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issue engineering in a classical sense implies theuse of organ-specific cells for seeding a scaffoldex vivo. The cell-based (but not necessarily stem

cell-based) nature of tissue engineering servedto d efine it, an d to distinguish it from guided

tissue regeneration in which a scaffold is designed toencou rage regenerat ion solely by cells residing at the site

of its transplantation. Irrespective of actual clinicalfeasibility, the experimental design and potential use of

tissue en gineering app roaches are endless, and range fromthe p reclinical gener ation of cardiac valve sub stitutes, to exvivo construction of nasal cartilages, to organ substitutes

such as the u rinary bladder (reviewed in r ef. 1).The list of tissues with the potential to be engineered is

growing steadily. This is du e in large part to recent p rogressin stem cell biology and recognition of the u nique biological

properties of stem cells, although not all stem cell-based

therapies (for example, some th at use n eural stem cells, assummarized by Temple, pages 112117) involve the con-

struction of tissue either ex vivo or in vivo. Non etheless, pilotstud ies in a variety of system s highlight great pr ospects for

future stem cell-based tissue engineering (Table 1). But

translation in to clinical reality has been reached so far in onlya few areas, and notably only in tho se areas in which there has

been lon g-stand ing insight into stem cell biology.Tissues that can now be engineered using stem cells

comprise a diverse range from epithelial surfaces (skin,cornea an d m ucosal mem bran es) to skeletal tissues. These

systems are inh erently different in their r ate of self-renewal

and physical structure, two impo rtant determinants of anyattem pt to reconstru ct tissues using stem cells (as discussed

by Spradling and colleagues on pages 98104). Apprecia-tion o f the inherent diversities of organ systems and cognate

stem cells is essential for developm ent o f adeq uat e str ategies

of inter vention in specific areas.Here we exam ine two ap plications o f stem cells to tissue

engineering. The first case is the regeneration of skin, whichinvolves the structural formation of two-dimensional

sheets. The second, m ore com plex examp le is the form ationof bone, which involves the reconstruction of three-

dim ensional shapes and inter nal architectures.

Engineering skin and other surfacesPermanent restoration of tissues characterized by highand co nt inu ou s self-renewal specifically requ ires extensive-

ly self-renewing stem cells. Haematopoiesis provides the

best paradigm of a stem cell-dependent, steadily self-

renewing system (see review by Weissman and colleagues,pages 105111). Among the hierarchy of haematopoietic

progenitors en dowed with varying capacities to self-renew,

it is only the small comp artm ent of long-term self-renewingstem cells that is necessary for permanent and complete

restoration of haematopoiesis after lethal irradiation 2.Likewise, progenitor cells represented in a keratinocyte

culture are also ran ked in a hierarchy, and u nd er clonogenicconditions form different types of colonies (holoclones,

m eroclones and p araclones) that vary significantly in theirability to self-renew and to generate a differentiated

epiderm is. On ly holoclones are the produ ct of a true stem

cell, as defined by their exceptional self-replication ability(>140 doublings), which is not ascribed to either

m eroclones (a popu lation of tran sient am plifying cells) orparaclones (a population of senescent or differentiating

progenitors) 3. In principle, a small but pure population of

holoclone-generating cells would b e all that is requ ired forgenerating epiderm al grafts. The recent iden tification of a

keratinocyte stem cell marker may provide a new tool forthis approach with respect to epidermis4.

These theoretical prediction s correlate with the clinical

outcom es of skin grafting. Skin auto grafts are produ ced bycultur ing keratinocytes to generate an epiderm al sheet, and

then transplanting this sheet along with a suitabledermis-like substrate (Fig. 1a) 5. But the success of these

procedu res has been variable, with graft failure resulting inmany cases after a promising initial engraftment.

Experimental conditions can cause depletion of the holo-

clone-generating compartment in cell cultures, similar tothe par tial depletion of the epidermal stem cell com part-

men t that can occur in vivo (for example, during ageing).Retention or depletion o f true stem cells in a keratin ocyte

population in vitro is significantly affected b y the n atur e of

the carrier or substrate used for culturing and grafting, andcould account for instances of graft failure in clinical

practice6. The long-term success of a skin graft thusdepends on appropr iate replenishm ent of stem cells in th e

graft. The specific techno logical challenge in r estoration of

epithelial surfaces in turn consists of the definition ofculture conditions, and of carriers that are designed

specifically to m aintain an ad equate stem cell com par tm entin the en gineered graft. Depend ence of epithelial surfaces

on relevant stem cell compartments is further highlightedby the successful recon struction of dam aged corneas using

stem cells derived from the limbus in conjunction with

an amniotic mem brane7,8.

