports cited above of mixed or undifferentiated stem andprogenitor cells that reported neuronal generation and integration alsoreported oligodendrocytes from donor cells.57,58


Kierstead et al.39 report on extensive experiments in which oligoden-drocyte progenitor-like cells (OPCs) derived from human embryonicstems cells were transplanted into rat spinal cords after contusion injuries,produced myelinating cells, and appeared to enhance neurological func-tion (i.e. a higher BBB locomotor score — two points), when transplantedat seven days, but not ten weeks, after contusion injury. These cells werederived from the H7 hESC lines at passage 32 via an extended (42-day)protocol in which neurospheres were isolated and exposed to thyroid hor-mone, FGF and other growth hormones. Purity, in terms of the presenceof oligodendrocyte progenitor markers was reported to be above 80%.Cao et al.62 used GRPs that were genetically modified to express D15A, amixed action neurotrophic molecule, to repair contusion injuries in ratcord, and found extensive remyelination by donor cells, accompanied byreturn of descending motor evoked potentials and improved behavioralrecovery.

In our own laboratory, Hill et al.54 showed that GRPs from hPLAPtransgenic rats could be acutely transplanted into large contusion injuriesof the rat cord, and appeared to form myelinating cells as well as providedanti-inflammatory and neuroprotective effects. However, neither thatstudy, nor a follow-up study transplanting GRPs at nine days after injury,produced substantial improvement in behavioral or physiological out-come measures. Thus, there is general agreement that GRPs and manyother progenitor and stem cell types can survive and remyelinate thespinal cord. Obtaining consistent functional recovery in animal modelsmay require optimization strategies, including the use of combinationtherapies. Nevertheless, remyelination has emerged as a major and nearterm target for cellular replacement therapy in spinal cord injury, and isbeing actively pursued as a clinical strategy.63

10. Keys to Future Progress

Stem and progenitor cells would seem to be a logical therapy for replace-ment of lost glial cells and neurons, and many laboratories have used avariety of cell types from CNS and elsewhere in attempts to providerepair. Indeed, tissue transplantation has a long history in spinal cordinjury, starting with Ramon y Cajal, and including the use of peripheral


nerve grafts, Schwann cells, and fetal tissue.26,34,64 It is only recently thatit has been realized that in many of these studies, the transplanted cells orfetal tissue underwent massive cell death early after transplantation, andthat the repair and replacement process was due to either replacement ofexogenous cells by endogenous Schwann cells65 or appeared to involvedie-back of the graft and repopulation of the host by proliferation of asmall number of surviving donor progenitor cells.34 This was simplybecause most studies did not examine the acute and sub-acute fate of thetransplanted cells, but rather, and understandably, heralded the presence offunctional grafts in more chronic stages. In addition, the use of geneti-cally-labeled donor cells has provided an easier method for trackingcellular transplants. The Hill et al.65 demonstration of variable Schwanncell survival with different transplant times and immunological suppres-sion, for example, used the transgenic hPLAP rat as a donor. There aremany examples of failure to thrive after transplantation of precursor orprogenitor cells, and it has been suggested that in many cases, the mostmultipotential cells are most vulnerable to early death, while morelineage-committed cells are more likely to survive.56 Thus the variablesuccess of different cellular transplantation strategies may be due to bio-logical features of both the host environment and the properties of thedonor cells. More information on the survival, proliferation, and differen-tiation of transplanted progenitor cells after grafting is needed.

The need for such information is driven not only by cellular trans-plantation studies in which there is a lack of enhancement of recovery,54,66

but even more so by remarkable hints at the prospects for dramatic suc-cess. Findings include evidence of enhanced function after transplantationof olfactory ensheathing cells, increased bladder and motor function aftertransplantation of mixtures of neuronal and glial restricted precursorcells,30 enhanced recovery with GRPs engineered to express neu-rotrophins,62 remyelination and recovery using oligodendrocyte progenitorcells derived from human embryonic stem cells (hESCs),39 remyelinationand some recovery with neural precursor cells derived from adult subven-tricular zone,67 glial-restricted precursor-derived astrocytes implanted intopartial cord transections,51 success with Schwann cell transplants if pairedwith cAMP elevations36 and many others. Many of these report that out ofseveral treatment strategies, only one was successful; i.e. these are not just


positive reports, but also contain data suggesting that many strategies areineffective. For example, the exciting report from the Fehlings lab67

reports that pilot studies showed that adult-derived neural precursorswere not successful unless transplantation was accompanied by the appli-cation of growth factors, immune suppressants, and anti-inflammatory(minocycline) treatment. Further, a careful counting of cells in some ofthe subjects suggested that less than 40% of the number of transplantedcells remained after eight weeks; proliferation rates were examined lateafter transplantation and found to be low, but were not examined early.Thus, the actual percentage of cells surviving is not clear. In addition,while 50% of the surviving cells were positive for oligodendrocyte pro-genitor cells or mature oligodendrocyte markers, and myelination wasseen, the remaining cells were astrocytes, or more often, unidentifiableby the markers used. And, enhancement of function was significant, butmodest (i.e. about two points on average on the BBB scale). Thus, thereis room for improvement even in this remarkable result. And, in contrast,the reports of a similar degree of success with OPCs derived fromhESCs39 required little if any “help” from combination strategies. In sum-mary, there is much to be learned about progenitor cell transplantation,and systematic studies of cellular interactions with the injury environ-ment should help to improve the ability to predict which strategies willbe most successful.

Further, there needs to be continued attention to improving preclinicalstudies and outcome measures, and an attention to possible adverse effectsof cellular transplantation. As noted, the thin rim of demyelinated axons incontusion lesions may be a target for remyelination and a substrate forregrowth, but it also may be damaged during transplantation procedures.Another consideration is the cells providing growth factors that enhancerepair mechanisms may also encourage the growth of sensory fibers and cir-cuits that can contribute to spasticity and pain,68 or autonomic dysfunction.69

11. Are Stem and Progenitor Cell Therapies Ready for Clinical Trials?

Given the questions above, it is pertinent to ask whether the field is readyto move into clinical practice. In truth, we are already there, with several


cell-based therapies being employed in SCI across the world.6 However,the issue involves identifying the pros and cons of moving cell-basedtherapies into controlled clinical trials in the US and Europe. TheProNeuron trial using activated macrophages,20,70 and the University ofFlorida experience with human fetal cell transplants34 at least show thatthese procedures can be accomplished without obvious harm. The tech-nical problems of using stem and progenitor cells have been at leastpartially solved, but questions surrounding immunogenicity, tumorogen-esis, and adverse effects such as chronic pain remain. Some would saythat the potential benefits already outweigh the risks, and there are anumber of planned trials including one focused on remyelination byoligodenderocyte progenitors derived from human embryonic stem cells(Geron, Inc.),71 which we have emphasized as a prime target in thisarticle. Risk benefit analysis should also include consideration of the cellsource and purity, and the route of delivery (e.g. injections versus opensurgical approaches72). The timing of therapy with respect to injury mayalso be important. There is much less positive data in chronic spinalinjury models than in acute or subacute (within one to two weeks). Eachcase therefore should be determined on its own merits with the excite-ment of new therapeutic opportunities balanced by the axiom of “do noharm.”

Acknowledgments

The Beattie and Bresnahan laboratory is supported by NIH Grants NS-31193 (MSB, JCB) and NS-038079 (JCB, MSB), The New York SpinalCord Injury Research Trust (UCSF subcontract to MSB and JCB fromContract #C019772, R. Ratan, P.I.), the Craig H. Neilsen Foundation andthe Roman Reed Fund of California.

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16

Potential of Tissue Engineeringand Neural Stem Cells in the

Understanding and Treatmentof Neurodegenerative Diseases

Caroline Auclair-Daigle and François Berthod

1. Introduction

Neurodegenerative diseases consist of an heterogeneous assembly ofpathological conditions in which specific regions of the central nervoussystem (CNS) relatively slowly and progressively deteriorate, resulting inmovement and/or cognitive impairments. The specific etiology and causesof those disorders mainly affect elderly people. On the economic level,these types of neurodegenerative disorders are generating economical costthat sum up, each year, to hundreds of billions of dollars in developedcountries. Given that the cause of most of these diseases remainsunknown, the in vitro reconstruction of tissue-engineered models thatmimic the diseases could be very helpful in understanding the subtle bio-logical alterations that occur in comparison to the controls. In addition,since neurons lack the ability to regenerate, stem cells therapy may offeran opportunity to replace cells lost through damage or degeneration.

321

2. Neurodegenerative Diseases and Their CurrentTreatments

2.1 Parkinson’s disease (PD)

The second most common neurodegenerative disease, inflicting debilitatingtroubles to close to 1% of the population aged 60 and over. The sufferingindividuals are mainly affected by motor symptoms such as rigidity (mus-cle stiffness), bradykinesia (severe uncontrollable movement disorder),and shivers while resting.1 PD is a progressive disease characterized by adegeneration of dopaminergic neurons in the substantia nigra, and a sub-sequent deficit of dopamine release in the striatum. The affectedindividuals are becoming severely disturbed and depressed, in addition tothe development of dementia over the span of the disorder (greater than70% in advanced PD patients).2 There is currently no cure and no effec-tive long-term treatment for PD, as the precise etiology of the neuronalloss is still unknown and the pathogenesis not fully understood.

The main treatment has been consisting, ever since the late 1960s, inthe pharmacological lessening of the striatal dopamine deficit by admin-istration of the dopamine precursor L-Dopa, which crosses theblood-brain barrier and enters neurons that convert it into dopamine.Alternative drugs were developed over the years, but failed to match theL-Dopa efficacy, which still remains the gold standard for the treatment ofPD. However, its chronic administration is associated with motor compli-cations reflecting fluctuations of the drug concentration in the plasma, andits efficacy lessens with time, with re-emerging of parkinsonian symp-toms.3,4 Neurosurgical procedures have also been used as alternativeapproaches, consisting of ablative procedures of specific regions of thebrain, deep brain stimulation using electrical currents,5 GDNF delivery inthe striatum (a neuroprotective factor) through osmotic pump, slow-release beads or gene therapy, and cell replacement of the diseasedneurons using fetal mesencephalic neurons.6,7

2.2 Alzheimer’s disease (AD)

A progressive neurodegenerative disorder of the cortical regions of thebrain, first affecting memory functions and then gradually affecting all

322 C. Auclair-Daigle and F. Berthod

cognitive functions with behavioral impairments, leading to the irre-versible loss of neurons and dementia. AD is the most common cause ofdementia worldwide, accounting for 50%–60% of all cases, some of itsrisk factors including older age, family history, lower education level, andfemale gender. In AD, some cholinergic neurons lose their ability to func-tion, reducing acetylcholine level. Current treatments include the use ofdrugs such as acetylcholinesterase inhibitors, in conjunction or not withNMDA-antagonists, but they have not been shown to delay institutional-ization or functional decline.8 Life expectancy following diagnosticusually range between three to 15 years, but may be more limited.

2.3 Huntington’s disease (HD)

An autosomal dominant hereditary neurodegeneration initially describedby psychiatrist G. Huntington in 1872. It consists of a disorder primarilyaffecting selective neuronal subtypes, and particularly GABAergicmedium spiny neurons, the main neuronal subtype in the striatum. Themutant gene encodes the huntingtin protein, and the disease is believed tobe due to a gain of toxic function of the mutant protein.9 The prevalenceof this condition varies in the range of two to ten cases every 100 000, andthe onset of the disease usually occurs in people aged between 30 to 50,but has also been observed in the elderly. At this point, there is a currentlack of effective treatment for HD. Since the abnormal huntingtin proteinand its cellular functions have been identified in 1993, drugs have beenelaborated to reduce the ailment’s magnitude. Strains of mice have beencreated with the identical gene responsible for HD in humans, leading tothe development of promising drugs, such as drugs that block the gluta-mine chains from clustering.10 The drug tetrabenazine proved to lowerdopamine release, limiting writhing movements.

2.4 Amyotrophic lateral sclerosis (ALS)

A progressive neurodegenerative disorder characterized by selective lossof lower spinal and brainstem motor neurons and upper motor neurons. Ofthe major physical consequences observed are paresis of skeletal andbulbar muscles, amyotrophies, fasciculations, spasticity and ultimately,

Stem Cells for Neurodegenerative Diseases 323

paralysis. There appears to have no identifiable underlying cause otherthan genetic basis in familial cases, and there is currently no effectivetreatment available. The disease normally progresses rapidly, and survivalrate rarely exceed three to five years after the onset. Death occurs as aresult of diaphragm weakness, or from pulmonary infections. The onlydrug treatment currently approved for ALS is riluzole, a glutamate-releaseinhibitor. The drug possibly will expand life from four to 18 months andpostpone the need for tracheostomy.11,12

2.5 Multiple sclerosis (MS )

An idiopathic primary demyelinating disease of the CNS, whose main con-dition generates destruction of normally developed myelin sheaths. It is achronic progressive disorder characterized by disseminated neurologicalsymptoms and, usually, several relapses during the course of its debut stage.MS was initially described by J. Charcot in 1866, defining the disease as agrouping of intention tremor, spastic paraplegia, speech impairment, visualloss and nystagmus. It currently stands as the most studied demyelinatingdisease. The most common theory for its presupposed etiology is multifac-torial, stipulating that MS develops due to the combined presence ofexogenous factors in genetically predisposed people, autoimmune response,and demyelinating lacerations in the white matter of CNS.9 This episodicneurological disease normally strikes people aged 20 to 40, although 10%are recollected in people aged over 50. Current treatment for relapsing-remitting MS are not curative, but include interferon-β-1a or interferonβ-1b, and glatiramer acetate (namely copolymer 1), a combination ofrandom polymers that mimic the amino acid composition of myelin basicprotein, which could both be postponing the onset of major disability. Otherpharmaceutical agents such as α-4 integrin antagonists, intravenousimmunoglobulin infusions and corticosteroids may limit the relapse rate.13

3. Tissue Engineering as a Tool to Better UnderstandNeurodegenerative Diseases

As previously discussed, the causes of most neurodegenerative diseasesdo remain unknown. The usual method to analyze such disorders is to


perform histological and immunohistochemical analysis on post-mortembrain biopsies obtained from the patients, compared to normal tissues.These observations revealed the formation of β-amyloid plaques in AD,Lewy bodies in PD or superoxide dismutase-1 (SOD-1) aggregates inALS, but they did not specifically point out the role of these structures asbeing either a cause or a consequence of the disease. Genetic studies canalso be performed with these post-mortem tissues to identify a potentialmutation as the cause of the disease, such as in HD.

In ALS, 20% of the patients have at least another family memberaffected by the disease, even though only approximately 10% of theseindividuals possess a SOD-1 mutation, the most well-characterized ALSmutation so far.14 As for the sporadic cases of ALS, a combination of envi-ronmental factors with potential genetic susceptibility, in addition to othercauses such as microtraumas, are currently being suspected.

The use of animal models that mimic the diseases can be of great help,but a minimal understanding of the etiology of the diseases need to bereached before those models can be generated. PD can be induced in ratsfollowing destruction of their substantia nigra, and ALS transgenic mousemodel can be generated by overexpression of the human mutated SOD-1gene. However, studying the effects of the disease on an entire animalremains complex and does not always bring a clear answer to the ques-tions raised. In ALS for example, it is still not well understood how theSOD-1 mutation induces motor neuron degeneration, even though manyresearch groups worldwide have been working on the transgenic mousemodel for more than a decade now.

Thus, in vitro culture systems could be very helpful to performdynamic studies at the cellular level. However, culturing neurons facesmajor limitations, because neurons are nearly impossible to extract fromthe adult brain or spinal cord, and these cells do not proliferate in vitro.This is why neurons are usually extracted from mouse or rat embryos atan early stage of development (E12–E14), before they have had the timeto establish too many connections. In addition, since neurons do not pro-liferate, cell extraction from embryos needs to be performed again foreach experiment.

Finally, a broad limitation for these cultures, in common with most ofthe other cell types, consists of the vast difference in environment that


occurs between a cell cultured on a plastic dish in two-dimensions, com-pared to the three-dimensional environment in situ. Both the cell-cell andcell-matrix contacts are totally different, as well as the global cell behav-ior, especially for the nervous system in which multiple connectionsbetween neurons are constantly established.

3.1 Two-dimensional in vitro models of neural cell culture

In vitro cultures of various types of neurons (hippocampal, motor, sympa-thetic, etc.) have been performed for several years now, by extracting cellsfrom the nervous system of fetuses. These culture systems allow for theisolation of purified neurons from their surrounding environment, whichgreatly facilitates studies such as mRNA or protein analysis. However, ithas been noted that neurons usually only survive over a short period oftime in the absence of glial cells.

Mixed culture of spinal cord cells can be maintained for an extendedperiod of time, and neurons can be microinjected with a plasmid to over-express a protein of interest (mutant or not) to study its impact on cellphysiology or survival.15

Double compartment cultures have been developed by using neuronscultured on a plastic dish, and glial cells attached to a glass slide and thenflipped over and put onto the neurons using spacers so that cells would beas close as possible from each other for studying the paracrine effectsoccurring without direct cell-cell contact.16

Some in vitro models have also been developed to mimic a traumaticinjury, through the stretching of cortical neurons cultured on elastic substratesand the subsequent analysis of their intracellular calcium concentration.17

3.2 Three-dimensional tissue-engineered models of thenervous system

Spinal cord or brain tissue slices can be maintained as organotypiccultures in vitro, preserving the three-dimensional organization of thenervous tissue with all of its cell-cell contacts. These models are veryuseful as they are highly physiological, allowing electrophysiologicalstudies to be performed only a few hours following the tissue harvesting.18


However, they do not provide any sort of control on the cell types presentin the tissue of interest, and the specific cells’ proportion and condition.

What could be really helpful to study neurodegenerative diseaseswould be to reconstruct the nervous system by independently combiningeach cell type. This will be particularly useful with the multiple transgenicmouse models developed to mimic various disorders, such as the overex-pression of the human mutant SOD-1 gene in the G93A ALS mousemodel. It would be even more interesting if we could be using the patient’sown stem cells differentiated into specific types of neurons and glial cells,providing that these cells would keep their diseased phenotypes over thefull course of differentiation.

Some three-dimensional neural constructs were developed by cocul-turing neurons with astrocytes in a 500–800 μm thick 3D Matrigel™.Cells within these constructs displayed extensive 3D process outgrowthsand a high viability over multiple weeks. In addition, neurons in thismodel can be tested through patch-clamp techniques, and were shown tobe able to display electrophysiological action potentials and functionalsynapse formation.19

However, a drawback with the use of Matrigel™ is that it consists of amaterial containing multiple active molecules, such as laminin, collagen IV,entactin, heparan sulphate and cytokines, in undetermined concentrations.

Other biological polymers characterized by better controlled composi-tions, such as collagen, fibrin, methylcellulose or agarose, have also beenused as culture scaffolds to reconstruct 3D neural tissues.19

We developed a tissue-engineered model of motor neuron culture tostudy the axonal migration and myelination processes in a three-dimensional environment. Since most of the motor neuron’s cell surfaceis located outside of the spinal cord, coupled with an axon that can bemeasured up to one meter long, the process of axonal migration representsa major issue in the study of motor neurons. Moreover, this axon is alsobeing myelinated by Schwann cells, the main glial cell type of theperipheral nervous system.

To closely mimic the 3D environment surrounding motor neurons, thisperipheral nerve migration aspect should be taken into account. To reca-pitulate the tissues through which nerves make their way to the muscle,we developed a 3D connective tissue made of fibroblasts cultured in a


collagen sponge and maturated for two weeks in order to promote extra-cellular matrix deposition. Mouse motor neurons extracted from E12mouse embryos were purified through density gradient centrifugation,20

and then seeded on top of the reconstructed connective tissue (Fig. 1). Themotor neurons formed a thick and dense cell layer over it. To promoteaxonal migration, the sponge was lifted at the air-liquid interface, and a


Fig. 1. Preparation of a tissue-engineered model to study axonal migration and myelina-tion of motor neurons through a 3D connective tissue.58

cocktail of neurotrophic factors was added to the culture medium under-neath. As shown by immunohistochemistry, a large number ofneurofilament M-positive neurites was observed migrating down from theneurons layer to the bottom of the connective tissue for over up to 1 mmlong in distance. In addition, when mouse Schwann cells were co-culturedwith the fibroblasts in the sponge, they migrated alongside with the neu-rites, as shown by the Myelin Basic Protein double-staining withNeurofilament-M.21 An analysis of these neurites, performed by transmis-sion electron microscopy, showed that a thick myelin sheath was wrappedaround some of the neurites after 28 days of in vitro maturation (Fig. 2).Thus, this tissue-engineered model was shown to promote axonal migra-tion, axon myelination by Schwann cells, and spontaneous myelin sheathformation for the first time in vitro.21

This model should greatly facilitate studies on diseases related tomotor neuron axon demyelination, such as MS or Charcot-Marie-Toothdisease of the peripheral nerves.

In addition, it could be very valuable to study ALS with such a model,by using different combinations of spinal cord cells obtained from SOD-1mutant mice versus wild-type mice, all the while measuring motor neurondegeneration.

Finally, this model could be even more interesting if it could be recon-structed using the patient’s own cells, to mimic the human disease in vitro.

Since living motor neurons cannot be extracted from an adult spinalcord, i.e. from post-mortem tissues, other alternatives should be exploredin order to generate these cells from the patients. One of the best solutionappears to proceed with the differentiation of motor neurons from adultstem cells.

In addition, the differentiation of neurons from autologous stem cellscould be applied for cell replacement therapy applications, as a novelapproach to treat neurodegenerative disorders by implantation of new andfunctional neurons.

4. Neural Stem Cells to Treat Neurodegenerative Diseases

After molecular biology, gene therapy, nanotechnology and tissueengineering, stem cells are now considered as representing the new



Fig. 2. (A) Fibroblasts and Schwann cells were co-cultured for 21 days in the collagensponge, and were observed by histology staining with Masson’s trichrome. (B) Mousemotor neurons were seeded on top of the sponge and cultured for an additional 14 days(C) The neurons layer (stained with an antibody against Neurofilament-M) wascultured at the air-liquid interface, but without the addition of neurotrophic factors;neurites did not migrate through the sponge. (D) The culture medium underneath thesponge was supplemented with neurotrophic factors, which promoted neurite migration

emerging field that could revolutionize the future of medicine. This is par-ticularly true in neurosciences, field in which the work with humanneurons is still impracticable, and for which the use of neural cells differ-entiated from human stem cells may be the new standard in a near future.To fully understand the current known potential and limitations of stemcells, as well as their diversity and specific related issues, a rapidoverview of fetal neurons, as well as a comparative standpoint of embry-onic versus adult stem cells, will be presented.

4.1 Human fetal neural transplantations, a proof of conceptfor cell replacement

Even though cell transplantation procedures have already been in clinicalpractice for numerous organs, attempts at replacing cells within the CNSdo remain experimental. PD readily represents a candidate disease ofchoice for cell replacement therapy since the vast majority of its lostdopaminergic neurons come from a circumscribed area, the substantianigra compacta.22 This specificity made it the first disease to be consid-ered and treated by cellular therapy. The first attempt was made usingheterologous fetal neurons obtained from aborted fetuses, mainly becauseonly fetal neurons can survive tissue extraction. This initial series of trans-plantation studies provided us with insights suggesting that the graftedcells could, at least partially, survive, integrate the brain, and improve thedisease’s symptoms for an extended period of time.

4.1.1 Parkinson’s disease

It was during the late 1980s that the very first methodical clinical trans-plantation trials using fetal dopaminergic neurons in patients with PD wereelaborated.23–25 Mesencephalic tissue (an area in the developing CNS rich inimmature dopaminergic neurons) of six to nine weeks old aborted fetuses


Fig. 2. (Continued) through the connective tissue. (E) A transmission electron micro-scopic picture of the sponge showed that some neurites were wrapped with thick myelinsheaths, observed with a higher magnification in (F) Bar in B, 60 μm for A–B; Bar in D,100 μm for C–D; Bar in E, 2 μm; Bar in F, 0.2 μm. (Modified from Ref. 58.)


was transplanted into the striatum.7 Various immunosuppression policieswere adopted, ranging from none,26 to cyclosporin A for six months27 and toa cocktail mix of cyclosporin A, steroids, and azathioprine.28

Of the open-labeled trials, without the presence of a control placebogroup or blinding procedures, the initial results following fetal nigralgrafts in PD patients showed graft survival and clinical improvements inthese PD patients.23–25 From that timeframe, over 400 PD patients through-out the world have been grafted with nigral tissue,29 and improvementswere reported from none to long-lasting dramatic benefits.30

Of the double-blind placebo controlled trials, in which there was pres-ence of a sham group consisting of the PD patients being anesthetized,their skull perforated but without the actual cell transplantation stepoccurring, both the patients and observers remained blinded to thiscondition. In one study, 19/40 patients received a tissue strand cell trans-plantation, after which a subjective global rating scale test was conductedone year post-transplantation.26

Slight improvement was noted in the transplanted group in comparisonto the sham one, but 15% of the patients ended up with severe off-dyskinesias. In another study, solid pieces of embryonic ventralmesencephalon have been transplanted into the putamen of both sides of thebrain. Eleven patients were sham controls. After two years, no net improve-ment was noted, and 56% of the patients resulted with off-dyskinesias.27

No real positive effect using cell transplantation was detected in thosestudies. Possible reasons for this poor outcome may be that the clinicalassessments were not standardized accordingly, or the tissue preparation,long-term in vitro storage, immunosuppression use, and surgical approacheshad design flaws.26,30

Interestingly, analysis of post-mortem tissues more than ten years aftergraft of fetal neurons showed that, whereas some of these cells underwentpathological changes similar to Parkinson disease (formation of Lewybodies), the majority of them did not display evidence of functionalimpairment or degeneration.31,32

4.1.2 Huntington’s disease

Fetal neural transplants have also been used to treat patients suffering fromHD.7 A recent study about fetal neural transplants reports cases of three HD

patients autopsies, performed ten years post-transplantation.33 In two out ofthose three patients, there seems to have been a differentiation into ade-quate cell types and proper glutamatergic and dopaminergic innervatingprojections from the recipients’ brain. Yet, degeneration was observed inthe grafted cells. The grafts also showed astrogliosis, inflammatory infil-trates, and microglial activation. Given that only temporary and minorbenefits, along with considerable graft degeneration were observed, thiswould limit the use of fetal cells for eventual studies conducted for HD.33

Human fetal neural transplantations applied to hundreds of PD patientsover the last two decades have set new standards for cell transplantation intothe brain. However, the risks linked with human fetal transplantation trialsmay very well be higher than initially expected. In fact, there are obviouslogistic problems linked with the amount of donors needed for each patient,on top of all of the ethical concerns that have been raised concerning the useof cells obtained from aborted fetuses. Put together, these factors are widelyrestricting the application of fetal tissue for neural transplantation.Nevertheless, even if this cell replacement therapy could be provensuccessful in only a few cases, by promoting cell survival and integrationinto the neuronal circuitry and, ultimately, for long-term benefits for thedisease’s symptoms, it still shows that cell therapy applications could besuccessful, once a better control of the graft parameters will have beenestablished.

4.2 Embryonic stem cells (hESC )-derivedneural precursor cells

Fetal neural transplantations are limited, not only by the availability offetuses but also largely by ethical concerns. One alternative could be thedevelopment of pluripotent stem cell banks generated from embryos at anearlier developmental stage (supernumeraries not used during in vitrofertilization), which may be not as ethically controversial as fetuses, andwhich could generate large amounts of various cell types due to their highproliferative and differentiation potentials (Table 1).

The use of hESC-derived NPCs led to some promising results in thetreatment of PD and HD. However, concerns similar to those related tofetal neural transplants have been raised for those cells: the hESC-derivedNPCs are heterologous and thus could be targeted by the host immune


system, while also facing major limiting ethical concerns. Meanwhile,hESCs undoubtedly hold the highest proliferative potential and the largestand most efficient differentiation capacity in comparison to adult NPCs.

4.3 Adult tissue-derived neural precursor cells (NPCs)

The idea of growing cells in culture is particularly attractive if the cellsconsist of adult tissue-derived NPCs, able to differentiate into variousneural cell types. Studies are currently being done to develop methodsaiming at modifying adult stem cells into precursor cells, opening up thepossibility to harvest a patient’s own cells and rendering them suitable fortransplantation into the nervous system (Table 1).

4.3.1 Brain-derived NPCs

NPCs were initially defined as the self-renewing, multipotent cells thatgenerate the main phenotypes of the nervous system, of both neuronal andglial subpopulations. Ever since the first subpopulation of mitotic NPCswas identified in the adult mice brain tissue,34 NPCs have been isolatedfrom various species including human.35


Table 1. Comparison of Human ESCs vs. Adult NPCs.

ESCs Adult NPCs

Pros• Pluripotency (extended potential) • Multipotency• Multilineage differentiation • Limited risk of tumor formation• Extensive self-renewal • Autologous cells• Access to early neural development stage • Availability (within tissues)• Ease for inducing stable genetic changes • Limited ethical concerns

Cons• Major ethical consideration • Fate-limited potentiality• Risk of tumor formation • Uncommitted• Risk of immune rejection by the host • Limited proliferative capacity• Limited availability of embryonic tissue • Limited amount of readily available

cells• Variability in results reproducibility

The maintenance of NPCs is ensured by the NPC niche, in whichmicroenvironmental cues, but also interactions implicating the extracellu-lar matrix and the cellular membranes, cell–cell interactions and theproximity to blood vessels, aid in maintaining cell proliferation, fate spec-ification and differentiation.36

In the past 15 years, research groups have put a lot of efforts ontoNPCs derived from various regions of the developing brain in order tostimulate the promotion of the functional recuperation process bydopaminergic differentiation in PD rat animal models.37 Even if charac-teristics such as graft survival, neuronal and astrocytic differentiation, cellmigration, and axonal extension have been demonstrated, these types ofstudies had not yet shown evidence that a significant dopaminergic dif-ferentiation had set place.38 However, given the actual location of theseNPCs within the brain, some limitations still prevent their use for appli-cations in cellular therapy.

4.3.2 Bone marrow-derived NPCs

Other cell types generated from non-brain derived tissues, such as bonemarrow, have been approached, having been shown to express, forexample, certain dopaminergic markers. Mesenchymal stem cells (MSC)obtained from adult rodent have been shown to differentiate, both in vitroand in vivo, into cells with mesenchymal, visceral mesoderm, neuroect-oderm, and endoderm characteristics.39 A subtype of these MSC wasisolated and termed multipotent adult progenitor cells (MAPC).Surprisingly, it has been reported that as many as 30% of mouse MAPCsdifferentiated in vitro into tyrosine hydroxylase (TH, the rate-limitingenzyme in the production of dopamine)-expressing neurons. Those cellswere presupposedly pluripotent, opening the possibility for MSC to be acell source of choice for neurotransplantation in PD. Human bone marrowstromal cells were also shown to be able to convert into a NPC-likepopulation that allowed for the expression of neuronal markers oncedifferentiated, of which about 11% of the cells were TH-positive and wereshown to release dopamine upon membrane depolarization.40 However,the functionality of those cells remains to be shown, and so additionalstudies in PD animal models are still necessary.


4.3.3 Skin-derived and adipose-derived NPCs

It has previously been shown that differentiation of NPCs isolated fromhuman adult skin could generate neural (and mesodermal) derivatives,while potentially providing a readily accessible source of human adultNPCs for transplantation: the skin-derived progenitor or precursor cells(SKP).41–43 These cells possess distinct surface markers in common withMSCs, while preferentially differentiating into neural cell types.It appears that these SKPs can be passaged for up to one year withoutshowing any significant senescence and seem to behave similarly to NPCsextracted from the brain in the way that they have the ability to form float-ing spherical colonies called neurospheres (Fig. 3).44 The achievability ofexpansion and possibility for long-term culturing of SKPs, coupled withtheir versatility, give them a certain appeal for use in therapy for disordersof the nervous system.45–47 SKPs have also been shown to generateSchwann cells. The differentiated Schwann cells were transplanted intorat spinal cords following an induced traumatic injury, and appeared tosurvive within the injured environment and to myelinate host axons, allthe while improving locomotor recovery.48

Adipose tissue has been identified in 2001 as an alternative source ofmultipotent stromal MSCs, which can be obtained by a less invasivemethod and in larger quantities compared with skin or bone marrow NPCs(through liposuction).49 These cells can also be a source of NPCs. Theability of Adipose-Derived Adult Stromal (ADAS) to neuroprotect orrestore function in an injured dopaminergic pathway was investigatedafter transplantation of naive or neurally-induced ADAS into the striatumof parkinsonian rats. ADAS failed to generate stable dopaminergic neu-rons in situ, but gene expression analyses showed that both naive anddifferentiated ADAS cells express neuroprotective and trophic factors atthe lesion site.50

4.3.4 Induced pluripotent stem (iPS) cells derived-NPCs

In recent years, several approaches have been developed in order to repro-gram differentiated adult cells into pluripotent stem cells, named inducedpluripotent stem (iPS) cells. Somatic cells have first been reprogrammed



Fig. 3. (A) Neurospheres-forming human skin-derived NPCs (phase contrast microscopy).(B) Human skin-derived NPCs differentiated in Neurofilament-M-expressing neurons(immunohistochemistry). (C) Human skin-derived NPCs differentiated in TH-expressingneurons. Nuclei were stained with Hoechst. Bar in A, 200 μ m; Bar in C, 25 μ m for B–C.

into iPS by viral transduction of four transcription factors, c-Myc, Oct4,Sox-2 and Klf451,52 and then by the expression of only Oct-4 and eitherKlf-4 or c-Myc.53 More recently, it has been shown that the generation ofadult iPS cells was achievable by no means of genetic modifications.54

A study has shown that iPS cells dedifferentiated from skin fibroblastscould undergo a differentiation into NPCs, then into dopaminergic neu-rons. After transplantation into the brains of parkinsonian rats, the graftedcells showed neuronal extensions throughout the neighboring brainregions, all the while lessening the PD-like symptoms.55

Autologous transplants using a patient’s own cells are now at handsreach for the potential development of human adult NPC-based therapiesfor neurodegenerative diseases. It would now be possible to generate pureand highly proliferative colonies of iPS cells that, in theory, would havean equivalent potential as embryonic NPCs, additionally to being autolo-gous, non-ethically restricted cells. But these avenues also come with amajor setback: they also hoist the likelihood to induce tumor formation.Taking that iPS cells may be proven to be just as or even more perform-ing than human adult NPCs, and all along being easier to extract, cultureand control, they may very well become a cell type of choice for thetreatment of neurodegenerative disorders.

4.4 Advantages and limitations of NPC culture

The main advantage with the use of human tissue-derived NPCs consistsof the possibility to isolate cells from an autologous tissue, whether it beskin, fat or any other somatic tissues for iPS cells. Patients can give theirinformed consent, hence circumventing the ethical concern. Harvestingskin, fat tissue or even bone marrow can be done quickly, through a min-imally painful procedure with local anesthesia. The next steps are morechallenging. First, NPC purification remains a major issue since no cellsurface antigen allowing for immunoprecipitation enrichment has beenidentified so far, but some interesting purification steps can readily beachieved through floating neurospheres selection (Fig. 3).47

Another limiting step that still remains is the NPC proliferation. Sincethe amount of autologous tissue that can be harvested is limited, the pro-liferation step is crucial to generate enough NPCs for transplantation


purposes. The steps for deriving and expanding human adult NPCs aredifficult to standardize, extremely complex and expensive to perform, andhave not been really efficient.

Also, once NPCs have been grown, they need to be differentiated intothe adequate subtype of neurons, depending on the neurodegenerative dis-order that needs to be addressed. In that regard, the differentiation potentialof human adult NPCs seems to still be more restricted than that of hESCs.56

Overall, human adult NPCs appear to be more complicated to cultureand consequently it is more challenging to control their fate while tryingto generate specific types of neurons.57 One of the next major challengeswould consist of generating enough human adult NPCs to use in thera-peutic replacement of damaged tissues in the nervous system. However,issues such as cell survival, appropriate synaptic integration into the hostbrain, behavioral recovery, and tumor formation need to be addressed.Nevertheless, these limitations may be compensated by the autologousnature of these cells, and the absence of ethical concern.

5. Conclusion

Human adult NPCs now represent a true possibility and a open door toseveral other studies to come on not only cell transplantation as a thera-peutic approach for various disorders of the nervous system, but also onthe creation by tissue engineering of 3D models to study the diseases.21

The combination of these models with the use of human adult neuronaland glial cells generated from the differentiation of NPCs isolated directlyfrom the patients and from readily accessible tissues sources (skin, fat,bone marrow) or iPS cells, will enable the development of powerful mod-els to better understand human neurodegenerative diseases. Lastly, withthe use of autologous human adult NPCs, therapeutic transplants maybecome one of the next best long-term hopes for reversing neurodegener-ative diseases.

Acknowledgments

Our work is supported by the Muscular Dystrophy Association(www.mda.org) and the Canadian Institutes of Health Research.


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17

High-Throughput Systems for StemCell Engineering

David A. Brafman, Karl Willert and Shu Chien

1. Introduction

Stem cells have the unique ability to grow indefinitely (proliferation) oradopt new cellular fates (differentiation). As such, stem cells represent aunique cell-based system to study and model human development and dis-eases, to screen safety and mode of action of novel drugs and to providethe raw material for cell-based therapies of presently incurable diseases.Human pluripotent stem cells (hPSCs), which are able to differentiate intoall cell types in the human body, have the potential to revolutionize thetreatment of many human disorders for which no effective therapiespresently exist. Experimental manipulation of these cells to affect prolifer-ation and differentiation is central to developing strategies for theproduction of defined and mature cell types that can be used to studydevelopment and disease progression, to perform drug screens, and to treata variety of degenerative disorders such as Alzheimer’s and heart disease.

Conventional cell culture methods are limited in their ability to screenthe vast number of factors that can influence stem cell behavior. The

347

establishment of high-throughput screening (HTS) technologies with stemcells is important for a broad range of applications from basic under-standing of the role of certain signaling networks in self-renewal to thedevelopment of novel therapeutic approaches, such as cell replacement ofdamaged, diseased or dead tissues. HTS technologies generally consist ofthree interrelated components: (1) platform fabrication, (2) data acquisi-tion and (3) data analysis and mining (Fig. 1). The behavior of stem cellscan be controlled either by altering their extracellular environment(extrinsic manipulation) or by interfering with intracellular signalingpathways and transcriptional networks (intrinsic manipulation). Thischapter discusses the emerging trend of using HTS technologies for theextrinsic and intrinsic manipulation and engineering of stem cells.

2. Sources of Stem Cells Suitable for High-ThroughputScreening Approaches

A stem cell is defined by its ability to (1) maintain its undifferentiated, or“blank slate,” state and (2) differentiate into mature and specialized celltypes. Broadly speaking, there are two types of stem cell populations,which vary significantly in their properties. Pluripotent stem cells can inprinciple be maintained indefinitely in an undifferentiated and highly pro-liferative state while retaining their pluripotency (i.e. the ability todifferentiate into all mature cell types). In contrast, adult stem cells arerestricted in their potency (multi- or uni-potent), giving rise to only a

348 D. A. Brafman, K. Willert and S. Chien

Platform Fabrication Data Acquisition Data Analysis and Mining

Fig. 1. Schematic of the three interrelated components of typical HTS approaches:(1) platform fabrication, (2) data acquisition and (3) data analysis and mining.

subset of specialized cell types. In addition, adult stem cells are extremelyrare and hence difficult to isolate in pure form and expand in an undiffer-entiated state in the culture dish. Given these limitations, high-throughputscreens are often not feasible with adult stem cells, and pluripotent stemcell lines provide a powerful alternative.

Until recently, human pluripotent stem cells were mainly derived bydissection of the inner cell mass from the blastocysts. The discoveries byTakahashi and Yamanaka ushered in a new era for human pluripotent stemcells: by introducing a combination of transcription factors into matureand specialized cell types, it is now possible to derive pluripotent stemcells, or induced pluripotent stem cells (iPSCs), that are nearly identicalto embryonic stem cells (ESCs). This technology permits the derivation ofpatient-specific stem cell lines. Together, embryonic and induced pluripo-tent stem cell lines provide a virtually limitless supply of human cells forlarge-scale high-throughput screens of any kind.

3. The Stem Cell Niche: A Cellular MicroenvironmentThat Controls Stem Cell Behavior

The in vivo cellular microenvironment is a complex mixture consisting offour distinct “components”: (1) Immobilized protein factors such as extra-cellular matrix proteins (ECMPs) interact with the cell through integrinbinding. (2) Soluble protein factors (such as growth factors, small mole-cules, and hormones) influence cell signaling pathways via theirappropriate extracellular receptors. (3) Mechanical forcers (such as stretchor shear stress) modulate intracellular signaling processes by activatingmechanoreceptors. (4) Neighboring cell types communicate with eachother by cadherin-mediated signaling (Fig. 2).1,2 Each of these four com-ponents include tens to thousands of “members” that influence numeroussignaling pathways, which perturb gene and protein expressions, andultimately affect cell fate and cell function.

Members of each of these components interact in complex manner toinfluence each other’s signaling ability, a phenomenon known as crosstalk.For example, endothelial cell attachment to fibronectin via α 5β1 integrinpotentiates α vβ3-mediated migration on vitronectin.3 Along similar lines,growth factors and ECMPs often have a reciprocal relationship — cell

High-Throughput Systems for Stem Cell Engineering 349

adhesion to ECMPs is required for activation of growth factor receptors,and growth factors are necessary to stimulate cell adhesion, migration,and the resulting integrin-dependent response. These interactions, whichcan be synergistic or antagonistic, are well documented in numerous bio-logical systems.4,5 For example, αvβ3 integrin associates with activatedinsulin growth factor (IGF) receptor and platelet derived growth factor(PDGF) receptor, and potentiates the biological activity of IGF and PDGF,respectively.6 Likewise, in human blood mononuclear cells, collagen-induced release of interleukin 1 (IL-1) through the binding of integrinα7β1 is potentiated by fibronectin binding to α5β1.7 These examplesdemonstrate the importance in crosstalk in regulating cell fate. Since theseinteractions are complex and not predictable from studies on individualmembers of the various components, high-throughput systematicapproaches to study crosstalk in stem cells are needed.


Immobilized FactorsECMPs (Collagen, Fibronectin, Laminin)

Proteoglycans (Chondrotin, Heparin)

Soluble FactorsGFs (WNT, TGFβ, FGF)

Small molecules (Steroids, Phenols, Alkaloids)

Salts (Na+, K+, Ca2+)

Mechanical StimuliShear stress

Compression

Stretch

Cell-Cell InteractionsCadherins

Notch ligands

Gap junctions

Signaling

pathways

Gene

Expression

Protein

Expression FunctionsProliferation

Self-renewal

Differentiation

Fig. 2. Cellular microenvironment. Four distinct components of the cellular microenvi-ronment that affect cell fate. (1) Immobilized proteins such as extracellular matrix proteinsinteract with the cell through most commonly through integrin signaling. (2) Cell-to-cellinteractions are mediated through cadherin signaling. (3) Chemical stimuli such as growthfactors and hormones interact with the cell through their respective receptors. (4) Mechani-cal stimuli such as shear stress or flow signal the cell through mechanoreceptors. Thesedistinct compartments have numerous members that interact in a complex manner, knownas crosstalk, to affect a variety of signaling pathways which in turn affect gene and proteinexpression and ultimately cell fate and function.

In vivo, stem cells reside in specialized microenvironments of organsand tissues called niches that regulate self-renewal and differentiation.8,9

The niche also serves to balance the choice of a stem cell to self-renew ordifferentiate: excessive self-renewal could lead to cancer,10 while differ-entiation could lead to depletion of a tissue’s regenerative potential. Thus,the niche must maintain a delicate balance between stem cell proliferationand differentiation.