Stem cells in tissue engineeringPaolo Bianco* & Pamela Gehron Robey

*D ipa rt im ento d i M edicin a Sp erim enta le e Patologia, Uni versi ta La S apien za, V iale Regin a Elen a 324, Rom a 00161 , Ita ly

(e-m ail: [email protected])

Craniofacial and Skeletal Diseases Branch, N ational Institu te of Den tal and Cran iofacial Research, N ationa l Institutes of Health, 30 Conv ent

Driv e M SC 4 320 , Bet hesd a, M ary lan d, U SA (e-m ail: probey @dir.n idcr.n ih .gov)

The concept of producing spare parts of the body for replacement of damaged or lost organs lies at thecore of the varied biotechnological practices referred to generally as tissue engineering. Use of postnatalstem cells has the potential to significantly alter the perspective of tissue engineering. Successful long-termrestoration of continuously self-renewing tissues such as skin, for example, depends on the use ofextensively self-renewing stem cells. The identification and isolation of stem cells from a number of tissuesprovides appropriate targets for prospective gene therapies.

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Engineering the corr ect shape and physical structure o f a graft isrelatively simple in epiderm is and other surface epithelial tissues, and

indeed can be achieved ex vivo. This reflects the essentially two-

dim ensional organization of skin an d oth er epiderm al surfaces, andis in shar p con tra st with system s such as the skeleton, where effective

engineering involves the reconstruction of complex shapes andarchitectures. These directly affect proper function, are normally

generated over a long per iod of time, and can not be achieved ex vivo.

Engineering the skeletonSkeletal stem cells (SSCs; also kn own as bon e-m arrow strom al stemcells, or mesenchymal stem cells) are found in the subset of

clonogenic adherent m arrow-d erived cells, and are able to un dergoextensive replication in culture. Upon ectopic in vivo transplantation

in m odel systems, all of the main tissues foun d in bo ne as an or gan

(bone, cartilage, adipocytes and haematopoiesis-supportingstroma) are formed (Figs 1b and 2a,f) (ref. 9, and reviewed in refs

10,11). SSCs obtained in cu lture mu st be com bined with app ropr iatecarriers before transplantation. These provide a three-dimensional

scaffold in which a vascular bed can be established, and tran splanted

progenitor cells can differentiate and form a bone/marrow organ.Many suitable mater ials are being generated and perfected, including

synthetic hydroxyapatite/tricalcium phosphates (Figs 1b and 2a),

and polyglycolic and polylactic acids12,13

. Most are effective insupporting bone regeneration, either alone or in conjunction with

growth factors such as bone morphogenetic proteins. However, sofar, little attention has been devoted to their compatibility with

long-term maintenance of stem cell properties, or to the ultimatefate of the bonebiomaterial composite being generated at the site

of t ransplantation.In th e sim plest procedure for bon e reconstruction , SSCs isolated

in culture from a limited volume of bone marrow are expanded ex

vivo, loaded onto an appropriate carrier, and locally transplanted(Fig. 1b). This pro cedu re results in th e effective repa ir of critical size

defects, which are larger than what can be repaired by resident cells,either spontaneously or as guided by an osteoconductive device

(Figs 1b an d 2b,c)1417. Convincing pr eclinical stud ies adopting th is

appro ach have led to preliminar y observational stud ies in hu m ans18

,and clinical trials are about to start in a number of centres. An

alternative approach h as led to the generation of fully vascularizedbone flaps of the desired shape in vivo, prior to their use for local

tran splantation ( Fig. 2d, e). In th ese experimen ts, SSCs loaded into

appropriate carriers and transplanted into a non-skeletal sitesurrounding an artery and vein; here they generate a vascularized

segment of bone, the size and shape of which are dictated by thecarrier geometr y19.

All of these applications can be categorized as surgical interven-tion thro ugh t ransplant ation of a biological prosthetic device

for th e cure of ph ysical defects in an o therwise nor m al skeleton. But

the existence of SSCs raises additional hopes and challenges fortreatment of more complex and generalized diseases, such as

crippling genetic diseases. Here, the concept of using SSCs toreplace misfunctioning bone cells with normal ones meets

another set of important obstacles, which further illustrate the

diversity of the skeleton an d its stem cells com pared with oth er stemcell-based systems. One is the route of systemic delivery of the

nu m ber of progenitors n eeded to pro du ce a biological effect. Som e

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NATURE | VOL 414 | 1 NOVEMBER 2001 | www.nature.com 11 9

(Seed with

dermal

fibroblasts)

Single-cell

suspension

(p63 selection?)

a Epidermis

b

Scaffold

Dermal substituteEx vivoexpansion conditions

to maintain 'holoclones'

(stem-cell population)

Full-thickness skin wounds

Potential

for genetic

engineering

Ex vivoexpansion

Attachment to hydroxyapatite/tricalcium phosphate particles

In vivotransplantation

into segmental defect

Skeletal

stem cell

Epidermal

stem cell

Bone

marrow

Bone matrix

Figure 1 Regeneration of two-dimensional (skin) and three-dimensional (bone)

tissues using stem cells. a , Skin autografts are produced by culturing k eratinocytes

(which may be sorted for p63, the recently described, epidermal stem cell marker)under appropriate conditions not on ly to generate an epidermal sheet, but also to

maintain the stem cell population (holoclones). The epidermal sheet is then placed on

top of a dermal substitute comprising devitalized dermis or bioengineered dermal

substitutes seeded with dermal fibroblasts. Such two-dimensional composites,

generated ex vivo, completely regenerate full-thickness wounds. b, Bone

regeneration requires ex vivoexpansion of marrow-derived skeletal stem cells and

their attachment to three-dimensional scaffolds, such as particles of a

hydroxyapatite/tricalcium phosphate ceramic. This composite can be transplanted

into segmental defects and will subsequently regenerate an appropriate

three-dimensional structure in vivo.