Stem cell niches of various tissues with regenerative potential, such asskin, stomach, intestine and blood, share certain structural and organiza-tional properties. The niche cells provide cell-cell contacts and paracrinesignaling that regulate the self-renewal of the neighboring stem cells. TheECM provides a scaffold for stem cell growth in the niche and can inter-act with soluble factors to regulate signal transduction. Additionally,glycoaminoglycans serve to locally concentrate and present solublecytokines. The physiochemical environment, including oxygen gradients,pH, matrix stiffness, and topography, also contribute to the regulation ofstem cell proliferation, self-renewal, and differentiation.11

The first stem cell niche identified in mammals was the hematopoieticstem cell (HSC) niche. Individual HSCs are multipotent and highly self-renewing, but yet proliferate quite slowly.12 However, the manner inwhich HSCs interact with their niche to promote self-renewal has not beenelucidated. Furthermore, the few existing culture systems that allow formaintenance and expansion of HSCs in vitro are not well defined.13 Morerecently, niches have been identified in a wide range of tissues such as theskin, brain, gut, and liver.14,15 Another well-characterized stem cell nicheis that of the intestinal stem cells. Intestinal stem cells can be isolated tonear homogeneity using the marker gene Lgr5, and these cells producein vitro an intestinal-like structure that generates its own niche cells tomaintain the Lgr5-marked stem cells. However, in most cases, expansionof stem/progenitor cell populations in vitro without loss of stem cellpotential is often difficult or even impossible.

HESCs and iPSCs are able to generate all derivatives of the three pri-mary germ layers — ectoderm, endoderm and mesoderm — a propertyreferred to as pluripotency.16 Unlike tissue-specific adult stem cells,hESCs are only transiently present during development and do not have astable niche in vivo. Nonetheless, various in vitro culture conditions for


hESC proliferation and differentiation have been developed. A com-bination of components, including ECMPs, soluble factors, and otherphysiochemical factors act to maintain and expand the stem cell popula-tion or promote particular differentiation programs. However, given themyriad of factors that may influence hESC proliferation and differentia-tion, the in vitro culture conditions and the cellular microenvironmentsthat either promote hESC expansion or their specific differentiation havenot been successfully developed and defined. Thus, the development ofHTS methodologies is necessary in order to identify culture conditions forthe maintenance, expansion, and directed differentiation of stem cells.

3.1 Traditional HTS for identifying modulators of stemcell fate

Traditional HTS uses microtiter plates along with robotics, liquid handlingdevices, and automated imagers to conduct rapidly thousands to millionsof conditions (Fig. 3A). A highly sensitive, robust and quick read-out iscritical, as is the case for any HTS method. Most HTS typically involvethe use of libraries consisting of hundreds of thousands or even millionsof small molecules that are created through combinatorial chemistryapproaches, in order to identify compounds that produce the desired phe-notype.17,18 These screens are a useful tool to address questions in basicstem cell biology and chemistry.

Many groups have used HTS of chemical libraries to identify smallmolecules that regulate mouse ESC (mESC) self-renewal. For example, atransgenic reporter mESC line that expresses GFP under control of Oct4,a marker of pluripotency, was utilized to screen 50,000 compounds underdifferentiation conditions free of leukemia inhibitory factor (LIF) andfeeder cells. This high-throughput cell-based screening approach wasused to identify several small molecules that promote the self-renewal ofmESCs in the absence LIF and feeder cells. Specifically, these authorsidentified a previously uncharacterized heterocycle, SC1/pluripotin, thatallowed for the propagation of mESCs in an undifferentiated, pluripotentstate in the absence of feeder cells, serum and LIF.19

These traditional HTS technologies have also been used to identifyvarious small molecules that promote mESC differentiation into multiple


lineages. A high-throughput phenotypic cell-based screen of kinasedirected libraries led to the identification of a synthetic small moleculeinhibitor of glycogen synthase kinase-3β (GSK-3β), TWS119, thatinduces neurogenesis in mESCs.20 Previously, a distinct GSK-3βinhibitor, called BIO, was shown to promote the undifferentiated growthof ESCs. Likewise, using a combinatorial library of peroxisome prolifer-ator-activated receptor (PPAR) ligands, novel molecules that promotedmesodermal differentiation of murine ESCs into beating cardiomyocyteswere identified.21 Along similar lines, a phenotypic cell-based screen of alarge combinatorial chemical library was utilized to discover a class ofdiaminopyrimidine compounds that efficiently induce mESCs to differentiateinto cardiomyocytes.22


A B

C

Fig. 3. Microtiter HTS for indentifying modulators of human embryonic stem cell fate.(A) Undifferentiated hESCs were dissociated into single cells and plated onto 384 well-plates.After 48 hours compounds were added and screened for their effects on hESC self-renewal or differentiation. After 7 days, automated immunocytochemistry for Oct4 was performed and images were captured and analyzed using an automated laser-scanning confocalmicroscope. Several compounds were identified to promote (B) maintenance of pluripotencyor (C) differentiation. (Figure and legend adapted from Ref. 23 with permission.)

The development of HTS for hESCs has been difficult because of chal-lenges involved in establishing suitable growth conditions. More recently,though, a strategy was developed to adapt hESCs to HTS conditions, andto screen 2880 compounds for their effects on hESC self-renewal ordifferentiation (Figs. 3B and 3C).23 Use of this HTS system resulted in theidentification of several drugs and natural compounds that promote short-term hESC maintenance and compounds that direct differentiation.23

A similar approach was used to identify a small molecule, stauprimide,that down regulates c-Myc and thus increases the efficiency of the directeddifferentiation of hESCs.24

3.2 Cellular microarray-based screening in stemcell research

Although HTS have greatly advanced modern biology and drug discov-ery, they are not practical for all stem cell related studies given the costand the large number of cells and reagents required. For example, typicalHTS require 25–50 × 104 cells per condition screened.23 Thus, HTS arenot feasible for screens involving adult stem cells, which are rare (e.g. onein 200,000 blood cells is a hematopoietic stem cell) and difficult to iso-late. HTS using conventional multi-well plates is cost-prohibitive andoften does not provide adequate quantitative information on cell function.Additionally, most HTS approaches only have the capacity to investigatethe effects of one factor at a time, often ignoring the complex crosstalksthat typically occurs in biological settings between combinations ofmolecules.

In order to overcome these obstacles, cellular microarrays have beenused for screening the effects of large numbers of biological molecules onstem cell fate.25 Typical cellular microarrays consist of a chip (e.g. glassmicroscope slide) where minute volumes (μL to nL) of various molecules(e.g. ECMPs, small molecules, cytokines, biopolymers) are deposited indefined locations and analyzed for their effect on cellular processes(e.g. changes in gene and protein expression levels). These arrays arefabricated using robotic spotting, photo-assisted, and soft-lithographyapproaches.


A major challenge in developing stem cell-based therapies is the iden-tification of conditions that specifically regulate and influence their fate.Being able to identify components that mimic the stem cell niche will aidin the expansion and differentiation of stem cells in vitro. Consideringthe complexity of the microenvironments in which stem cells reside, it isimpractical to test proliferation and differentiation conditions using cur-rent established HTS methods. Cellular microarrays have advantagesover traditional well-based HTS in that they provide more informationfrom smaller sample volumes in a rapid, efficient, and cost-effectivemanner.

3.2.1 Combinatorial protein arrays for studying stemcell microenvironments

Combinatorial protein arrays consist of immobilized biological signalingmolecules on a surface onto which cells are seeded (Fig. 4). The first suchplatform was used to screen ECMPs and their effects on stem cells in acombinatorial fashion.26 Specifically, with the use of a DNA robotic spot-ter, different ECMP combinations were spotted, and the commitment ofmESCs towards an early hepatic fate was evaluated. Several combinationsof ECMP components that influenced stem cell differentiation and hepa-tocyte function were identified. This platform was further developed toinvestigate the interactions between ECMPs and soluble growth factorson stem cell fate.27 Several ECMPs and growth factors were found toinfluence mESC differentiation towards the cardiac lineage.

Similar robotic spotting techniques were used to print arrays ofECMPs along with growth factors and adhesion molecules to evaluatetheir effects on the proliferation and differentiation of human adult neuralprecursor cells,28 and the results revealed significant effects of certain sig-naling molecules (such as BMPs, Wnts, and Notch) on the extent anddirection of differentiation into neuronal or glial fate. For example, it wasfound that Wnt and Notch co-stimulation maintained the cells in an undif-ferentiated state. Along similar lines, a photo-assisted patterning processwas used to create matrix-growth factor arrays to identify materials thatdirect neural stem cell growth and differentiation.29



A

B D

E GC

F

Fig. 4. Combinatorial protein array technology for manipulating hESC fate. (A) Typicallayout of arrayed cellular microenvironments. Arrays of pre-mixed combinations of extra-cellular matrix proteins (ECMPs) and signaling molecules are printed onto acrylamidecoated glass slides using a contact microarray printer. Human embryonic stem cells(hESCs) were cultured on the arrays for 5 days. Cells were imaged live (B–C) and thenfixed and stained for DNA (D–E) and Nanog (F–G) to identify conditions that hESCattachment, proliferation, maintenance of pluripotency and differentiation. (Figure andlegend adapted from Ref. 30 with permission.)

Although these platforms were used to elucidate the role of certainmicroenvironmental components on stem cell fate, they have limitations.Specifically, these platforms either relied on the addition of signaling mol-ecules to the surrounding media, thereby limiting the throughput and thecomplexity of the microenvironments that could be screened, or involvedthe covalent attachment of the signaling molecules to the chip, thus affect-ing their biological activity. Additionally, the throughput of these systems(<100 conditions per chip) was significantly lower when compared toconventional HTS systems. In order to overcome these limitations, anintegrated array platform was developed in which ECMPs, growthfactors, and small molecules were non-covalently arrayed on acrylamide-coated slides, thereby creating comprehensive microenvironments thatclosely resemble the in vivo microenvironment in which cells reside(Fig. 4).30 This technology platform was used for the real-time simultane-ous screening of thousands of physiochemical parameters on stem cellattachment, proliferation, differentiation and gene expressions.30,31

Specifically, through the systematic screening of ECMPs and other sig-naling molecules, a completely defined culture system for the long-termself-renewal of three independent hESC lines was developed.30 In anotherstudy, this technology was used to investigate the effects of microenvi-ronmental modulations on the fate of hepatic stellate cells (HeSCs), aprogenitor cell that resides in the liver.31 It was determined that differentcomponents of the microenvironment differentially influence other com-ponents to influence the HeSCs phenotype. For example, it was found thatthe influences of Wnt signaling molecules on HeSC fate are dependent onthe ECMP composition in which they are presented. Furthermore, thisarray platform technology was validated by the finding that data obtainedfrom these experiments were indistinguishable from data obtained fromtraditional multi-well-based assays.

3.2.2 Polymer arrays for screening of biomaterials thatinfluence stem cell fate

Biomaterials have been used for the expansion of many human adult stemcell and progenitor populations. Mesenchymal stem cells (MSCs), forexample, were maintained and differentiated on several different classes


of synthetic and natural biomaterials.32–36 Unmodified and methyl-modified silane surfaces were used to enhance MSC proliferation.32

Likewise, neural stem cells (NSCs) were maintained and expanded on 3Dscaffolds composed of amino polymers such as poly(D-lysine).37 Therehas been some progress in the application of polymers for expansion andmaintenance of ESCs. For example, aliphatic poly(α-hydroxy esters) suchas poly(D,L-lactide) and poly(glycolide) were used to propagatemESCs.38 Nonetheless, biomaterial-based expansion and differentiationof stem cells has been slow coming. This has been mainly due to the inef-ficiency of the iterative nature of biomaterials-based research in whichmaterials are fabricated, tested, and redesigned.39

In order to establish a set of principles or properties that can assist inprediction of which polymers would influence stem cell fate, high-throughput array-based approaches have been implemented. In one study,an array-based biomaterials screen was used to identify specific functionalgroups that promote human MSC (hMSC) differentiation,40 it was foundthat phosphate surfaces promoted osteoblast formation, while t-butyl-modified surfaces promoted adipocyte formation. A similar approach wasimplemented to study the effects of 576 synthetic materials on stem celldifferentiation (Fig. 5).41 Using this method, several classes of polymers that promoted high levels of differentiation into epithelial cells wereidentified. More recently, this technology has been used to identifybiodegradable polymers that support the growth and expansion of hMSCsand neural stem cells.39

3.2.3 Microwell approaches to analyze physical cuesregulating stem cell fate

By using soft lithography methods, microwell arrays with defined dimen-sions can be fabricated (Fig. 6). For example, microfabrication was usedto create an array of approximately 10,000 microwells on a glass cover-slip for the parallel, quantitative analysis of single ESCs.42 Furthermore,the well dimensions could be adjusted over ranges of 10–500 μm in heightand 20–500 μm in diameter, and the platform was compatible with tradi-tional light and fluorescent microscopy. The microwell platform was usedto investigate the effect of cell density on the proliferation dynamics of ratneural stem cells (NSCs).



A

B

Fig. 5. Biomaterial microarrays for exploring polymer-stem cell interactions.(A) Monomers were mixed at a 70:30 ratio pairwise in all possible combinations.Monomer combinations were printed with a radical initiator onto a layer of poly(hydrox-yethyl methacrylate) (pHEMA), on top of an epoxide-coated slide. (B) Day 6 embryoidbodies were dissociated and seeded onto polymer arrays in the presence of retinoic acid,the absence of retinoic acid and with a 24-h pulse of retinoic acid for 1 or 6 d. Cells werethen stained for cytokeratin 7 (green), vimentin (red) and DNA (blue). (Figure and legendreproduced from Ref. 41 with permission.)


A B

C D

E

Fig. 6. Microwell platform for controlling the size of embryoid body (EB) formation.(A) Schematic of the process for generating microwell patterning Matrigel (MG) on a cellculture substrate, and subsequently seeding cells onto MG-patterned substrate. (B) To gen-erate size-controlled micropatterened (MP) EBs, hESCs are dissociated to single cells andplated at high density onto patterned Matrigel islands and cultured to confluence. Intactcolonies are then detached using a cell scraper and transferred to suspension in differenti-ation medium. Quantitative demonstration of EB size control in EBs generated fromsize-controlled human embryonic stem cell colonies (C) by EB diameter and (D) by com-paring cell number in colonies to cell number in generated EBs. (E) The effect ofmicropatterened human embryonic stem cell (MP-hESC) colony size and cell compositionon mesoderm and cardiac induction in MP-embryoid bodies (EBs). Gene expression lev-els measured for mesoderm markers Brachyury (Bry) and Mixl1, and cardiac markerα-Actin in day-8 (d8) EBs plotted with respect to the Gata6/Pax6 gene expression ratio inthe corresponding MP-hESC starting population, and as a function of MP-hESC colonysize demonstrate that mesoderm and cardiac induction of hESCs was significantly higherat larger EB sizes. (Figure and legend reproduced from Ref. 47 with permission.)

Directed differentiation of ESCs through formation of embryoid bod-ies (EBs) has been enhanced by using microwell array techniques.43–47

EBs are typically formed using the hanging drop method48 or in suspen-sion culture.49 The resulting EBs are heterogeneous in shape and size. Asa result, cell populations obtained from EBs formed by these methods canvary considerably. To provide more uniform microenvironments to EBsand thus more uniformly direct EB differentiation, microwell approacheswere implemented (Figs. 6A and 6B). For example, poly(ethylene glycol)(PEG) microwells were used to create EBs of homogenous size and shape(Figs. 6C and 6D).45–47 More recently, such approaches were used to con-trol hESC differentiation trajectories (Fig. 6E).47 Specifically, it wasobserved that mesoderm and cardiac induction of hESCs was significantlyhigher at larger EB sizes.

Microwell arrays were utilized for the parallel manipulation and quan-titative analysis of stem cells at the single cell level. For example,combined biomimetic hydrogel matrix technology with microengineeringwas used to fabricate a microwell array which can used to control NSCfate and neurosphere formation.50 Using this technology, the authorsenhanced the viability and control the size of neurospheres formed from asingle founding cell.

Microwell approaches provide precise control over microenviron-mental parameters such as shape and size. However, in contrast to mostmulti-well formats, all wells in the microwell approach share the sameculture media. Thus, it is difficult to vary other parameters of themicroenvironment in a high-throughput manner.

3.2.4 Microfluidic array approaches to study stemcell biology

Microfluidic devices are powerful tools that have the ability to control thesoluble and mechanical properties of the cell culture environment.Microfluidic devices are fabricated by the casting poly-dimethylsiloxane(PDMS) over a prefabricated-mold (Fig. 7A). The advantages of microflu-idics include (1) decreased reaction rates and analysis times, (2) reducedconsumption of reagents, (3) reduced production of harmful by-products,and (4) ability to run multiple experiments on a single chip.51


Recently, microfluidic approaches were used for the analysis of signalsthat affect stem cell fate. A microfluidic device was developed for ana-lyzing 16 unique mESC cultures in parallel.52 Using this platform, theauthors identified an optimal flow rate that enhanced mESC colony for-mation, proliferation, and maintenance of pluripotency (Figs. 7B and 7C).Along similar lines, a micro-bioreactor array was fabricated using softlithography that contains 12 independent micro-bioreactors.53 Each micro-bioreactor was perfused with independent culture media containingdifferent biological molecules. Using this platform, a correlation betweenvarying flow patterns and hESC differentiation into vascular lineages wasestablished. Recently, an integrated microfluidic platform was designedthat allows the screening of individual hESC colonies in real time usingsix individual cell culture chambers.54 Such approaches provided


A B

C

Fig. 7. Microfluidic arrays for culture of hESCs. (A) Fabrication of 4 × 4 microfluidic arraydevice. (B) Culture of mESCs in microfluidic array under different flow rate conditions.Chambers with high flow rates display large, round colonies, suggesting a favorable growthenvironment. (C) Mean colony area vs. flow rate mESCs grown under different flow rates.The mean colony areas are larger at higher flow rates while the mean colony areas are smallerat the lowest flow rate. (Figure and legend reproduced from Ref. 52 with permission.)

important information about the degree of hESC colony heterogeneity.Although these microfluidic platforms enable the multiplexing ofexperiments, they have the disadvantages of: (1) relative low throughput,(2) difficulty of fabrication, (3) inability to perform on chip immuno-cytochemistry, and (4) lack of compatibility with conventional,high-magnification light and fluorescent microscopy.

4. High-Throughput Intrinsic Systems forStem Cell Investigations

Each of the microenvironment factors acts individually and in combina-tion to perturb intrinsic cellular signaling networks which in turninfluence stem cell functions and ultimately stem cell fate. As a result, avariety of HTS systems have been developed to manipulate the intrinsicsignaling networks of stem cells. Such systems generally utilize largelibraries comprised of small molecules, RNAi molecules or expressionsystems carrying shRNAs or cDNAs.

4.1 High-throughput RNA interference studies toinvestigate gene functions in stem cells

Elucidation of gene function is a critical aspect of advancing stem cellresearch. Functional genomics typically involve gain- and/or loss-of-functionstudies, which reveal the molecular mechanisms of a cellular phenotype,but these are difficult to implement at the genome-wide scale in culturedstem cells. Thus, most gene-silencing studies are restricted to knockoutstrains of model organisms such as yeast, flies, and mice.

Recent advances in RNA interference (RNAi) have aided the field offunctional genomics by allowing for loss-of-function studies in mammaliancells without the need for germline inactivation of the gene being studied.55

RNAi occurs through the effect of the ribonuclease (RNase) enzyme Diceron double stranded RNA. Dicer cleaves the dsRNA into double-strandedsmall interfering RNAs (siRNAs) which can act either through the RNA-induced silencing complex (RISC) to degrade complementary mRNAsequences or through the RNA-induced transcriptional silencing (RITS)complex to repress transcription and modify DNA and histone methylation.56


RNAi screens have been used to study the effect of genetic control ele-ments on stem cell behavior. For example, a subtractive RNAi libraryapproach was used to indentify multiple genes involved in the regulationof expression of Oct4 and of self-renewal.57,58 However, large-scale cell-based RNAi screens have been hampered by the demands and inefficiencyof traditional HTS. Recently, RNAi cell microarrays were used for effec-tive gene knockdown in hMSCs (Fig. 8).59 These technology platformscould offer an efficient approach for carrying out high-throughput loss-of-function studies.60 Arrays can be fabricated by spotting eitherlentiviruses that express short hairpin RNA (shRNA) to silence geneexpression through RNAi61 or peptide transduction domain — doublestranded RNA binding domains (PTD-dRBDs) that carry siRNA across thecell membrane and knockdown gene expression.59,62 Such RNAi arrays willaid in the rapid functional annotation of stem cell genomes and in the iden-tification of genes involved in stem cell self-renewal and differentiation.

4.2 High-throughput generation of geneticallymodified stem cell lines

The ability to obtain information about gene function has been greatlyaided by the generation of genetically modified stem cell lines. Several


A B

Fig. 8. siRNA microarray for high-throughput screening of gene function. (A) Co-transfection of an EGFP vector and anti-EGFP siRNA into hMSCs and co-transfection ofan EGFP vector and scramble siRNA into hMSCs. (B) Quantification of the observedeffects demonstrates the efficiency of the spotted siRNA gene knockdown. (Figure andlegend reproduced from Ref. 59 with permission.)

techniques have been implemented to create genetically modified stemcells in a high-throughput manner. For example, gene-trap mutagenesis, atechnique that randomly generates loss-of-function mutations, was used tocreate more than 8000 mutagenized ES cell lines.63,64 More recently,clonal microarrays were used to create genetically modified stem celllines (Fig. 9).65 Clonal microarrays are produced by seeding stem cells onmicrofabricated surfaces generated using soft lithographic techniques.Stem cells grown on these arrays can be infected with DNA constructs andthen isolated after assaying in parallel for various parameters, such asproliferation, signal transduction, and differentiation.

5. Conclusions and Future Trends

The technologies presented here are capable of screening and perturbingseveral components of the stem cell microenvironment and can greatlyadvance the engineering of defined cell types from stem cells. However,additional factors, including oxygen and salt concentrations, mechanical


Fig. 9. Formation of neurosphere microarray. Schematic of procedure used to generatearray. Patterned array of neurospheres of adult rat hippocampal neural progenitor cellsexpressing green fluorescent protein and mouse embryonic CNCs. (Figure adapted andlegend reproduced from Ref. 65 with permission.)

forces, matrix stiffness, and dimensionality, also play critical roles in themicroenvironment and determination of cell fate. In vivo, the complex net-work of signaling and matrix molecules is subject to mechanical forces(such as pressure, fluid shear stress, and stretch), which play importantroles in specifying embryonic polarity66 and tissue development.67 Recentstudies have shown that application of shear stress to mESCs induces cellproliferation and endothelial cell (EC) lineage differentiation with theexpression of marker genes indicative of ECs and the enhancement ofendothelial functions.68–70 Application of cyclic stretch may induce differ-entiation toward smooth muscle lineage,70 and compression induceschondrogenesis.71 Substrate compliance is also known to influence cell fatedecisions. For example, human mesenchymal stem cells (MSCs) showedhigher rates of growth on stiffer substrates.72 Recently, it was demonstratedthat lineage-specific differentiation of MSCs is induced by matrix stiffnessthat matches the respective tissue — soft matrices are neurogenic, rigidmatrices are osteogenic, and intermediate matrices are myogenic.73 Finally,in vivo, cells often reside in 3D as opposed to 2D microenvironments. Infact, three-dimensionality has been shown to play a critical role in themicroenvironment and can affect cell function.74,75 In the future, these high-throughput tools can be enhanced to screen such additional components,thereby creating more in-vivo-like screening conditions.

It has recently been demonstrated that stable genomic integrationand high expression of four factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28, can reprogram fibroblast cells into induced pluri-potent stem cells (iPSCs).76,77 This ability to generate patient-specificpluripotent stem cells will have a major impact in regenerative medicine.However, the use of iPSCs in cell-based therapies is limited because of thepresence of virally transduced transcription factors and oncogenes.Furthermore, current protocols to establish iPSCs are extremely ineffi-cient (<0.1%). The technology platforms presented here could be used toidentify microenvironment components, such as ECMPs, growth factors,and small molecules, not only for the purpose of enhancing reprogram-ming efficiency, but also identifying factors that could replace the need forvirally transduced transcription factors and oncogenes.78,79

The HTS technologies described here could find usage in the pharma-ceutical industry by aiding in crucial steps of drug development: toxicity


screening, target identification, and lead assessment. The current methodof target identification involves the use of 96- or 384-well microtiterplates with monolayer cell cultures.25 However, the multi-well plate for-mat suffers from several limitations, most notably the cost of the relativelylarge amounts of reagents and cells needed. The HTS technologiesdescribed here, by increasing the parallelism and efficiency of screeningcompound libraries, offer an attractive solution. Additionally, these tech-nologies could be used to identify potentially toxic compounds earlier inthe drug development process. For example, an array-based high-throughput system was recently developed that can be used to mimic theeffects of human liver metabolism and simultaneously evaluate the cyto-toxicity of small molecules and their metabolites.80 Along the same line,the use of high-throughput technologies in conjunction with stem cellsand their differentiated progenitors could provide more realistic in vitromodels for predicting the effectiveness and toxicity of drug candidatesand chemicals in humans. Most cell lines currently used in drug screeningparadigms are virally transformed cells of tumor origin that are not repre-sentative of the disease to be investigated. The derivation of hPSCs hasmade it possible, for the first time, to study various aspects of humandevelopment and disease using cells representative of these conditions.

In summary, hPSCs represent an infinite supply of cellular “raw-material” that can be used to generate more realistic disease models fordrug discovery. The ability to manipulate hPSCs and adult stem cellsusing high-throughput technologies will enable the production of largeamounts of specialized cells needed for applications in regenerative med-icine and drug discovery.

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18

Microscale Technologies for TissueEngineering and Stem Cell Differentiation

Jason W. Nichol, Hojae Bae, Nezamoddin N. Kachouie, Behnam Zamanian, Mahdokht Masaeli and Ali Khademhosseini

1. Introduction

There is a severe shortage of tissues and organs for use in repair of dis-eased or otherwise defective tissues. Tissue engineering has emerged as afield to fabricate tissues in sufficient quantities to repair damaged tissues.1

While tissues such as skin2 or bone3 can repair a small injury in a reason-able amount of time, tissues such as cartilage4 and myocardium5 cannotregenerate, and will continue to degenerate and decrease in function with-out intervention.

Early “top-down” approaches comprised a majority of tissue engi-neering approaches, whereby biodegradable polymer scaffolds, such aspoly (glycolic acid) (PGA),6 were seeded with the different cell types. Inthis approach, it was expected that the cells would proliferate and migratethroughout the scaffold and secrete the appropriate extracellular matrix(ECM) often aided by growth factors,7 perfusion,6 and mechanical8 orother stimulation.9 However, despite advances in scaffold technology,such as surface patterning10 or decellularization techniques for native

375

ECM structures,11 it is still difficult to recreate the intricate tissuemicrostructure necessary to create functional tissues.

The “bottom-up” approach aims to recreate the tissue microarchitec-ture by designing individual building blocks with specific microstructuralfeatures and assembling these blocks into larger engineered tissues. Thereare a number of techniques to fabricate these building blocks, such as fab-rication of cell-laden microgels,12,13 self-assembled aggregation,14 creationof cell sheets15,16 or tissue printing technologies.17 After creating the buildingblocks, larger engineered tissues can be fabricated from these modules bya variety of approaches such as stacking,18,19 random packing,20 or selfassembly.14 The bottom-up approach attempts to mimic nature as manytissues are largely composed of repeated units with similar functions andarchitectures, including the lobule of the liver.21 Recapitulation of thenative microarchitecture of naturally repeated units of tissues could leadto improved function at the microscale, creating biomimetic engineeredtissues with improved functional properties.

One primary objective of bottom-up approaches is control of thecellular microarchitecture to better direct cellular function and ultimatelytissue morphogenesis.22 In this respect, many microscale technologies andtechniques used to create cell-laden building blocks can also be used asinvestigative models for determining and controlling cell behavior.23

These techniques have been employed recently for driving stem cell dif-ferentiation and function down specific lineages,24 which could bebeneficial for use in a variety of tissue engineering and regenerative med-icine applications. As many tissues are comprised of cells that typically donot undergo extensive self-replication, such as cardiomyocytes in cardiactissue, the ability to control and dictate stem cell behavior is crucial to thefuture success of engineered tissues. In addition, recent research has con-firmed the existence of stem cells residing in niches that are unique to thetissues and organs, which contain highly ordered microarchitectures andcellular compartmentalization and arrangement.25 Microscale technolo-gies have the ability to recreate many of these complex features, givinggreat promise to creating in vitro microenvironments with the ability toquickly and effectively direct both adult and embryonic stem cell (ESC)behavior bringing us closer to the ultimate goal of clinical regenerativemedicine applications.

376 J. W. Nichol et al.

The following chapter will highlight the current techniques for usingmicroscale technologies both for creating engineered tissues using the bot-tom-up approach and for investigating and directing stem cell behavior.These two applications of microscale engineering are crucial to the futureof regenerative medicine. In addition, as one field progresses and experi-ences new breakthroughs, similar analogous techniques can drive researchin the other field. The future clinical success of regenerative medicine ishighly dependent on successes in both tissue engineering and stem celldifferentiation, suggesting that the more these fields can interact and usesimilar techniques the faster development will occur hopefully shorteningthe time needed to bring these techniques to the clinic.

2. Control of Cellular and Tissue Microarchitecture

The ability to control the cellular and tissue microarchitecture is key toboth stem cell differentiation and bottom-up tissue engineering tech-niques. Through restriction of the cellular geometry, controlledenvironments can be created to investigate cell behavior.26 These geomet-rical restrictions can be performed by a number of methods, such as cellseeding in microscale channels27 or microwells28 or creating cell-ladenhydrogels using micromolding.12

2.1 Hydrogels

Engineering an environment in which cell proliferation, differentiationand function can be tightly controlled is of great importance to re-generative medicine.29 As the cellular microenvironment has a profoundeffect on stem cell physiology, including proliferation and differentiation,the ability to precisely control the cellular microenvironment could allowfor better control over directing stem cell behavior.30 Similarly, thecellular microenvironment, such as cell-cell and cell-ECM and cell-soluble factor interactions, have a substantial effect on the function andtissue morphogenesis of engineered tissues.31 Due to their characteristics,including biocompatibility, flexible methods of synthesis and a widevariety of physical characteristics, hydrogels have long been employed asscaffolding materials for tissue engineering.32

Microengineering for Tissue and Stem Cell 377

Hydrogels are composed of networks of hydrophilic polymer chains,which are the product of chemical or thermal interactions between pre-polymer chains. These chemical bonds could be permanent (such ascovalent and ionic bonds) or temporary bonds (such as hydrogenbonds).33 Such structures make it feasible to control the crosslinking andnetwork formation through manipulation of the reaction process, ulti-mately leading to better control over the microenvironment of thehydrogel.32 Adjusting the temperature of the hydrogel or manipulatingthe reaction energy by tuning the ultraviolet light (UV) exposure timeand power are examples of such manipulations. In terms of ioniccharges, based on the groups incorporated into the hydrogel backbone,hydrogels could be neutral, cationic, anionic or ampholytic.34 However,it is observed that charged hydrogels tend to have a greater potential forcell attachment as compared to neutrally-charged hydrogels. As a result,charged hydrogels are potentially better candidates for engineeringtissue scaffolds.35 Many of the currently available hydrogels aresynthesized from monomers which are either ionized or ionizable,improving the possibility of cell attachment and proliferation withincell-laden hydrogel scaffolds. Furthermore, incorporation of variouscell-binding peptide domains, such as arginine-glycine-aspartic acid(RGD), into the hydrogel can dramatically increase cell binding andspreading within the gel.36

The physical properties of hydrogels are another key factor makinghydrogels attractive for tissue engineering applications. To optimize cellviability, having a microenvironment in which cells can easily exchangenutrients and waste products, oxygen and other soluble factors is of greatimportance. Diffusion is a key factor in solute transport in hydrogels,however in hydrogels with microscale pore structures or forced flowconditions, convection can be a critical factor as well.37 Experimentalresults have demonstrated that the hydrogels pH, temperature, mesh sizeand environmental conditions could significantly affect diffusion in ionichydrogels.38 In addition, as will be detailed more thoroughly later in thetext, hydrogels have been demonstrated to be highly amenable to anumber of techniques to create structures with controllable features on themicroscale. Microfabrication technologies have been applied to hydrogelsto mimic the complex spatiotemporal in vivo ECM environment leading


to engineered tissues with biomimetic properties and microarchitectures.The ability to exert tighter control over the cellular microenvironmentthrough microfabrication of hydrogels could greatly enhance the ability tocontrol cell and tissue behavior ultimately leading to functional tissuestructures.

2.2 Cell seeding in microwells

Control of cell aggregation through seeding in fixed geometrical spaces isan effective means of restricting aggregate size and dimensions to directtissue morphogenesis. Seeding cells in non-adherent microwells formedspheroid structures,28 while larger organoid structures were created by cul-turing cells in linear channels39 leading to improved cell alignment andresulting tissue function. A major advantage of these techniques, for com-patible cell types, is the ability to allow the cells to remodel and recreatethe microarchitecture using only natural materials while allowing the cellsto dictate the pace and organization of the tissue morphogenesis.

Taking this one step further, researchers have used micromolds to gen-erate shape-controlled cellular aggregates, in geometries such asspheroids,40 individual and connected tori or honeycomb structures.41

Cells were then seeded onto the molded hydrogels, leading to self assem-bled cellular organization within the shape restricted templates.

2.3 Cell-laden hydrogels

To create microtissues researchers combine cells with hydrogels to createtissue-like structures of specific mechanical properties and geometries.Some common techniques mix cells within polymer hydrogels, such aspoly (ethylene glycol) (PEG)21 or other photopolymerizable materials,42

self-assembling peptide gels43 or temperature sensitive hydrogels.5 In thisapproach, cells are combined with the hydrogel precursors, pipetted intomicromolds and polymerized using incubation5 or UV light.28 An alterna-tive approach creates cell-laden hydrogels by directly passing UV lightthrough a patterned mask containing specific shapes, which, when opti-mized, only polymerizes the hydrogels where the UV light is able topenetrate the mask.14


One example of using cell-laden hydrogels for tissue engineering cre-ated rings of primary cardiac cells within molded collagen or Matrigel.These cell-laden rings were arranged in contact with other rings and cul-tured under cyclic mechanical stretch forming a composite tissue, withimproved contractile function and cell alignment. In vivo implantation ofthese tissues in a rat myocardial infarct model displayed significantimprovements in cardiac function demonstrating the ability to create func-tional tissues using assembled macroscale cell-laden hydrogels.5

2.4 Microarray systems

Microscale technologies have emerged as a powerful tool for high-throughput cellular and biological studies as well as a means for manipu-lating biological systems and miniaturizing experiments.44 A microarray isa fabricated device composed of a specified number of microwells ofdefined shape and size which can be used to culture cells and/or to depositcombinations of different materials such as collagen, laminin, andfibronectin to enable the study of cell behavior in a high-throughput man-ner. These miniaturized arrays can be used to segregate cells for single cellanalysis and multiple cells studies, such as forming embryoid bodies.45 Onemajor advantage of microarray systems is that the throughput issignificantly higher than is possible using traditional cell culture techniquesas these arrays can contain hundreds or thousands of microwells. Inaddition, due to the microscale size, expensive reagents and soluble factors,such as growth factors, cytokines or drugs, are conserved making large-scale combinatorial experiments feasible. Using time-lapse microscopyand immunostaining, the fate of several hundred single stem cells can betracked simultaneously making this a powerful tool for in vitro determina-tion of the impact of exogenous factors on cell and tissue behavior.

3. Microscale Technologies to Investigate and ControlStem Cell Behavior

The derivation of ESCs from both mouse and human46 has opened up newpossibilities for cell-based therapies. However, the use of human embryosto generate ESC lines is controversial and therefore recognized as


ethically problematic.47 A breakthrough to this problem is in the form ofreprogrammed somatic cells.48 These induced pluripotent stem (iPS) cellshave been shown to be functionally and molecularly similar to ESCs49 andoffer new opportunities in regenerative cell therapy applications.50

The ability to reliably direct stem cell differentiation to ectodermal,mesodermal, and endodermal lineages is a potentially powerful methodfor generating functional tissues, as tissues emerge from well-organizedsequences of cell renewal, differentiation, and assembly under normalphysiological conditions.51

Stem cells are sensitive to a variety of microenvironmental stimulithat regulate both self-renewal and differentiation.52 Thus, throughmicroscale engineering, cell differentiation can be guided by controllingthe interplay between regulatory factors down to the individual cellularlevel.

3.1 Microarray analysis of ESC differentiation

ESC differentiation can be directed through different combinations offactors that influence the microenvironment. Spatiotemporal microen-vironment signals may be influenced by the size of embryoid bodies(EBs), co-culture of different cell lineages, small molecules present inthe microenvironment and ECM materials in combination with otherunknown parameters. To study the effect of all stem cell differentiationfactors is an enormous combinatorial problem that would be extremelydifficult to analyze without the facilitation of high-throughput analysis,screening, and imaging. This goal can be achieved much more readilythrough the use of microarray systems for analysis and screening ofmultiple factors individually and in combination. Because of thesignificant number of possible combinations, high-throughputapproaches can be used to rapidly test and discover important combi-nations of parameters in ESC differentiation. Parameters such as ECMmolecules, soluble factors (e.g. cytokines, growth factors), and bioma-terial interactions can influence various biological pathways thatinfluence ESC differentiation. To improve our understanding of ESCdifferentiation, studying the combined influence of parameters thatmay regulate stem cell expansion and specialization in an efficient and


cost effective manner using high-throughput analysis, screening, andimaging may prove beneficial.

In addition to growth factors and cell-secreted factors, syntheticsmall molecules can direct ESC differentiation. Small permeable,naturally occurring molecules such as vitamin C, sodium pyruvate,dexamethasone, thyroid hormones, and retinoic acid have been used toregulate stem cell fate.53 Similarly, new synthetic, heterocyclic smallmolecules that can alter stem cell fate have recently been studied tocontrol stem cell differentiation. Ding et al. reported ESC differentia-tion into various cell types employing small molecules.54 One mannerby which synthetic molecules can be used to selectively control andregulate stem cell differentiation and proliferation is through adjustingthe activities of proteins. Such processes have been successfullyemployed to cause controllable neurogenetic and cardiomyogeneticinduction in murine ESCs, osteogenesis induction in mesenchymalstem cells (MSC), and skeletal muscle cell differentiation.55 Furtherinvestigation into small molecule discovery could play a substantialrole in furthering the ability to reliably differentiate stem cells downspecific pathways.

The interactions between ESCs and the surrounding matrixenvironment can profoundly influence stem cell behavior. To assessthis parameter Anderson et al. used a biomaterial library comprised of576 different acrylate-based polymers to analyze stem cell behavior.56

Combinations of the different polymers were mixed in 384-well platesand were robotically printed on coated glass slides. After printing, theslides were exposed to long wave UV to initiate polymerization, dried,sterilized, and washed with PBS and cell culture media. Embryonicstem cells were then seeded onto the slides and the influence of thematerials on their differentiation was analyzed. Based on these exper-iments, it was concluded that ESC differentiation may be inducedtoward epithelial cell lineage through interactions with specific com-binations of materials. This study demonstrated the potential forcreating a library of materials to study ESC-material interactionswhich could be used to better design scaffolds or culture conditions forcontrolling the behavior of ESCs, or other stem or differentiated celltypes.


3.2 Microwell fabrication

As briefly described above, microengineered wells can be a useful tool tocontrol the geometry and homogeneity of cell aggregates. To fabricatemicrowell arrays, UV-photocrosslinkable PEG prepolymer containingphotoinitiator can be placed on a glass slide that has been treated to be adhe-sive to the hydrogel. The precursor solution was crosslinked between thesurface-treated glass slide and a coverslip with a photomask on top to con-trol the spatial geometry of the crosslinking (Fig. 1). Microscopy cover slipswere used as spacers between the glass support and the coverglass to define


Fig. 1. (Left) PEG microwell fabrication through UV crosslinking. (Right) Localizingcells within arrays of microwells using a wiping technique. This method produces cellseeding densities that vary consistently with microwell geometry and cell concentration.(Copyright (2009) Wiley. Used with permission from Ref .63.)

the depth of the microwells. The PEG precursor solution was then irradiatedthrough a bright field photomask with UV light of 350–500 nm to generatethe microwells. The PEG precursor solution only underwent polymerizationin the areas where UV light was able to pass through the photomask, whileall other areas remained in the liquid prepolymer state. Following polymer-ization of the polymers, the coverglass was carefully removed and theuncrosslinked PEG macromers were removed with deionized water.

3.3 Microfluidic systems

The field of microfluidics could be useful for controlling the interactionof stem cells with their surrounding soluble environment as it allowsmanipulation of microliter volumes of fluids within microchannels.57

Microfluidic systems have been used in a number of different applicationsfor controlling the cellular microenvironment such as micropatterning ofcells, subcellular localization of media components, high-throughput drugscreening, introduction of a large range of laminar-flow rates, and creationof soluble factor gradients.58,59

A unique aspect of microfluidic systems is the ability to control mixingand shear stress in which the microfluidic devices can produce alogarithmic scale of flow rates and a logarithmic concentration gradient.60

This ability has been utilized to study ESCs where high flow rates resultedin increased proliferation with minimized usage of media as compared totraditional macroscale experiments.61 This important advantage may beapplied for the screening of media components, conditioned media, ordifferent chemical formulation with minimized consumption andoptimized cell behavior. The use of microfluidic systems in controlledflow rates or concentration gradients have been shown to be important forregulating cell proliferation and differentiation.58

3.4 Controlled microbioreactors

One of the main challenges in regenerative medicine is obtaining ampleamount of specific cell types for capable transplantation. However, theseefforts have been hindered by limited proliferative capacity of the desiredcells. Incorporation of bioreactor systems with ESCs is an active area of


investigation since ESCs can be differentiated into cell lineages of allthree primary germ layers.51 ESCs can greatly benefit from bioreactors asbiological processes can be carried out under tightly controlled (oxygen,nutrients, or other molecular and physical regulatory factors) environ-mental conditions to account for controlled nutrient transfer. Theseconditions will help minimize the batch to batch variability, making theprocess sufficiently reproducible in regenerative medicine. Bioreactorsystems may better facilitate the transition from laboratory to clinicalscale production due to high level of control, while providing a means toquantitatively study cell behavior in response to various stimuli. Some ofthe essential requirements to be considered during design are: (1) rapidand controllable expansion of cells; (2) enhanced cell seeding of 3D scaffolds (at a desired cell density, highyield, high kinetic rate, and spatial uniformity); (3) efficient localexchange of oxygen, nutrients, and metabolites; and (4) implementationof physiological stimuli.52

4. Assembly Techniques for Creating Engineered Tissuesfrom Microscale Building Blocks

Just as ESC behavior can be directed and manipulated through microscaletechnologies, engineered microtissues can be engineered by microgelswith specific microarchitectural features. The field of bottom-up tissueengineering aims to create macroscale engineered tissues with tightlycontrolled microarchitectural structures to better recapitulate the nativestructure and function of the tissues we are aiming to repair or replace. Themajor challenge of tissue engineering using bottom-up techniques isassembling macroscale engineered tissues from microscale buildingblocks. For optimal function in vivo, engineered tissues must recreate thenative microarchitecture, while also possessing robust mechanical proper-ties for appropriate interactions with the surrounding tissues and towithstand the mechanical environment. Some specific challenges to over-come for this field are: determination and recapitulation of nativemechanical properties, building block integration to form robust tissues,creation of integrated microvasculature, scale-up technologies to movefrom the laboratory to the clinic at the appropriate length scale and


successful demonstration of in vivo functional improvement in diseasedtissues.

4.1 Layer by layer production using cell-laden hydrogels

Layer by layer assembly of tissue sheets can be used to generate larger3D structures that mimic tissues. In one approach to create engineeredtissues using this layer by layer approach, arrays of cells and ECM/polymer were photocrosslinked in an additive manner.62 Through addi-tive photopolymerization, multilayer engineered tissues were fabricatedfor hepatic tissue engineering using cell-laden hydrogels. Hepatocytesfrom adult Lewis rats were mixed with PEG prepolymer that had beenfunctionalized with cell-adhesive RGD motifs. The cell-PEG mixturewas pipetted into a custom designed sterile chamber and polymerizedthrough the application of UV light exposed through one of three pho-tomasks, the first of which was in the shape of three pointed stars. Next,the spacer height was increased to create a second subsequent layerwhich extended the full spacer height partially resting on top of the ini-tial layer (Fig. 2). Finally, the spacer height was increased a final time,and a honeycomb enclosure was created around each two-layer unit, cre-ating engineered microtissues with microarchitecture and functionsimilar to native hepatic tissues. Using this technique Bhatia and col-leagues were able to demonstrate many benchmarks of functional hepatictissue, such as urea production, while also greatly improving hepatocyteadhesion both by inclusion of RGD molecules as well as through reca-pitulation of the native microarchitecture of the liver lobule. While thereare many clear advantages of this technique, such as recreation of thenative microarchitecture combined with positive functional propertiessimilar to the native tissues, one potential problem with this technique isthe inability to tightly control the layer thickness. There is no simplemethod for restricting subsequent cell-PEG mixtures from filling thechamber, making it difficult to have multiple layers that only occupy oneplane. For some tissues using different cell types on each layer, thistechnique would have difficulty recapitulating the native cellulararrangement. Clearly the positive aspects outweigh the negative,however in an effort to create more complex patterns and mimic more


complex tissues, addressing these shortcomings could make this tech-nique applicable to more tissues and applications.

4.2 Packing of co-cultured building blocks forcapillary filtration

A major challenge in creating engineered tissues that can function andintegrate in vivo, is the ability to create tissues containing a functionalmicrovasculature to integrate with and ensure perfusion through and aroundthe surrounding tissues. Recently an approach has been demonstrated thataims to create a perfusable tissue without creating microvasculature.


Fig. 2. Photopatterning of hepatocytes to improve nutrient transport and cell viability.(A) Control — hepatocytes encapsulated in 1.5 mm thick by 10 mm diameter hydrogel.MTT stain (black) demonstrated mitochondrial activity that was stronger nearer the out-side edge. Scale bar = 500 μm. (B) Hepatocytes micropatterned at the same density andoverall hydrogel dimensions demonstrated good viability with no diffusive limitation.Scale bar = 1 mm. (C) Quantification of MTT demonstrates the reduced viability in thecenter of the control construct that is alleviated by micropatterning. (D) Layer by layerdemonstration of micropatterning technique. Scale bar = 500 μm. (Copyright (2007) byFederation of American Societies for Experimental Biology (FASEB). Reproduced withpermission of FASEB.21)

To create perfusable tissues analogous to capillary filtration, a randomhydrogel packing technique was developed to create tissues with tortuous,perfusable networks of capillaries.20 Cylindrical tissue units were createdthrough packing of cell-laden collagen hydrogel building blocks withinperfused silicon tubing. To create the building blocks, collagen was mixedeither with HepG2 cells, a model hepatic cell line, or without. Followinggelation in cylindrical tubing, cylindrical cell-laden building blocks werecreated by chopping the long cylindrical tissue into pieces. The cell-ladengels were collected in media, then were combined with HUVEC cells toform a confluent endothelial layer around the perimeter of the hydrogelblocks. The HepG2-HUVEC modules were perfused into tubing contain-ing a porous plug to both trap the gels and also to allow the modules toaggregate and compact together. Over time the co-cultured constructscompacted further and remodeled together to form a porous, perfusabletissue. The engineered filtration devices were perfused with blood todemonstrate both high viability of the encapsulated HepG2 and the sur-face coated HUVEC cells, and the ability to perfuse without clotting, foruse as clinical blood filtration devices (Fig. 3). While, this relativelysimple technique for creating perfusable tissues could potentially be effec-tive in replacing tissues which primary function as filtration systems, suchas the kidney or liver, other tissues would potentially not be possible dueto the lack of a self-contained enclosure as well as poor mechanical


Fig. 3. Packing of cell-containing building blocks in a perfusable reactor. HepG2 livercells were encapsulated in collagen, cast into modular units then coated with a confluentlayer of HUVECs and packed inside a perfused tube to generate a perfusable filtration unit.(Reprinted with permission from the Proceedings of the National Academy of SciencesUSA.20)

properties. In addition, the encapsulated cells would have to retain theirviability and function while being surrounded by endothelial cells, poten-tially limiting the available cell types appropriate for encapsulation.

4.3 Directed assembly of cell-laden hydrogels

Recent work in our laboratory has used directed assembly to demonstratethe ability to create ordered tissue structures using cell-laden microgels asbuilding blocks.14 This technique demonstrates one method by whichhigher order structures can be created when the building block materialsare fragile or difficult to handle. In addition, this technique also demon-strates the potential for scale-up or automated techniques, as theengineered tissues can be created almost entirely without intervention.

This technique was made possible by harnessing the properties of sur-face tension with regard to hydrophilic hydrogels to assemble cell-ladenbuilding blocks into tissue-like structures (Fig. 4). For the first step, rec-tangular PEG hydrogels of varying aspect ratios were created containingencapsulated cells through direct UV photopolymerization using spe-cially designed photomasks. These building blocks were then placed intomineral oil, which is hydrophobic, to cause the hydrophilic microgels toaggregate. These aggregates were then exposed to a brief, second UVexposure to crosslink the assembled microgels. The ultimate shape andoverall dimensions of the tissues were demonstrated to be controllable toa certain extent based on the aspect ratio of the building blocks used, withthe total number of microgels increasing with the aspect ratio. Evengreater control of the process, and the potential for making more complexstructures, was demonstrated through use of spatially controllable lockand key geometries, suggesting a potential way to further increase thesize and intricacy of tissues created using this technique. While there areclear advantages to this technique, such as the ability to create tissues ofspecific sizes based on the geometries of the building block materials andwithout complicated manipulation or assembly techniques, there are alsopotential drawbacks to this technique. For instance, the ultimate size ofthe assembled tissues was on the order of millimeters, making a clearclinical application difficult to envision, unless these tissues were used asthe basis for a secondary assembly process. In addition, a large fraction



Fig. 4. Directed assembly of shape controlled hydrogel building blocks using a two-phase reactor. Lock (A) and key (B) shaped hydrogel modules were created directly byphotopolymerization using UV through a photomask, then allowed to aggregate and self-assemble in a hydrophobic media (mineral oil), into single (C, D), double (E, F) and triple(G, H) arrangements demonstrating the possible control of assembled co-cultured struc-tures. Scale: 200 μm. After assembly a second UV polymerization solidified the structures.(Reprinted with permission from the Proceedings of the National Academy of SciencesUSA.14)

of the microgels assembled randomly instead of in a controllable manner,suggesting that optimization must be performed to reduce the variabilityin the system. Current research into adapting this technique to be per-formed on a surface, rather than as a 3D immersion, are beginning toovercome many of these challenges and show great promise towards cre-ating engineered tissues with controlled co-culture on a clinicallyrelevant length scale.

5. Conclusions and Future Directions

Some major challenges affecting both the fields of microscale tissueengineering and stem cell differentiation in the future are improving theoverall spatial resolution leading to smaller size structures withenhanced biomimetic functionalities. Control on an even smaller scaleof structures and stem cell will allow the investigation of interactions ona broader spectrum than what is possible with current techniques. Themajor challenge for bottom-up tissue engineering approaches will be theimproving the ability to create tissues with controllable microarchitec-tures, yet, on a clinically, and biologically, relevant length scale for invivo implantations. One of the greatest challenges of microscale stemcell technologies is the ability to direct proliferation and differentiationmore reliably. The combination of these fields contains great potentialfor the advancement of the field of regenerative medicine. Theimprovements in the fields mentioned above will make the possibility ofregenerating degenerated or defective tissues much closer to reality, pro-viding hope for millions of current and future patients with no otheralternatives for cure.

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19

Quality Control of AutologousCell- and Tissue-Based Therapies

Nathalie Dusserre, Todd McAllister and Nicolas L’Heureux

1. Introduction

Cell- and tissue-based therapies can be broadly defined as the treatment ofhuman diseases using human or animal cells. Original examples of suchtherapies were introduced with the intent to restore the blood and immunesystem of patients. In its simplest version, this approach dates back to thefirst successful blood transfusion in 1818 by James Blundel. Later,E. Donnall Thomas performed the first syngeneic (1954) and then allo-geneic (1969) bone marrow transplants, pioneering work for which hereceived the Nobel Prize in Physiology and Medicine in 1990.1,2 Morerecently, stem cells of a different origin have been successfully used forsimilar purposes.3 Varying the nature of the starting cells or tissue and themethod or extent of any subsequent processing has generated a stunningrange of applications for cell- and tissue-based therapies. Some of theseapplications currently include diabetes regulation, osteoarthritis treat-ment, immune system modulation, regeneration of neural cells throughinjection of stem cells into the spinal cord, and replacement of wholeorgans (cornea, skin, blood vessels) through tissue engineering.

397

Cell sources for cell- and tissue-based therapies include differentiatedcells extracted from the tissues of living adult humans or cadavers, adultstem and progenitor cells, and embryonic and fetal stem cells. For mostapplications, the cells or tissues used require some level of ex vivo pro-cessing. Some applications require only minimal manipulation for cellpurification and storage. For others, a more extensive manufacturingprocess may include the proliferation, differentiation, selection, phar-macological treatment, or genetic modification of the cells. Ex vivomodifications are made to expand a specific cell type, restore a specificcell function, or build a specific tissue structure from the cells (tissue-engineered products). Personalized cell- and tissue-based therapies add tothis level of complexity by customizing the cell-based treatment to indi-vidual patient needs.

Regulations are in place to ensure the safety of the patients bycontrolling the quality of the cell- and tissue-based product. They aimto control all aspects of the manufacturing process from the selectionof the cell and tissue source, to its extraction and subsequent process-ing, and ultimately its implantation or re-injection into the patient. Thespectrum of possible cell- and tissue-based therapeutic applications iswide enough that regulations applying to the quality control of oneapplication might not apply to another. This chapter will focus on thequality control of therapeutic products derived from autologous cellsor tissues and requiring extensive ex vivo manipulation. Such productsmeet the FDA’s definition of autologous somatic cell therapyproducts.4

2. Regulations Pertaining to Quality Control of Cell- and Tissue-Based Products

Regulations pertaining to quality control of autologous cell- and tissue-based product manufacturing are based on preexisting regulationsdesigned for drugs and pharmaceutical products. These preexisting regu-lations were adjusted to meet the specific challenges associated with themanufacture of therapeutic products derived from cells or tissues manip-ulated ex vivo. Not surprisingly, the regulatory burden varies with theperceived risk associated with each product.

398 N. Dusserre, T. McAllister and N. L’Heureux

In the current Good Tissue Practices (cGTP) Final Rule (21 CFR1271), the last version of which was issued in 2005, the FDA categorizedcell- and tissue-based products into three classes based on their relativesafety risk.5 This risk assessment is based on the extent to which therequired processing alters the initial biological characteristics of the cellsor tissues used in the product. Products requiring only minimal manipula-tion and where the cells or tissues perform the function for which theywere biologically intended are considered low risk and are not subject to21 CFR 1271 rules.

In contrast, cell- and tissue-based products that either:

• require significant ex vivo manipulation such as cell expansionthrough tissue culture,

• are combined with another article,• are intended for a use that is different from their initial biological

purpose,• or have a systemic effect and are dependent upon the metabolic

activity of live cells for their primary function,

are considered high-risk and are subject to more stringent regulations (see Table 1 for more details on three-tiered cGTP classification).Products derived from autologous cells or tissues and requiring extensiveex vivo manipulations belong to this high-risk category. As such, theseproducts are regulated as biological drugs under the Federal Food, Drug,and Cosmetic Act and Section 351 of the Public Health Services (PHS)Act (42 U.S.C. 262, “351 products”).6 They must be evaluated through aBiologic License Application (BLA) pathway before they reach the mar-ket, an Investigational New Drug (IND) application must be granted bythe FDA before they are clinically tested, and their manufacturing processis subject to current Good Manufacturing Practices (cGMP) (21 CFR 210and 211) and applicable parts of 21 CFR 1271 (subparts A, C and D).4,7,8

They are also subject to the various regulations listed in Tables 2 and 3.Similar pathways and risk assessment-based classifications exist in

Europe where autologous cells or tissues manipulated ex vivo andintended for medical applications are labeled as Advanced TherapyMedicinal Products.9–13

Quality Control of Autologous Cell Therapies 399


Table 1. Three Tiered Approach of cGTP Final Rule (21 CFR 1271).

Risk Assessment Criteria Met by Products

Low risk, not subject to Requiring only minimal manipulation and are meant to 21 CFR 1271 rules replace a function for which the cells or tissues

were biologically intended (homologous use)

Higher risk, subject to All of the following:21 CFR 1271 rules and • Minimally manipulatedSection 361 of PHS* Act • Intended for homologous use only

• Manufacture does not involve combining cellsor tissue with another article, except for water,crystalloids, sterilizing, preserving, or storageagents that do not raise new clinical safety concerns

• Either:

— Does not have a systemic effect and is notdependent upon the metabolic activity of livecells for their primary function; or,

— Has a systemic effect and is dependent uponthe metabolic activity of live cells for itsprimary function, and is for:

a. Autologous useb. Allogeneic use in a first- or

second-degree blood relative, orc. Reproductive use

High risk, subject to Any of the following:21 CFR 1271 rules and • Μοre than minimally manipulated: expanded,Section 351 of PHS*Act activated, genetically modified

• Combined with another article (except some preserving and storage reagents)

• Intended for a use that is differentthan its initial biological purpose

• Has a systemic effect and is dependentupon the metabolic activity of live cells forits primary function, and is not for:

a. Autologous useb. Allogeneic use in a first- or second-degree

blood relative, orc. Reproductive use

*Public Health Services Act.

3. cGMP, cGTP and Quality System

The objective of cGMP and cGTP is to ensure patient safety by control-ling the identity, purity, stability, viability, and consistency of both theintermediate and final products. In order to ensure compliance with cGMPand cGTP requirements, every establishment processing “351 products”must devise its own Quality System. This system is a comprehensive pro-gram developed by the investigator or manufacturer and is tailored tomonitor specific cell- and tissue-based products. According to 21 CFR1271 (1271.3 (hh)), this program should be designed, among other things,to prevent, detect, and correct deficiencies that might increase the risk ofintroducing, transmitting, and spreading communicable diseases. While


Table 2. Regulations that Apply to “351” Products While Under IND.

Regulation Title

21 CFR 312 Investigational New Drug (IND)Application

21 CFR 210/211 Current Good Manufacturing Practices21 CFR 50 Protection of Human Subjects21 CFR 56 Institutional Review Boards21 CFR 1271 Subparts A, C, and D

Table 3. Regulations that Apply to “351” Products Once Licensed asBiologics (BLA).

Regulation Title

21 CFR 201 Labeling21 CFR 202 Advertising21 CFR 210/211 Current Good Manufacturing Practices21 CFR 600 Biological Products; General (includes Reporting

of Adverse Experiences and Biological Deviations)21 CFR 610 General Biologics Standards21 CFR 1271 Subparts A, B, C, and D

there are multiple organizational variations, the quality program typicallyincludes:

• A set of Standard Operating Procedures (SOPs): written documentsthat describe all activities performed.

• A Quality Control Program: a program that monitors certain charac-teristics of the process and product.

• A Quality Assurance Program: a program that utilizes audits ofrecords, revision of SOPs, personnel training, deviation monitoringand investigation, implementation of corrective and preventiveactions (CAPA), and review of the final-product release process toensure that all activities are performed according to SOPs and that allquality control results meet preset criteria.

Figure 1 succinctly illustrates how these three basic elements interact tocontrol the manufacturing and clinical testing of cell- and tissue-basedproducts.

4. Core Requirements of a Quality Program

4.1 Facilities

(§ 1271.190(a) and (b)): Any facility used in the manufacture of cell therapyproducts must be of suitable size, construction, and location to prevent con-tamination of the product with communicable disease agents and to ensureorderly processing without mix-up. The facility must be designed so thateach manufacturing step has a dedicated area, whose cleanliness matches thesafety risk associated with that step. A cleaning program, supported by appro-priate environmental monitoring, must be developed, implemented, andcarefully documented. The flow of supplies, personnel, products, and wastesin the facility must be conceived so as to minimize the risk of contaminationor cross-contamination of any of the products manufactured in the facility.

4.2 Environmental control and monitoring

(§ 1271.195(a)): Where environmental conditions could reasonablybe expected to cause contamination or cross-contamination of the


product or equipment, they must be adequately controlled. The fol-lowing control activities or systems should be provided as needed:temperature and humidity controls, proper ventilation and air filtra-tion, adequate cleaning and disinfection of rooms and equipment toensure aseptic processing, and maintenance of equipment used tocontrol conditions necessary for aseptic processing operations.Documents such as the FDA’s Guidance for Industry: Sterile DrugProducts Produced by Aseptic Processing-cGMP14 and the EU’sAnnex 1 (from Guidelines to GMP for Medicinal Products for HumanVeterinary Use)15 can be used to determine the appropriate level ofcontrol needed in each production area. This level will depend on


Fig. 1. This figure illustrates how the Quality System monitors the manufacturingand clinical testing of cell- and tissue-based therapy products. It also depicts how itsthree basic elements: Standard Operating Procedures (SOPs), Quality Assurance(QA), and Quality Control (QC) function together within the Quality System frame-work. Activities typically controlled by QA and QC are highlighted in red and blue,respectively.

factors such as the manufacturing steps involved, whether they areperformed in an open or closed system, etc.

Monitoring of controlled environments must be performed regu-larly, but the appropriate frequency is defined by the manufacturer orinvestigator. Monitoring can include non-viable and viable particu-late air monitoring, clean-are positive pressure levels, as well assurface and personnel monitoring. USP ⟨1116⟩16 and ISO 14644-217

contain useful information to help determine the type, method, andfrequency of environmental monitoring needed for any specificcontrolled environment. Records of environmental control and mon-itoring activities must be maintained. It is important that relevantprocedures define alert and action levels for environmental monitor-ing results and specify relevant corrective actions when these levelsare exceeded.

4.3 Equipment

(§ 1271.200(a)): Equipment design, location, and installation shouldfacilitate all operations, including those pertaining to maintenance andcleaning. Cleaning, sanitizing, and maintenance of all equipment shouldbe performed according to well-defined procedures and schedules. Allequipment used for inspecting, measuring, or testing must be demon-strated to be capable of producing valid results and calibrated accordingto established procedures and schedules. This includes, for example,equipment used to monitor the temperature of a storage unit or equip-ment used to measure air particle counts inside a controlled environment.Calibration accuracy should be in accordance with accepted standards(e.g. National Institute of Standards and Technology). Although §1271.200 does not specifically require equipment qualification and certi-fication, doing so is strongly recommended, particularly as part ofprocess validation.18–20 Equipment in which aseptic operations are per-formed, e.g. laminar flow hoods and biological safety cabinets, should becertified according to a strict schedule. Records of maintenance, clean-ing, sanitization, calibration, certification, and use for each piece ofequipment used during the manufacture of any batch of product shouldbe kept on file.


4.4 Supplies and reagents

(§1271.210 (a) and (b)): All materials used during manufacturing,whether or not they come in direct contact with the processed product, areconsidered supplies or reagents. Examples of supplies include steriledrapes, gloves, pipettes, cell culture flasks, and shipping containers.Examples of reagents include culture medium, saline, antibiotic solutions,cleaning agents, and chemicals used in processing.

A system must be implemented that will ensure that supplies andreagents are not used until they have been verified to meet predeterminedspecifications. These specifications are designed to diminish the risk ofintroducing, transmitting, or spreading communicable diseases, and toprevent toxicity. The establishment using the supply or reagent can eitherverify that it meets the requisite specifications through relevant testing orby obtaining proper documentation from the manufacturer (typically aCertificate of Analysis for reagents or a Certificate of Compliance for sup-plies). Each supply or reagent must go through an approval process duringwhich accompanying documentation is checked to verify that it corre-sponds to the predetermined specifications. Prior to inspection, itemsshould be stored in a quarantine location according to manufacturerinstructions. Accepted items must be moved to areas dedicated to the stor-age of approved items (1271.26(a–d)). They should then be stored andused according to the manufacturer instructions. Reagents produced inhouse should also undergo a verification process to ensure they meet thepredetermined specifications (e.g. solutions are sterile, have the properconcentrations, and fall within a specific pH range). The water usedthroughout the manufacturing process (to rinse equipment or parts or pre-pare reagents) must be of appropriate cleanliness and, if produced in thefacility, properly monitored. To limit the possibility of endotoxin contam-ination, water used for preparing reagents is usually Water for Injection,USP grade. Processes used to produce these reagents should be validated.

Keeping information data sheets listing specifications for all suppliesand reagents is a good way to ensure consistency during the approvalprocess. These sheets should be updated when products change and anarchive of previously used data sheets should be kept. Under §1271.210 (d),records must be maintained for receipt of each supply or reagent, including


the type, quantity, source, lot number, date of receipt, and expiration date.Records of the approval process for each supply or reagent, including testsresults or Certificates of Analysis from the vendor, should be kept on file.Records of all lots of supplies or reagents used in the manufacture of eachbatch of product should also be kept.

4.5 Manufacturing process validation

The ability of an established manufacturing process to effectively andreproducibly provide intermediate and final products of predeterminedspecifications must be documented through adequate validations.18,19

There are three possible validation types: prospective, concurrent, or ret-rospective. Prospective validation is the preferred approach and should becompleted prior to commercial distribution of the final product. The num-ber of process batches to be included in a validation is a function of thecomplexity of the manufacturing process. For prospective and concurrentvalidation, three consecutive, successful production batches should typi-cally be used. More batches may be necessary to prove the consistency ofa manufacturing process if it is particularly complex or requires prolongedcompletion times. For retrospective validations, data from ten to 30 con-secutive batches should generally be reviewed.

Critical parameters should be controlled and monitored during processvalidation studies. Such studies should include all critical process stepsand be performed using qualified equipment and facilities according to awritten validation protocol. The quality assurance team should regularlyreview validated processes. If the reviews confirm that the process con-sistently produces products meeting specifications, no revalidation isnormally needed.

Additionally, the aseptic status of a manufacturing process should bevalidated through a medium fill simulation.15,16,20 This is done by simulat-ing each of the critical manufacturing steps using a growth medium(broth) that promotes the growth of bacteria and fungi instead of any liq-uid reagent regularly used. The broth is subsequently incubated andobserved to detect any microbial growth. Whenever feasible, simulationsshould be performed at the end of a production shift to capture the worst-case scenario conditions in terms of microbial contamination risk. For the


initial (start-up) validation, three medium fill simulations should typicallybe performed per manufacturing step. One simulation per manufacturingstep can be performed during each subsequent validation. Subsequentvalidations should be performed once every six months.

5. Tailoring Quality Control to the Manufacturing Process

5.1 Quality control: Safety, consistency, and traceability

A proper quality program should address critical safety issues specific tothe various steps of the manufacturing process. This is easiest to achievewhen the quality system framework is developed in parallel with the man-ufacturing process during the product development (a concept coined as“Quality by Design”).21 This is realized by thoroughly assessing the safetyrisk associated with each manufacturing step and by implementing specificrisk control measures to reduce the identified risk to acceptable levels.13

The adequacy of the risk control measures should be regularly reviewed aspart of risk control management.22 Importantly, the level of risk associatedwith the manufacturing process and the resulting final product must becommunicated to all interested parties (including regulatory authorities andpatients, when relevant) throughout the product life cycle.23

To ensure consistency, proper ways to execute and control each man-ufacturing step must be described in written documents, called StandardOperating Procedures (SOPs). It is also critical that personnel be regu-larly trained to follow SOPs. Furthermore, every step of the manu-facturing process, as well as all results from quality control tests, shouldbe documented in manufacturing Batch Records that undergo regularscrutiny by the quality assurance staff (21 CFR 211.188) and must bethoroughly reviewed as part of the process leading to the final productrelease (21 CFR 211.192). According to 21 CFR 211.188, batch recordsshould include full traceability of patient material through all steps of themanufacturing process, including initial biopsy, final-product shipping,and eventual disposal. They should also contain the identification of allequipment, reagents, and supplies used during the manufacturing process.Personnel performing, supervising, or controlling any significant step ofthe process should be listed in the batch record. Any quality assurance


investigation made during the process (according to 211.192) should alsobe documented.

To thoroughly control the safety and consistency of a manufacturingprocess, a quality control program should at least assess:

• starting material (cell or tissue) sourcing and raw materialsqualifications,

• characteristics of pre-production cells,• critical process steps through In-Process Control testing,• and final product properties through Final Release testing.

5.2 Cell and tissue sourcing

Quality control of the manufacturing process starts with cell and tissuesourcing, also known as “collection.” To prevent the transmission ofcommunicable diseases from the cell donor to the recipient of the therapyproduct, determining donor eligibility, donor screening, and testing are allrequired by cGTP regulations (§ 1271.215). This is especially importantfor allogeneic cell therapy applications. For autologous applications,where the donor and the recipient are the same individuals, donor eligi-bility determination or donor screenings are not requested (21 CFR1271.90(a)). However, if manufacturing procedures have the potential toincrease the risk of propagating pathogenic agents that may be present inthe donor, then the donor should be tested for these pathogens and theresults should be documented. Typically, autologous cells and tissues areconsidered potentially infectious and manipulated as such.

Cross-contamination between cells of different patients is a keysafety issue. To address this issue, a proper quality control programbegins with specific, documented, and controlled labeling of the speci-mens harvested from the patient. For privacy issues, a unique patientidentifier is assigned to each patient and recorded with his or her nameon a log accessible only to the clinical staff. The labeling follows thepatient material whenever it changes containers. Forms requiring a wit-ness’s signature or the use of a bar code labeling system can ensurestringent traceability from the initial biopsy to the final product. At thepatient bedside, the final product labeling is reconciled with the patient


identity before implantation or injection. Very strict segregation is alsorequired for different batches of cells or tissues being processed simulta-neously. This can be achieved through dedication of specific equipmentto specific batches as well as by thorough and validated cleaning ofshared equipment before each use.

Unless processing occurs in a closed unit at the patient bedside, whichis unlikely for applications that involve extensive ex vivo manipulation,collected cells or tissues must be transported to the processing site, usu-ally by shipping. There are many requirements to properly control theshipping of the specimens. Each patient specimen must be packaged in adedicated, properly labeled, shipping container whose specificationsshould prevent the contamination or cross-contamination of the materialshipped. The containers should be sterile, endotoxin-free, and leak-proof.In addition, the surrounding shipping package should provide environ-mental conditions (e.g. temperatures ranges) that ensure that thebiological characteristics and viability of the material shipped will beproperly preserved. Both the shipping containers and the package shouldcomply with the Pressure Differential and Thermal Shock requirementsoutlined in the Department of Transportation’s Title 49 CFR (§173.196(a)(6) and (a)(7))24 and IATA regulations, and be validated to maintaintheir specifications in any shipping environment. Optimally, environmen-tal conditions inside the package should be recorded during the wholeshipping process and documented.

5.3 Cell culture

Cell extraction is usually the next step of the manufacturing process.Extraction typically requires the use of enzymes of animal origin, like col-lagenase or trypsin. These enzymes, as well as any biological rawmaterial of animal or human origin, used in any manufacturing step,should be demonstrated to be free of adventitious agents, including bacte-riae and fungi, mycoplasmas, mycobacteriae, and viruses.25

Further requirements apply to biological raw materials. For instance,processes used to remove or inactivate potential infectious contaminantsfrom biological raw materials should be validated. Sourcing, donor screen-ing data (for human sourced-reagents26), and the results of adventitious


agents testing must also be fully documented. Useful recommendationsregarding the testing of all materials of animal origin (e.g. enzymes, adhesionfactors, albumin, growth factors, etc.) can be found in Refs. 25 and 26. Thetype of testing necessary depends on the animal species from which thematerials are sourced. Because of the risk of Bovine SpongiformEncephalopathy transmission, reagents of bovine origin are of special con-cern.27,28 Interacting with regulatory agencies early during the developmentof the manufacturing process is valuable for identifying questionablereagents and either finding alternative reagents or determining appropriateactions to minimize the safety risk associated with those reagents.

The composition and purity of raw materials of non-biological originshould also be thoroughly documented. It is very advantageous to usematerials that are already licensed for human use.

Whether extracted cells are subsequently expanded, banked (storedfrozen) or immediately used for further production, it is required that pre-production cells (or bulk cells) be characterized (see 21 CFR 610 forrelevant required tests).29 The objective of this characterization is toverify that the cells are suitable for further production. Proper bulk cellcharacteristics increase the likelihood of manufacturing a final productwith adequate biological properties.

Generally, bulk-cell identity, purity, viability, and microbial safetymust be assessed.30,31 However, the consequences that a drift in the bulk-cell characteristics might have on the properties of the final product mustbe evaluated. Results from this risk evaluation are necessary to determinethe extent of necessary cell characterization and will dictate whether addi-tional testing might be necessary (e.g. tumorogenicity, etc.). Therefore,tests should be established during the development of the product to helpidentify which cell characteristics are necessary for the production of anadequate product. Based upon historical data, a set of acceptance criteriacan then be determined for pre-production cells. Those criteria are subse-quently used as a means to control the quality of these cells.

In many instances, extracted and expanded cells are banked. Mostoften, banking cells for cell therapy applications involves establishing aMaster Bank. Working Banks are then created by expanding cells from theMaster Bank and then using them to manufacture the product. For autol-ogous applications, however, the number of cells can be limited and it


may not be possible to create a Master Bank. Regardless of the cell-bankingstrategy, viability and phenotypic stability of the cells during cryostorageshould be established.

Assessing microbial safety of pre-production cells entails testing forsterility (absence of bacteriae, yeast and fungi per USP⟨71⟩32 or 21 CFR610.12), endotoxin amount (per USP⟨85⟩),33 and mycoplasma (per FDA’s“Points to Consider in the Characterization of Cell Lines used to ProduceBiologicals”).34 To prevent spreading microbial contaminations to otherpatient cells, sterility and mycoplasma should be tested before the cellsare transferred to liquid nitrogen storage containers for cryopreservation.In liquid nitrogen storage, viruses (Hepatitis viruses) can spread from onesample to another.35,36 Therefore, vapor-phase nitrogen storage is pre-ferred. Note that testing for the presence of specific human viruses,including CMV, HIV-1 and -2, HTLV-1 and -2, EBV, B19, HBV, andHCV, is not required for autologous cells banks. However, for cells thathave been exposed to bovine or porcine components (e.g. serum, serumcomponents, trypsin), it is appropriate to test for bovine and/or porcineadventitious agents.37

5.4 In-process testing

Quality control tests should be performed along each critical step of themanufacturing process through In-Process Control testing. During themanufacturing of cell-based products, microorganisms and impuritiescan be introduced from multiple sources, including components ofbiological and non-biological source, poorly controlled process steps, orfailures in aseptic processing techniques. To thoroughly control themanufacturing-process quality, each step of the process must be assessedto identify any potential routes of microbial contamination or impurities.This type of risk assessment should take into consideration differentparameters, such as:

• materials sources, • level of manipulation (e.g. multiple or few manual processes, auto-

mated processes, no manipulation), and the processing path (e.g.open, partially closed, closed, sealed),


• cell culture or incubation duration (longer times are riskier), • cell culture vessel (e.g. open (flask, tube, dish), partially closed (sterile

collection bag), or closed (bioreactor) system).

Results from this risk assessment should be used to determine criticaltime points for testing, the most appropriate samples to harvest, and thetests to be performed at these times. For example, in-process sterility test-ing might be performed during an extended period of culture or aftermanufacturing steps involving manual processing.

Similarly, it is advisable to test an intermediary-step product forpotency (i.e. measuring the product characteristics and biological activitythat contribute to its function) or identity immediately after a manufactur-ing step that is designed to (or that could) modify those parameters, e.g. cell expansion, differentiation, gene modification, etc.

Typically, in-process materials should be tested for sterility, endotoxin,identity, strength (concentration and potency), quality, and purity, asappropriate. The test methods used for in-process control are at the dis-cretion of the manufacturer. However, regulatory authorities will reviewthe testing procedures, the justification of their choice, their limits ofacceptance, and the rationale for these limits. Most importantly, valid in-process specifications for such characteristics should be consistent withthe final product specifications. They should be derived from previousprocess averages and variability estimates, where possible, and deter-mined by the application of suitable statistical procedures, whereappropriate. In-process control tests are the responsibility of the qualitycontrol team and their results are part of the batch records. The qualityassurance team will audit them as part of the final-product release.8

As specified in 21 CFR 211.10, written procedures (SOPs) detailingeach manufacturing step must be established and followed carefully at alltimes. They should include the description of the in-process controls, thetests or examinations to be conducted, and the appropriate samples ofin-process materials to be tested for each batch. In-process control pro-cedures are useful in monitoring the output and validating theperformance of the manufacturing steps that may be responsible for caus-ing variability in the characteristics of in-process materials and the finalproduct. Performing such tests during the manufacturing process allows


for the early identification and rejection of compromised batches of prod-ucts downstream and immediate investigation of the integrity of themanufacturing step.

5.5 Final release testing

At the end of the manufacturing process, according to 21 CFR 211.165,each batch of product to be released should be appropriately tested todetermine satisfactory conformance to final specifications, includingidentity, strength (or potency), purity, and freedom from contaminatingmicroorganisms. For aseptic products, sterility and absence of endotoxinsare essential qualities (21 CFR 211.167). For products whose biologicalproperties depend on the presence of specific viable cells, testing for via-bility and purity of the cell population is also critical. Sampling andtesting plans for final release should be established and described in writ-ten procedures that should include the method of sampling and thenumber of units per batch that will be tested. For each test, or each spec-ification, the acceptable quality level must be set based on a well-documented record of developmental data. The selection of these levels,also called acceptance criteria, is very important since they will be usedin making the decision to accept or reject batches of final products.Typically, acceptance criteria are revised and refined as a historical datarecord is established, however, they cannot be changed to justify theacceptance of a batch already produced (e.g. during a clinical trial).

The quality control team ensures that batches of products meet eachacceptance criteria necessary for their approval and release. The accuracy,sensitivity, specificity, and reproducibility of the test methods employedmust be established and documented. Such validation and documentationmust be in accordance with 21 CFR 211.194 (a)(2). For cell- and tissue-based therapy products, the tests necessary for final release are listed inTable 4. Note that cell therapy products are exempt from the GeneralSafety Test requirement (per 21 CFR 610.11).

Significant challenges are associated with the final release of cell- andtissue-based products. One of the challenges for small-sized autologouscell therapy batches is that the sampling size might be different fromthose specified in the regulations, which were originally developed for


large-sized batches of pharmaceutical drugs. Acceptable sampling sizeand strategy will have to be negotiated on a case-by-case basis with theappropriate regulatory authorities. Other challenges stem from the factthat most cell-based products are manufactured using aseptic manipula-tions because they cannot undergo sterile filtration or terminalsterilization. This makes the assessment of sterility, absence of endo-toxin, and freedom from mycoplasma paramount. However, manycell-based products cannot be cryopreserved or otherwise stored withoutaffecting viability and potency. Thus, their shelf life is shorter than thestandard test read out times. Test durations for sterility and mycoplasmatesting (per CFR 610.12) have been standardized as 14 and 28 days,respectively.29,34

Until recently, in the absence of rapid and effective microbiologicaltesting that avoided administrating the final product before the final steril-ity test results became available, the FDA recommended a programtailored specifically for these products.38 This program combined standardmicrobiological testing (sterility, endotoxin, and mycoplasma) initiated afew days (48 to 72 hours) before the final release of the products with aGram staining performed at the time of shipping or implantation. Anothermicrobiological sample was also harvested at the time the final productwas shipped. No-growth results from the 48- to 72-hour sterility test andthe negative Gram stain were used for final release criteria. During the last


Table 4. Tests Required for Final Release.

Required Test Relevant Biologics Standard Test Method

Potency 21 CFR 610.10 Not specifiedSterility 21 CFR 610.12 SpecifiedPurity (pyrogenicity) 21 CFR 610.13 SpecifiedIdentity 21 CFR 610.14 Not specifiedConstituent materials 21 CFR 610.15 Not specifiedMycoplasma* 21 CFR 610.30 SpecifiedCommunicable diseases 21 CFR 610.40 Not specifiedViability Not specifiedPhenotype Not specified

*Only required for cells that are cultured.

couple of years, alternate microbiological tests have been developed andare now available for cell therapy products with a limited shelf life. Rapidmicrobiological methods are designed to provide performances equivalentto the sterility testing methods described in 21 CFR 610.12, whileproviding results in significantly less time. Those alternative tests are stillnot standardized and must be validated, as required for most methods offinal product testing under 21 CFR 211.165(e) and 211.94(a)(2). The prin-ciples of validation specific to rapid growth-based microbiologicalmethods are described in Guidance for Industry Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellularand Gene Therapy Products.39 Recently, rapid mycoplasma detection tech-niques using Polymerase Chain Reaction (PCR) have been developed aswell. Prior to licensing, data demonstrating that the PCR test selected hasa sensitivity and specificity similar or greater than the standard test mustbe provided.

Guaranteeing final product safety by assessing the potency of the finalproduct constitutes another huge challenge for autologous biologicalproducts. Potency of an autologous product often varies from one patientto another. Live cell- and tissue-based therapies frequently rely on theactivity of cell populations that are not 100% pure or on the combinedactivities of several cell populations that can have complex mechanismsof action. The characteristics of autologous cell therapy products thatmake the development of potency tests difficult are described in Table 5.40

Historical developmental data, relevant preclinical information, and earlyclinical study results can be used to determine which product attributes aremost relevant for measuring potency.

For products that contain more than one active ingredient, potencymeasurements should be designed to assess the biological activity of allactive ingredients. The potential for interference and synergy betweenactive ingredients should be considered. A collection of assays (biologicaland/or analytical assays) can be developed for products with complexmechanisms of action or presenting multiple biological activities. At leastone quantitative assay must be included in the combination. Analyticalassays can evaluate immunochemical, biomechanical, and molecularattributes of the products. Their data should be correlated with a relevantproduct-specific biological activity.



Table 5. Autologous Cell- and Tissue-Based Product Characteristics that Make theDevelopment of Potency Test Difficult.

Challenges to PotencyAssay Development Examples

Inherent variability of starting • Donor variabilitymaterials • Cell line heterogeneity

Limited lot size and limited • Single-dose therapy using cells suspendedmaterial for testing in a small volume

• One single lot per patient

Limited stability • Viability of cellular products

Lack of appropriate reference • Autologous cellular materialsstandards

Multiple active ingredients • Multiple cell lines combined in finalproduct

• Heterogeneous mixtures of immune-modulating cells

Potential for interference or synergy • Multiple cell types in cell preparationbetween active ingredients

Complex mechanism of action(s) • Multiple characteristics required forappropriate functions (e.g. biomechanicalresistance and anti-thrombogenic propertiesfor an autologous tissue-engineered blood vessel)

• Multiple potential effector functions of cells

In vivo fate of product • Migration from site of administration• Cellular differentiation• Remodeling of tissue-engineered construct• Invasion by recipient own cells• Immune response to components

introduced during manufacturing process

Modified from [Guidance for Industry. Potency Tests for Cellular and Gene Therapy Products, CBER, 2008].

To market a biological product, a validated potency assay withdefined acceptance criteria must be described in the BLA (21 CFR211.165(e)). The acceptance criteria, intended for subsequent finalrelease testing, should be based on knowledge gained through manufac-turing experience and data collected during all phases of productdevelopment and clinical investigation. Reference material used as con-trols for the assays should be available and can include well-characterized clinical lots. Several resources are available for analyticalmethod validations.41–43

5.6 Shipping the final product

After final release, biological products are either stored or shipped to thepatient bedside. Requirements similar to the ones pertaining to biopsy ship-ping, labeling and packaging, apply to the final product shipping.Supplementary labeling (e.g. “For Autologous Use Only”, “Not evaluatedfor infectious diseases”, “Warning: advise recipient of communicable dis-ease risk”, “Biohazard”, etc.) might also be required.44 In addition, under21 CFR 211.166, an adequate number of batches must be tested to deter-mine the product stability under specific storage and shipping conditions.The results of such stability testing are used in determining appropriatestorage and shipping conditions as well as in establishing the product shelflife. Stability of such products should be assessed in the worst shippingconditions possible (duration, temperature) in the same container system inwhich the product will be marketed. For further information, refer to ICH5C, Q1A(R2), and Q1E.45–47 Typically, living autologous cell- and tissue-based therapy products will have a short shelf life.

6. Conclusion

Cell- and tissue-based therapy applications, especially those requiringextensive, and sometimes lengthy, ex vivo cell manipulation present anadditional challenge for both regulatory authorities and the companiesdeveloping them. The challenge lies in the necessity to strike a balancebetween the quality control official’s need to ensure product safety and thecompany’s ability to comply. More simply put, the extent and number of


the quality control steps, which tend to multiply with the complexity andlength of the manufacturing process, need to guarantee patient safety,but should not prevent a much-needed treatment from becoming clini-cally available. Although many autologous somatic cell therapyapplications have been developed so far, only a few are commerciallyavailable. In the United States, only two autologous cell therapy prod-ucts requiring extensive ex vivo manipulation and regulated through theBLA pathway have yet reached the market. Carticel®, produced byGenzyme Tissue Repair, became available in August 1997 andProvenge®, from Dendreon Corporation, in April 2010. Over the lastdecade, a significant discrepancy has existed between the rapid devel-opment of cell- and tissue-based therapies and their slow access to themarket. This discrepancy has incited various companies to develop andtest new applications outside of the United States. Hopefully, a moreprogressive political context will allow the FDA to accelerate the rate ofapproval for such therapies so that more US patients can safely benefitfrom them.

Acknowledgments

The authors thank Marissa Peck and Corey Iyican for their help in revis-ing and editing this chapter.

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for human use. EMEA/CHMP/96268/2005 (2005).

23. ICH Q9 Quality Risk Management (2005).

24. 49 CFR 173.196. General Requirements for Shipments and Packages:

Infectious Substances (2002).

25. CBER/FDA: Guidance for Industry: Characterization and Qualification of

Cell Substrates and Other Biological Starting Materials Used in the

Production of Viral Vaccines for the Prevention and Treatment of Infectious

Diseases (Draft Guidance) (2006).

26. 21 CFR 640: Additional Standards for Human Blood and Blood Derivatives.

27. FDA. Proposed Rule: Use of Materials Derived from Cattle in Medical

Products Intended for Use in Humans and Drugs Intended for Use in

Ruminants. 72 FR 1581 (2007).

28. EMEA/CHMP. Note for Guidance on the Use of Bovine Serum in the

Manufacture of Human Biological Medicinal Products. CPMB/BWP/

1793/02 (2003).

29. 21 CFR 610: General Biological Products Standards.

30. CBER/FDA. Guidance on Applications for Products Comprised of Living

Autologous Cells Manipulated Ex Vivo and Intended for Structural Repair or

Reconstruction (1996).

31. ICH Q5D Derivation and Characterization of Cell Substrates Used for

Production of Biotechnological/Biological Products (1997).

32. United States Pharmacopoeia ⟨71⟩: Sterility Testing. In: USP 32-NF 27

(2009).

33. United States Pharmacopoeia ⟨85⟩: Bacterial Endotoxin Testing. In: USP 32-

NF 27 (2009).


34. CBER/FDA. Points to Consider in the Characterization of Cell Lines Used

to Produce Biologics (1993).

35. R. S. Tedder, M. A. Zuckerman, A. H. Goldstone, et al., Hepatitis B trans-

mission from contaminated cryopreservation tank, Lancet 346: 137–140

(1995).

36. A. E. Hawkins, M. A. Zuckerman, M. Briggs, et al., Hepatitis B nucleotide

sequence analysis: linking an outbreak of acute hepatitis B to contamination

of a cryopreservation tank, J Virol Methods 60: 81–88 (1995).

37. 9 CFR 113: Detection of extraneous viruses by the fluorescent antibody tech-

nique. Animals and Animal Products.

38. CBER/FDA. Guidance for Reviewers: Instructions and Template for

Chemistry, Manufacturing, and Control Reviews of Human Somatic Cell

Therapy Investigational New Drug Applications (2003).

39. CBER/FDA. Guidance for Industry: Validation of Growth-Based Rapid

Microbiological Methods for Sterility Testing of Cellular and Gene Therapy

Products (Draft Guidance) (2008).

40. CBER/FDA. Guidance for Industry. Potency Tests for Cellular and Gene

Therapy Products (2008).

41. CBER/FDA. Guidance for Industry: Analytical Procedures and Methods

Validation Chemistry, Manufacturing, and Controls Documentation (Draft

Guidance) (2000).

42. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology

(2005).

43. United States Pharmacopoeia ⟨1225⟩: Validation of Compendial Procedures.

In: USP 32-NF 27 (2009).

44. CBER/FDA. Eligibility Determination for Donors of Human Cells, Tissues,

and Cellular and Tissue-Based Products (HCT/Ps) (2007).

45. ICH Q5C Quality of Biotechnological Products: Stability Testing of

Biotechnological/Biological Products (1995).

46. ICH Q1A(R2) Stability Testing of New Drug Substances and Products

(2003).

47. ICH Q1E Evaluation of Stability Data (2003).


20

Regulatory Challenges for Cell-BasedTherapeutics

Todd McAllister, Corey Iyican, Nathalie Dusserreand Nicolas L’Heureux

1. Introduction

Interactions with the Food and Drug Administration (FDA) or other regu-latory agencies typically conjure images of a bureaucratic quagmire atbest, or an adversarial fight at worst. In all fairness, these regulatory bod-ies are tasked with an incredibly challenging situation: trying to balancethe mutually exclusive demands for a “least burdensome process” fromindustry with a “zero risk” expectation from the patients and overly eagerlitigators behind them. Industry is quick to criticize the FDA for itslengthy approach to review and approval, and the lay press is quick tochastise the agency over rare clinical debacles such as those associatedwith Vioxx (Merck) or TBN 1412 (TeGenero). Few people, however, laudthese same regulatory agencies for presiding over nearly 50 years of med-ical innovations in the modern era of drug and device regulation. Indeed,in the post-thalidomide era, the average life expectancy has increasedfrom roughly 70 years in 1962 to nearly 80 years in 2003.1 In effect, we

423

can thank the FDA for safely adding more than two hours to each 24-hourday that we live.

While there have been some dramatic failures of drugs and devices,none in the modern era of regulation have approached the scope of thethalidomide tragedy. When viewed in light of the billions of devices anddrug doses that have been safely administered, the overall performance ofthe worldwide regulatory bodies is commendable. Safely managing thetransition of medical innovations from bench-top research to clinical usehas driven this improvement in longevity and quality of life. This is evenmore remarkable in light of the funding disparity between the regulatoryagencies and the scientific communities. Fueled by an annual budget ofapproximately US$30 billion from the National Institutes of Health (NIH)and billions more from industry, medical innovation has leapt forward atunfathomable rates. The FDA is understandably hard pressed to keep pacewith this innovation on an annual budget of just over US$2 billion. As lateas the early 1960s, considered the dawn of the modern era of drug anddevice regulation, the structure of DNA was still unknown, catheter-basedcardiovascular interventions were essentially non-existent, and drug thera-pies for cancer and cardiovascular disease were in their infancies. Thus, arealistic and sympathetic approach should be sustained when setting expec-tations for regulatory agencies to keep pace with innovations such as genetherapies or Regenerative Medicine.

While it is appropriate to acknowledge the unenviable tasks assigned tothe FDA and other regulatory agencies, it is clear that there are unnecessaryburdens in each system, particularly with respect to new therapies, in par-ticular Regenerative Medicine and cell-based therapeutics. Indeed, there aredramatic regional disparities in the data required to support submission, thetolerance of risk, and the subsequent time required to receive the studyapprovals. Additionally, there are significant differences in costs associatedwith clinical studies and access to patient populations. In this chapter wewill explore some of these regional differences and provide insights intomore efficient management of the regulatory processes, irrespective of loca-tion. As evidenced by the dramatic increase in the number of clinical trialsperformed outside countries like the US, France, Germany and the UK, it isclear that developing a cohesive global clinical trials strategy has become acritical priority for a burgeoning Regenerative Medicine company.2

424 T. McAllister et al.

Specifically, in this chapter we will address:

• Key regulatory considerations for each clinical trial phase• Strategies that target certain geographical regions for specific

developmental advantages• Use of a Clinical Research Organization (CRO)• Universal regulatory considerations for cell-based therapeutics

2. Regulatory Challenges at Each Phase of ClinicalDevelopment

2.1 Clinical trials design considerations

While it may be obvious, it should be stated at the outset that a wellthought out clinical trials strategy is perhaps the most important elementto consider in the process of transitioning to human use. It is difficult tomake sweeping recommendations relating to protocol design, as eachtrial will be driven by its own particular requirements. It is worth empha-sizing, however, that the appropriate selection of endpoints can make thedifference between approval and rejection of the protocol from a regula-tory perspective, and more broadly, can make the difference betweenperceived success and perceived failure of the study. While the percep-tion of the latter point is beyond the scope of this chapter, one shouldrealize that setting aggressive endpoints can result in adverse interpreta-tions from the Data Safety Monitoring Board (DSMB) or the regulatoryagencies once the data is reviewed. For example, in our own Phase I/IItrials involving hemodialysis access, a cell-based vascular graft was usedin a subset of “worst case scenario” patients. This type of protocol isquite common with radically new technologies. Data relating to the stan-dard of care, typically reported across a much broader patient population,predicts a life expectancy of nearly six years. So, in the context of thestandard of care, it is tempting to set Phase II endpoints at 12 or even18 months, as is typically done with the standard patient population.However, with the short life expectancy unique to our subset of patients(one to two years), we would have been unlikely to meet these endpoints.This same argument can apply to Phase I studies in Regenerative

Regulations and Cell-Based Therapies 425

Medicine, as often there are secondary endpoints that relate to longer-term efficacy. Regenerative Medicine trials rely upon complex biologicalmechanisms, and it is difficult to accurately predict the outcomes and themost appropriate endpoints. To minimize the likelihood of studies beingsuspended due to outcomes that are unexpected or difficult to interpret,one strategy we have employed successfully is defining clear failure cri-teria and continuation criteria in the original protocol. By negotiatingfailure and continuation criteria upfront, there is less pressure on theDSMB or regulatory agencies to suspend a study that has an unexpectedresult.

While there is no question that the regulatory challenges facing theclinical development of cell-based therapeutics are onerous, there aremany shortcuts that can, and should, be made to streamline the overallapproval process. It is important to understand that philosophically, theFDA is completely risk averse and depends largely upon a strategy ofexhaustive data collection to demonstrate a thorough review. While spon-sors tend to shy away from direct confrontation with the FDA orsimilarly established regulatory agencies, these agencies, particularly inEurope, are actually quite responsive to rational arguments againstunnecessary data collection. So, while the guidance documents mayencourage detailed, standardized testing pulled from every conceivableprecedent, many of these tests can be eliminated from lot release criteria(or at a minimum be done a single time as a validation study) if there issupporting rationale. Moreover, as detailed below, some experimental“requirements” can be delayed until Phase II or even Phase III studies areto be performed.

The classic lines defining Phase I, II, and III trials are often blurred inRegenerative Medicine. While some simple cell-based injections(myocardial regeneration, for example) may require a classic Phase I dos-ing study, even these initial safety studies have efficacy endpoints. Thus,we tend to call our initial studies Phase I/II studies, which are clearly dis-tinct from controlled Phase III studies. So while the study phases may bedefined differently for specific applications, the recommendations belowmay accelerate clinical studies. These strategies, however, should be con-sidered as a part of a detailed risk analysis to ensure that these “shortcuts”do not appreciably compromise patient safety.


2.2 Phase I/II clinical testing

Most companies do not fully appreciate or take advantage of the relaxedmanufacturing requirements for Phase I trials. While the FDA does notencourage “garage” manufacturing during pre-IND (Investigational NewDrug) meetings, the guidance documents very clearly establish that thereis no requirement for Good Manufacturing Principles (GMP)/Good TissuePractices (GTP) manufacturing at this stage of clinical development.3 Thisis not the case for Europe, as GMPs are required for products manufac-tured for human clinical trials.4 However, even in Europe, the regulatorybodies tend to offer some leeway in co-developing a company’s variousGMP processes early on, especially if it is an SME (small and medium-sized enterprise) and/or the company does not yet have a product on themarket. Given the fact that early stage money is extraordinarily expensive(that is, early valuations are low, making early stage expenditures dispro-portionately “expensive” from a capitalization perspective), it makes littlesense to implement overly extensive quality assurance/quality control(QA/QC) procedures for Phase I trials. For most Phase I cell-based thera-peutics trials, the key manufacturing elements that affect patient safety(functionality, uniformity, sterility, avoidance of cross-contamination,etc.) can be consistently performed in any reasonably organized universitylab as long as the appropriate release criteria are employed. Relativelybasic documentation of the key manufacturing steps usually suffices forinitial studies. These basic systems can then be filled in as the study pro-gresses with more detailed procedures and hardware. Indeed, our ownapproach to Phase I clinical trials was to establish GMP-level manufac-turing controls for the key, high-risk steps in the manufacturing process,as well as for those repeated frequently, such as change of cell culturemedium. This more focused GMP effort significantly augments productsafety without unnecessary early stage expenditures. Additional safe-guards, such as stringent functional release criteria, conservativeenrollment rates, and long post-treatment surveillance periods, wereemployed. We felt that these safeguards probably did more for overallpatient safety in Phase I trials than the more costly efforts later required tobring the full production process up to GMP standards. Our experiencewith eventually building a GMP-compliant manufacturing facility was that


80% of the cost was applied to the final 20% of the facility. This final 20%focused on relatively obscure details that had little impact on the overallrisk analysis for our small, early-stage trials. This recommendation of astreamlined QA/QC process should not be confused with a poorly definedmanufacturing process. It should be noted that even minor changes to themanufacturing process itself can result in the necessity to repeat Phase Istudies (or similarly perform a validation study to demonstrate equiva-lence between the processes). Therefore an important strategy in Phase Itrials is to therefore to make every attempt to finalize and document thedetails of the manufacturing process. Similarly, in vitro functional assaysshould be developed that can be used in lieu of clinical validation studiesto demonstrate equivalence in the likely event that process changes aremade as the technology evolves through Phase II and Phase III studies.

Some aspects of manufacturing controls or lot release criteria aredeemphasized in regions with emerging clinical programs. It is temptingto take advantage of these relaxed requirements in a globalized clinicaltrials strategy. However, since FDA or Western European reviewers willanalyze the enrollment and manufacturing safeguards in place for earlierstudies before accepting human data, it is essential to design the clinicalstudy as if it were being conducted in Western Europe or the US. The prin-cipal advantage to a globalized clinical trials approach is primarilyassociated with decreased study costs and a quicker process for receivingstudy approval, as discussed later in this chapter.

A final point worthy of mention for Phase I studies is the use ofClinical Research Organizations (CROs). A CRO is a third-party companythat provides a variety of services for clinical trials sponsors, includingclinical trials management, medical and safety monitoring, and data man-agement and analysis. The common perception among RegenerativeMedicine companies is that regulators require the use of CROs. Theseentities may seem omnipresent in all stages of the clinical trials process,but careful review of the guidance documents in most countries revealsthat while outside, independent monitoring is encouraged, it is notrequired (a DSMB is required for each trial, in part to provide independ-ent consideration for major trial decisions). While the CROs do provideconvenience and some guidance, the use of a CRO ultimately becomes animportant issue when cost is factored in. Even in relatively inexpensive


regions, CROs seem to have uniformly elevated their fees to Americanlevels. Most clinical cost estimates we have seen show that more than50% of the overall clinical costs for all studies are associated with regu-latory submissions, legal representation, and clinical monitoringperformed by CROs. Moreover, budget overruns associated with trialsoutside the US and Western Europe are almost always associated with theCRO and not the clinical study itself. While additional discussion on thistopic can be found later in the chapter, overall, we recommend careful costanalysis and vigilant contractual navigation when partnering with a CRO.

2.3 Phase II/III clinical testing

Transitioning to Phase II/III trials brings with it the responsibility of fullGMP and GTP manufacturing compliance and all of the associated costs.3

While the requirements for GMP manufacturing are discussed elsewherein this book, there are two key elements to Phase II/III clinical trials thatshould be carefully considered: endpoints that demonstrate geographicdistribution and cost-effectiveness.

2.3.1 Geographic distribution

There is clear pressure from investors to gain early access to the large USand EU markets, regions that are also the most likely to support the pricepremiums required from cell-based therapeutics. While this makes sensefor classic drug and device business models, the model is less relevant forcell-based therapies, where the overall sale volumes have been low. Eventhe most successful commercial products, such as Organogenesis’Apligraf ® or Advanced BioHealing’s Dermagraft®, sell to a very smallfraction of the wound healing population. There are no cell-based thera-pies that sell in sufficient volumes to claim a penetration rate of more thana few percentage points. Said another way, even these icons of industrialsuccess, which are both clinically efficacious and profitable, sell littlemore than a few tens of thousands of units per year. Moreover, currentmanufacturing capacities by most Regenerative Medicine companies can-not service significant penetration in these largest markets. Thus, one canmake the argument that a slow-growth business model, even in a smaller


market, could provide the basis for a successful outcome. Our own clini-cal trials strategy is to continue with Phase II/III clinical studies in EasternEurope and South America to lower the overall cost and expand to a sin-gle Western EU country to facilitate eventual commercialization inEurope. For our clinical indication, which addresses an extremely largepatient population, even modest penetration into a single country likeGermany would represent a major commercial success for a cell-basedtherapy and would likely exceed our current manufacturing capacity.Thus, the typical strategy of concurrent regulatory and commercial activ-ities in both the US and Western Europe should be carefully evaluated.

2.3.2 Cost-effectiveness

While clearly outside the scope of regulatory considerations, clinical tri-als design should also be mindful of the increasing pressure todemonstrate cost-effectiveness. While Medicare policy does not acknowl-edge the requirement for demonstrating cost-effectiveness, reimbursementfor cell-based therapeutics will absolutely require convincing data oncost-effectiveness. Treatments for life- or limb-threatening diseases ororphan diseases, where few or no clinical options exist (certain cancervaccines, for example), may be immune. In general, however, our indus-try will be required to demonstrate that the cost of our treatments cancompete with the standard of care or that higher costs are justified by anincreased life span and/or improved quality of life. This realization is crit-ical for clinical trials design, as a rigorous sponsor will tailor their trials toboth overcome short-term commercialization hurdles and demonstratecost-effectiveness in the long-term. For example, in the United States, theCenters for Medicare and Medicaid Services (CMS) is the institution thatselects healthcare products that are reimbursed by the government; thus,demonstrating cost-effectiveness to CMS is as significant a task asearning a commercialization permit from the FDA.

3. Regional Considerations for Clinical Trials

Historically, smaller medical device companies and RegenerativeMedicine companies have focused on early clinical trial approvals in the


US and Europe. These approvals are seen as key milestones, as they areassociated with access to larger markets and can lead to a subsequentincrease in valuation. While this is more relevant for Phase III trials, PhaseI and II trials, particularly for an SME in the field of RegenerativeMedicine, should be driven more by cost and speed considerations. Thekey valuation inflection point for fledgling Regenerative Medicine compa-nies with a limited funding horizon is first-in-man studies. As long as GoodClinical Practices are followed (suggesting the data would ultimately beadmissible as a part of later stage EU or US regulatory applications), itwould seem appropriate to avoid spending expensive early-stage capital bychoosing a region with lower costs and more rapid approvals. Indeed, theappeal of lower cost and faster study approval has driven a significantglobalization of clinical trials.5 That is, there has been a shift towards per-forming early-stage clinical trials in such countries such as Russia, Brazil,China, and India. Interestingly, the US provides an unparalleled entrepre-neurial and scientific environment for innovation, yet the US’s completeaversion to clinical trials risks and associated litigation problems are driv-ing access to cutting edge clinical treatments out of the country.

In deciding where clinical trials should be performed, a balance must bestruck between the overall cost, speed of study approval, access to an appro-priate patient population, quality of care, and the perceived quality of thedata produced (e.g. does clinical data from India impart the perception ofsufficient quality to a regulatory agency in Germany?). Trumping all ofthese questions, however, should be the selection of a Principal Investigator(PI) and a CRO. Ultimately, the value of the clinical study is directly relatedto the quality of the data collected, and it is the PI and the CRO that willcreate, collect, and manage this data. Table 1 summarizes by region thedifficulty ranking of the various regulatory hurdles discussed in this section.

3.1 United States

The United States market is a double-edged sword for RegenerativeMedicine. The US offers a single, centralized regulatory agency for bothclinical trials and commercialization, with a now well-established regula-tory framework for cell-based therapeutics. A very clear set of guidancedocuments, with “Guidance for Human Somatic Cell Therapy and Gene


432T. M

cAllister et al.

Table 1. Ranking by Region of Difficulty of Completing Various Regulatory Processes for Cell-Based Therapeutics.

Clinical Trial Relative Cost to Perceived Value Commercialization ReimbursementApproval Process Complete Clinical Trial of Clinical Data Approval Process Process

United States Difficult High High Difficult FairEuropean Union Moderate Moderate to high High Moderate Moderate to difficultSouth America Low to moderate Low to moderate Low to moderate Moderate Fair to difficultEast Asia Low to moderate Low to moderate Low to moderate Moderate Fair to difficult

Therapy” and “Sterile Drug Product Produced by Aseptic Processing” atits core, define this framework.6,7 In addition, the US offers both a highrate of reimbursement and a huge potential market for sales, with a popu-lation of roughly 300 million people and a large fraction of elderlyinhabitants.8 However, due to a highly litigious society and huge politicalpressures to avoid scandal, the FDA is shifting more and more to a policyof complete risk avoidance. Thus, the FDA’s approval processes are wellestablished and predictable, but they are inflexible and require large vol-umes of experimental data. While the FDA may attempt to evaluateproducts on a case-by-case basis, the truth is that the regulations arelargely fixed and the agency typically errs on the side of extreme caution.Indeed, the US system seems to encourage the rise of globalized clinicaltrials for first-in-man studies. Even in the medical device markets, com-panies now tend to seek commercialization in Europe first, and then later,if at all, in the US. As an example, there are approximately 15 repairdevices for abdominal aortic aneurysms (AAAs) CE-marked for sales inthe EU, but only about five available for use in the US.

3.2 European Union

The European regulatory framework is advantageous due to its uniquecombination of regional and centralized controls. Clinical trials areapproved and performed under regional authority, taking advantage ofcost/approval advantages in countries such as Poland. Commercializationto all EU member states is then controlled by a single centralized entity,the European Medicines Agency (EMA), following the framework set upby the Advanced Therapy Medicinal Products (ATMP) directive and gov-erned by expert committees, such as the Committee for Human MedicinalProducts.9 This myriad of regulations can be difficult to navigate. Forexample, in Germany, a company must apply for a clinical trials approvalthrough one state agency, approval of its GMP processes through another,and then a manufacturing permit through the EMA. In France, a companyproducing cell-based therapeutics must be registered as a national tissuebank, requiring the manufacturer to set up a facility in France. A flowchartsummarizing the regulatory process in Europe is presented in Fig. 1. Whilethe regional bureaucracies can create a complicated process, overall, the



EU CommunityLevel (Inter-state)

EU MemberState Level State Level, Non-EU

Legend

Clinical Trials

Sponsor’s Quality andPre-Clinical Data

Commercialization

Marketing authorization (EMA)

Advisory Bodies (Pre-Commercialization)

EMA - CAT Committee for Human-Based MedicinalProducts (CHMP)

Advisory Bodies (Pre-Clinical Trials)

Committee for AdvancedTherapies (CAT)

CT Regulatory BodyEU Member State 1

CT Regulatory BodyEU Member State 2

Clinical Trial, Non-EU Country 1

Clinical Trial, EUMember State 1

Clinical Trial, EUMember State 2

Fig. 1. Regulatory pathway for cell-based therapy clinical trials and marketing authori-zation in the European Union. Note that this pathway refers to advanced therapy medicinalproducts (ATMPs) placed on the market in Europe after Dec 31, 2008 (and all ATMPs onthe market in Europe after Dec 31, 2012). This pathway is not applicable for therapies des-ignated as medicinal devices. Also, this pathway does not include potential post-marketingPhase IV clinical trials that may be requested by the EMA.

access to initial clinical trials and then a large commercial market (over500 million) can be significantly more efficient and less costly than theUS pathway.

3.3 South America

Unlike Europe and US, there is no centralized regulatory body in SouthAmerica. The advantage to running studies in South America is that theseregulatory bodies are significantly more accepting of risk and less liti-gious than the US or even Western Europe. Thus, South America’svarious approval processes, especially for clinical trials, usually are com-pleted much faster than the US and EU. In our experience, it tookapproximately six months to obtain approval to perform a Phase I trial inArgentina, versus the two plus years it has taken in Germany and threeplus in the US. However, this lack of a rigid system can also be a detri-ment, as the various regulatory systems do not necessarily correspondwith one another. Thus, the pathway to approval for clinical trials or com-mercialization in Argentina will not be the same as Brazil or Ecuador.Finally, reimbursement rates in these countries is much lower than in theUS or Western Europe. As such, it has been our experience that it is farmore attractive to perform clinical trials in these regions, but to hold offon commercialization.

4. Use of a Clinical Research Organization

The primary driver for conducting clinical studies in emerging countrieslike Brazil, Russia, or India is cost. While many sponsors are hopeful thatthese countries represent significantly accelerated access to human use,the actual review processes are typically more thorough and take longerthan some may think. When one factors in the additional work of repli-cating regulatory efforts in Western Europe or the US, the overall timebenefit is marginal. So, while there may be some near term advantages inrapid initial access to human trials (Phase I/II), the long-term benefitsrelate to cost savings. Somewhat surprisingly, the costs of running a clin-ical study in South America or Eastern Europe can be dominated by thefees paid to a CRO. Careful selection of the CRO and the responsibilities


they will be given is an excellent way to significantly reduce the overallcosts associated with the regulatory approval process and the clinicalstudy itself. There are two basic drivers that force the use of a CRO. Thefirst is a general requirement in most countries for legal representation tosubmit regulatory documents, import medical products, or conduct clini-cal studies. The second is a general requirement to have a clinical monitorto check source data. While established CROs often perform or managethese roles, there are a variety of other options, detailed below.

Given the uniqueness and complexity of running RegenerativeMedicine studies, in our experience, most CROs are ill-equipped to han-dle trials outside the scope of the classic drug or device. Even those thatpromote themselves as experts in Regenerative Medicine often have littlereal expertise in the regulatory, clinical, or logistical requirements of run-ning a study that is based upon the use of a living product. While thisknowledge gap is closing as more and more clinical studies are performedin this field, it is safe to say that the sponsor should expect to play a directand engaged role in the day-to-day management of the clinical trial.Indeed, we have hired our own local employees to help accelerate andmanage our regulatory and clinical activities performed outside the US. Inmost instances, these same local employees, under the tutelage and man-agement of the sponsor’s clinical trials manager, can perform all of theroles normally performed by a CRO at a fraction of the cost. It is impor-tant to note that this includes data monitoring. There is generally norequirement to use a CRO or even an independent monitor to performthese tasks. However, the risk of inadvertent mistakes that can be perpet-uated when the sponsor is directly responsible for both study design anddata monitoring should be evaluated and balanced against the cost savingsassociated with this strategy. Similarly, the potential for a regulator’s nega-tive perception of an “internal” biased monitor must be evaluated.

CROs can also provide localized legal representation. As an alterna-tive to large global CROs, there is a growing number of smallercontractors that perform this role. We have used entities such as MARESLtd. in Western Europe and Access Medical Research in South Americaand Poland with success. This provides a low-cost alternative to a full-time local or regional CRO, but puts significantly more responsibility andworkload on the sponsor’s clinical and regulatory teams. On one hand,


this may result in additional direct costs and delay for the sponsor; on theother hand, it will reduce indirect costs paid to a CRO or similar thirdparty company, allow the Sponsor more control over timeline andexpenses, and develop clinical expertise in-house that can be used infuture trials. Given the relative inexperience of CROs within RegenerativeMedicine, we have found this to be an appropriate trade-off.

5. Universal Regulatory Considerations for Cell-BasedTherapeutics

Tissue Engineering and cell-based therapies have been hailed as the thirdpillar of future medicine, alongside devices and drugs. While the field asa whole has tremendous support in academia and has reached importantmilestones over the last decade, translation to widespread clinical use willbe crippled by the current regulatory framework. It is incumbent upon usall to collectively lobby for rational regulations that are more relevant toRegenerative Medicine and result in an appropriate risk-benefit profile forthe patient. The regulatory framework in almost every country with estab-lished guidance documents on cell-based therapeutics has been derivedfrom aseptic drug manufacturing requirements. The fundamental prob-lems with this approach are the dramatic disparities in cost per “dose” andlot sizes between pharmaceuticals and cell-based therapeutics. With lotsizes in the hundreds of thousands and cost per dose of a few cents, thedrug industry can easily absorb the QA/QC costs associated with exten-sive manufacturing controls and lot release testing. In cell-basedtherapeutics, however, even most allogeneic products have lot sizes of afew thousand at most, and costs per dose that are orders of magnitudehigher than pharmaceuticals. While companies like Organogenesis andAdvanced Biohealing have brought manufacturing costs down to thepoint of profitability (if one ignores the hundreds of millions of dollars inR&D expenses forgiven by bankruptcy), many companies are burdenedwith more complex products, smaller lot sizes, difficulties in cell sourc-ing, etc. While it is not in the FDA’s mandate to establish a regulatorysystem that promotes profitability for industry, it is their responsibility toenable patients to have access to new therapies. The question, of course,


is what level of risk are we willing to tolerate in order to bring QA/QCcosts down?

There is a valid argument to be made that the current environment ofcomplete risk aversion does a disservice to both patients and industry. Forexample, an immuno-compromised, terminally-ill cancer patient wouldlikely accept the small risk of death from a contamination in order to haveaccess to the potentially life-saving benefit of the treatment. We arerequired to test for sterility, mycoplasma presence, and endotoxin pres-ence for our tissue-engineered grafts at three separate time points in themanufacturing process. In nine years of production, about half of whichwas performed under non-GMP certified conditions and the remainderunder GMP conditions, we have had one test come back as “inconclu-sive.” Thus, it would seem that our process controls have more thanadequately controlled the risk of contamination and that GMP certifica-tion did not significantly improve upon this. Similarly, many hospitallaboratories have been producing cultured epithelium for burn patientsunder non-GMP conditions for years without issue.

Ironically, a variety of cell-based products manufactured and/orimplanted with reduced QA/QC requirements are considered the standardof care today. For instance, organ transplants are typically performed “asis,” with limited QC evaluation. In addition, “in vitro surgeries” arecommon, in which the living tissue of a patient is manipulated (often withsurgical methods and instruments) and transformed into a construct that isimplanted. Also, as previously stated, hospitals currently produce culturedepithelium for burn victims. Obviously, these products are not subjectedto the same manufacturing controls as commercial cell- and tissue-basedproducts. Furthermore, all implantable products are implanted in an oper-ating room environment that does not meet the cleanliness standardsrequired for aseptic manufacturing. We certainly could apply the sameQA/QC standards required for cell-based therapies to the products listedabove to ensure “zero risk” of contamination and error. The result wouldbe that the already exorbitant cost of interventions would continue toswell and further decrease the portion of patients who could afford theprocedure. The truth is that this “zero risk” attitude is already a contribut-ing factor to the steep rise of healthcare costs in the US. Thus, if we wantto see the day when complex tissue-engineered organs can be produced


and implanted at reasonable cost, regulatory agencies will have to acceptsome safety trade-offs to reach an acceptable balance of cost and risk.

Another fundamental problem is that the regulatory agencies demandthat the sponsor perform every test that has cumulatively encumberedother applicants. The responsibility then lies with the sponsor to whittledown this all-inclusive list that gives an appropriate level or risk-benefitfor the particular patient population. While the regulators have the benefitof knowing all the data from prior applications, industry has not typicallyshared regulatory or manufacturing data so that we can turn the tables andargue for the least common denominator. In other words, knowing whatlot release criteria, for example, are required to demonstrate safety andefficacy for a similar product might have dramatic implications in a suc-cessful argument for a similarly streamlined lot release strategy. Despitethe secrecy that has historically shrouded the development of medicaldevices and pharmaceuticals, we believe it is critical to the survival of ourindustry to begin an active data sharing program with respect to regula-tory QA/QC requirements. Whenever possible, the FDA and otheragencies should provide the data on which they base their regulatory andguidance policies and lay out their decision-making processes to thepublic.

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INDEX

Index Terms Links

2D culture 216

351 product 399 401

A

acceptance criteria 410 413 417

adipose derived stem cell

(ASC) 51 170 217

adipose tissue 216

brown 216

white 216

adipose-derived mesenchymal

stromal cell 259

Advance Therapy Medicinal Products

(ATMP) 399 433 434

advantage 217

adventitious agent 409 411

aggrecan 214 216 218 234

241 242

alcian bluepicrosirius staining 220

alginate 144 146 147 214

alkaline phosphatase 221

Index Terms Links

alternate microbiological test 415

Alzheimer’s disease 322

amyotrophic lateral sclerosis 323

angiogenesis 135 136 138 139

141 144 148 217

animal model 214

anterior cruciate ligament

(ACL) 221

ACL derived cell 221

anti-angiogenic factor 217

anti-inflammatory drug 211

array 356 361 362 365

367

biomateria/polymer 357

cellular 354 355

combinatorial protein 355 356

microfluidic 361 362

microwell 358 361

articular cartilage 181 189 197 199

201 211 218 219

221 222

ascorbic acid 214

autologous chondrocyte implantation

(ACI) 212

autologous chondrocyte

stimulation 185

autologous graft 212

Index Terms Links

autologous somatic cell

therapy 398 418

axonal migration 327

B

back pain 232 244

basic fibroblast growth factor

(bFGF) 139

batch records 407 412

bilaminar cell pellet (BCP) 241

bioactive

molecule 213

regulator 212 214

biocompatible 212

biodegradable 212

biologic license application

(BLA) 399 401 417 418

blood 31 37

blood vessel 72 74 115 117

119 120 123 127

bone 73 74 77 81

82 84 251 259

260 262 269

formation 214

regeneration 277 279 284 286

skin 54 56 59 60

Index Terms Links

bone marrow (BM) 32 37 38 50

185 186 188 192

193 201

aspirate 213

BM derived mononuclear

cell 126 127

stimulation 185 186 188

stromal cell 216 217

bone marrow mesenchymal stem cell

(BMMSC) 213 220 221

bone marrow stromal cell

(BMSC) 301 303 306 307

bone morphogenic protein (BMP) 85 87 88 212

215 216 218

BMP-2 215 218

BMP-6 216

bovine spongiform encephalopathy

(BSE) 410

bulk cell 410

C

cAMP 305 310

canine model 214

cartilage 51 52 54 56

60 61 81 181

196

damage 211

Index Terms Links

cartilage (Cont.)

defect 212 214 219

regeneration 212 219

cartilage oligomeric matrix protein

(COMP) 214 218

cartilage-like tissue 214

cartilaginous phenotype 214

cell

and tissue sourcing 408

banking 411

carrier 237

fate 13 23

pellet 213

source 3 8 212 213

219 220

therapy 211 222 333

cell based application 1 7

cell based therapeutics 423 429 437

cell based therapy 212 213 397 398

415 418

cellular

allograft 280 281 284 291

292

cardiomyoplasty 96 102 104

microarchitecture 376

Centers for Medicare and Medicaid

Services (CMS) 430

ceramic 85 86

Index Terms Links

certificate

of analysis 405

of compliance 405

characterization 410 411

chitosan 190 200

chondrocyte 182 195 198

chondrogenesis 193 199 213 214

216

chondrogenic 181 189 193 194

198 212 216

differentiation 214 218 219

phenotype 216

chondroinduction 216

chondroinductive ability 216

chondroitin sulfate 182 196

Clinical Research Organization

(CRO) 425 427 431 435

clinical trial 424 425 427

design 425 430

phase I 425 431 435

phase II 425 426 428

phase III 426 428 431

clongeniclineage-specific gene

marking 222

cluster of differentiation (CD) 213

co-culture 240 241

Index Terms Links

collagen 73 78 182 187

191

gel 214

type II 214 216 219 234

235 241

type X 221

collagenase digestion 216

collagen-mimetic peptide 195

colony forming

efficiency 221

fibroblast frequency 220

Committee for Human Medicinal

Products 433

composite 77 85 87

congenital pathology 211

consistency 401 405

continuation criteria 426

contusion lesion 302 303 305 307

308 311

corrective and preventive action

(CAPA) 402

cost-effectiveness 429 430

critical

manufacturing step 406

safety issue 407

cross contamination 402 408 409

current good manufacturing practice

(cGMP) 399 401 408 409

Index Terms Links

current good tissue practice

(cGTP) 399 408

D

Data Safety Monitoring Board

(DSMB) 425 426 428

deep zone 221

degenerative joint disorder 211

delivery vehicle 71 78 80 81

88

demyelination 299 300

dental implant 278 282 284 290

dermal substitute 79 80

dexamethasone 214

dextran 144 147

disintegrin and metalloproteinase

with thrombosondin motif 182

distraction osteogenesis 253 255

drug development 366 367

E

ectoderm 220

electrospun 198 199

embryoid body (EB) 137 138 144 145

147 148 360

Index Terms Links

embryonic stem cell (ESC) 13 136 137 141

145 212 298 306

310 312 333 349

353 356 360

endoderm 220

endothelial cell (EC) 136 139 143 147

148 151

endpoint 425 426 429

environmental control and

monitoring 402 404

epidermal stem cell 166

European Medicines Agency

(EMA) 433 434

ex vivo manipulation 398 399 409 418

expandable 212

extracellular matrix (ECM) 52 98 100 102

137 138 141

protein 214 221

F

failure criteria 426

Federal Food, Drug, and Cosmetic

Act 399

fibrin 74 81 144 149

188 190 199

glue 216

Index Terms Links

fibroblast 214 217 219 303

308

fibroblast growth factor-2 (FGF-2) 214 218 221

fibrocartilage 211 214

hyaline cartilage 211 215

fibrous connective tissue 214 219

final release 408 413 414 417

Food and Drug Administration

(FDA) 398 403 411 414

418 423

full thickness femoral condyle

cartilage defect 219

funding 424 431

G

gene expression 217

gene therapy 233 236 237

genetically modified stem cell 364 365

glial restricted precursor cell

(GRP) 306 307 309 310

glial scar 299 303 307

glucosamine 189 190 200 201

glycosaminoglycan (GAG) 182 189 214

good manufacturing practice

(GMP) 427 429 433 438

good tissue practice (GTP) 427

Index Terms Links

growth factor 233 236 244 303

306 311

H

haematopoietic ossicle 213

Hayflick limit 214

heart 52 54

congenital defect 116

disease 115

hematopoietic stem cell

(HSC) 31 36

expansion 37

high throughput screening 348 364

human bone marrow mesenchymal

stem cells (hBMMSC) 213 218 220 221

human dental pulp stem cell

(HDPSC) 219

human umbilical cord 220

human umbilical cord perivascular

cell (HUCPVC) 220

Huntington’s disease 323 332

hyaluronan 189 214

hyaluronic acid 144 148 182 185

189 196 217

hydrogel 73 74 83 84

87 143 146 151

193

Index Terms Links

hydrogel (Cont.)

cell-laden 377 386 389

hypertrophy 214

hypoxic

condition 233

environment 217

I

identity 401 409 410 412

in vitro 212 216 219 221

222

expansion 221

model 326

surgery 428

in vivo bioreactor 218

induced pluripotent stem cell

(IPSC) 14 16 140 172

260 298 336 349

366

inflammation 298 303 307

injectable 72 73 82 83

in process

control 408 411 412

testing 411

insulin growth factor-I (IGF-I) 218

interleukin 184

Index Terms Links

International Organization for

Standardization (ISO) 404

International Society for

Cytotherapy 213

intervertebral disc degeneration 232

invasive procedure 212

investigational new drug

(IND) 399 401

J

joint fusion 211

joint replacement 211

K

keratinocyte 78

L

labeling 401 408 417

lactic acid 233 234

ligament fibroblast 221

living skin equivalent 80

lymphocyte 216 218

Index Terms Links

M

macrophage 300 303 304 312

major histocompatibility complex

(MHC) 212 216 218

manufacturing 427 433 437

process 398 399 405 416

418

master bank 410 411

matrix metalloproteinase 182 184 195

matrix remodeling 233

mechanical factor 212

medium fill simulation 406

mesenchymal progenitor cell

population (MPC) 220

mesenchymal stem cell

(MSC) 49 53 58 62

71 139 140 150

152 170 186 188

192 193 212 213

216

adult 238

source 222

mesoderm 220

mesodermal lineage 217

metabolic activity 211

microarray 217

microbial safety 410 411

Index Terms Links

microenvironment 349 355 361 363

365 366

microglial activation 299

micromass 213 217

middle zone 221

milk teeth 219

mosaicplasty 185 186

multiple sclerosis 324

multipotent 212 217 218 222

myocardial infarction (MI) 95

N

National Institutes of Health

(NIH) 424

neovascularization 139 140

neuron 321 325 335

neuronal restricted precursor cell

(NRP) 306 307

neuroprotection 300 302 303 305

NF-κB 184

inhibitor of κB 184

niche 349 351 355

Notch-1 221 222

nucleus

pulposus 231 233 234

regeneration 234

Index Terms Links

O

olfactory ensheathing glial cell 301 306

oligodendrocyte progenitor cell

(OPC) 309 310 311

osmotic pressure 234

osteoarthritis (OA) 181 183 185 201

211 221 222

osteochondral autograft transfer

system 185

osteoprogenitor cell 252 253 258 261

262 267

P

Parkinson’s disease 322 331

partial thickness articular cartilage

defect 219

patello-femoral joint 214

periosteal cell 218

periosteum 213 214 217

periosteum derived stem/progenitor

cell (PDSC) 217

peripheral vascular disease 115

permanent molar teeth 219

plasticity 212 214 216

platelet rich plasma 81 85

Index Terms Links

platelet-derived growth factor

(PDGF) 138

plethora 213

pluripotency 13 348 351 356

362

pluripotent 212

stem cell 34 36 37 40

41

poly(ethylene glycol) 193 194

poly(glycolic-co-sebacate) acrylate

(PGSA) 145 149 150

poly(lactic-co-[glycolic acid])

(PLGA) 145 149 150 214

poly(lactide-co-glycolide) 75

poly(l-lactic acid) (PLLA) 145 149 150

polycaprolactone 151

polyester 191 192 198

polylactic acid (PLA) 214

polymer 73 82

potency 412

precursor cell

neural 333 334

skin derived 336

principle investigator (PI) 431

process validation (prospective,

concurrent, retrospective) 404 406

Index Terms Links

progenitor cell 217 218 220 221

298 305

endothelial 125 126

proteoglycan 182 183 189 197

Public Health Services (PHS)

Act 399

pulp tissue 219

purity 401 410 412

Q

quality assurance (QA) 402 403 406 407

412 427 428 437

quality by design 407

quality control (QC) 397 398 402 403

407 408 411 417

418 427 428 437

quality system 401 403 407

R

rabbit 214 217 219

model 217

raw material 408

regeneration 298 306

regulation 397 401 408 409

413

Index Terms Links

regulatory

challenge 423 425 426

pathway 434

reprogramming 13

risk 423 424 426 431

433 435

assessment 399 400 411 412

risk control management 407

RNA interference 363

S

scaffold 212 216 222 252

254 260 263 266

biomaterial 96 102

cell-3D construct 213

fibrous 197 199

porous 212

Schwann cell 301 305 310

secondary injury 297

self-renewal 212 348 351 357

364

serum free culture medium 214

shipping 405 407 409 414

417

single cell lineage 217

sinus augmentation 284 286 292

skeletal reconstruction 253 266

Index Terms Links

skin 78

SKP 305

small and medium-sized enterprise

(SME) 427 431

smooth muscle cell (SMC) 119 121 123 124

126 136 138 141

Sox9 214 218

spinal cord injury (SCI) 297 301 305 306

308 309 312

stability 401 411 416 417

standard operating procedure

(SOP) 402 403 407 412

stem cell 2 211 216 218

277 279 289 290

297 298 304 312

adult 211 348 349 351

354 357 367

differentiation 35 36 39 49

58 181 187 189

192 196 234 237

239 244 347 351

360 364 375 381

382 391

neural 321 329

STRO-1 219 221 222

superficial zone 221

surface marker 212 213 217

surgical intervention 211

Index Terms Links

synovial

chromatosis 218

fluid cavity 218

membrane 218

nodule 218

synovium 214 218 219

synovium derived mesenchymal stem

cell (SMSC) 218 219

T

T-cell 216 218

The United States Pharmacopeia

(USP) 404 405

tissue 49 54 55

tissue based therapy 397 398 415 418

tissue engineering 1 5 8

bone 219

cartilage 211 222

functional articular

cartilage 222

functional 212

microscale 391

sheet based 123

skeletal 251 257 263 266

268 269

vascular 116 117 120 125

Index Terms Links

tissue stiffness (tissue

mechanics) 100

traceability 407 408

transcription factor 214

trans-differentiation 212

transforming growth factor β-3 (TGF

β-3) 212 214 216 218

220 221

trauma induced injury 211

traumatised muscle tissue 220

trophic

effect 213

factor 300 303 305 307

tumor necrosis factor 184

U

umbilical cord blood (UCB) 33 34 36 39

UCB derived stem cell 220

unrestricted somatic stem cell

(USSC) 220

V

vascular 135

vascular cell adhesion molecule-1

(VCAM-1) 221 222

vascular endothelial growth factor

(VEGF) 138 143 147

Index Terms Links

viability 401 409 413 414

416

W

Wharton’s Jelly 220

working bank 410

wound

healing 80 81 159 163

164 169 171

repair 159 160 162 165

170 172

Y

yucatan pig 219

Z

zero risk 423 438

501-Stem Cell and Tissue Engineering-Song Li Song Li Nicolas L'Heureux Jennifer Elisseeff-9814317

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Transcript of 501-Stem Cell and Tissue Engineering-Song Li Song Li Nicolas L'Heureux Jennifer Elisseeff-9814317