Table 1 Current approaches to tissue engineering

Stem cell-based tissue engineering Non stem cell-based tissue engineering1

Blood vessels23,29 Liver31 Bladder MeniscusBone1417* Pancreas 41 Cartilage (ear, nose Oral mucosaCartilage39 Nervous tissue42* and joints)* Salivary glandCornea7,8* Skeletal muscle24,27 Heart valves TracheaDentin40 Skin5,6* Intestine UreterHeart muscle28,29 Kidney Urethra

*In clinical trials or clinical observational studies.



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experimental evidence is available for limited engraftment of

osteogenic progenitors that are infused (as is don e with haem atopoi-

etic stem cells or HSCs) through the systemic circulation 20. Butevidence for a biologically significant effect of the system ic infusion

of SSCs is not available, and cu re of systemic skeletal diseases usingapproaches borrowed simplistically from haematology is not in

sight. D efinitive preclinical work in anim al mo dels, com pliant with

strict criteria for assessment of engraftment, must be conductedbefore these types of procedures reach clinical trials11, although

preliminar y clinical stud ies are under way21.Another p ertinen t point is that renewal in a po stnatal skeleton is

markedly slower than in skin or blood (the whole skeleton being

comp letely replaced only about three tim es in a lifetime, whereas theskin is replaced once a month). Consequently, we would expect

replacem ent o f skeletal tissue with in fused SSCs to occur o ver longertimescales compared to rapidly self-renewing tissues, even if issues

related to efficient cell delivery and systemic engraftment wereresolved. Altern ative mean s for local int ervent ion in genet ic diseases

are conceivable. For examp le, engineered SSCs could b e tran splanted

locally at the site of a clinical event ( such as a fractur e or deform ity) in

genetic diseases of osteogenic cells, such as osteogenesis imp erfecta

(caused by mu tations in genes encoding collagen type I) o r fibrous

dysplasia of bone (caused by activating missense m utation s of theGNAS1 gene). But ho pes for a system ic cure of genetic disorders of

the skeleton will rely on m ore im aginative avenues. In som e casesthese might include in u tero tran splantation, whose feasibility with

SSCs has been shown in preliminar y stud ies22. As with the p reviou sly

described techniques, caveats related to efficiency of engraftmentand biological effects remain to b e addr essed.

Heterotopic stem cells for tissue engineeringThe m arrow is at centre stage for future techno logical developmen ts

in tissue engineering, not o nly as the only organ in which at least twotypes of stem cells (HSCs and SSCs) reside, but also as the organ in

which progenitors for a number of distant tissues can be found.Recent studies indicate that the traditional wall separating the

haem atopoietic and m esoderm al tissue systems and lineages is beingdemolished. Cells capable of regenerating blood vessels, skeletal

mu scle and cardiac mu scle are found in the m arrow2325. The unex-

pected potential for myogenesis and cardiomyogenesis has been

12 0 NATURE | VOL 414 | 1 NOVEMBER 2001 | www.nature.com

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Figure2 Bone regeneration by

marrow-derived skeletal stem

cells (SSCs). a, SSCs

transplanted in conjunction with

appropriate vehicles (v) such as

hydroxyapatite/tricalcium

phosphate generate an network

of bone (b) and stroma composed

of adipocytes and reticular cellsthat support haem atopoiesis (hp).

b, Critical size defects that do not

heal spontaneously, such as

those as in the calvaria, can be

completely regenerated by

SSCvehicle constructs (c); ft,

fibrous tissue. d,e, In addition, by

wrapping SSCvehicle constructs

around an artery and vein (bv) in

conjunction with Gore-Tex (g) to

prevent collateral vascularization

(dotted box in d), fully

vascularized bone flaps of desired

shape can be constructed.f, Under appropriate conditions,

SSCs can also form c artilage (c)

as demonstrated by alcian blue

staining and type II collagen

immunohistochemistry (not

shown).



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ascribed to both H SCs and strom al (mesenchymal) stem cells in th emarrow2629. SSCs can p erhaps give rise to n euron s or glia30, pu rified

m ouse H SCs can regenerate liver cells31, and cells able to r egenerate

bon e are also foun d in blood 32. Perhaps what we have referred to asthe H SC is in itself much m ore a tr ue m ultipotent stem cell with

transgermal potentials2,31, nor m ally devoted to haem atopoiesis as aresult o f local cues.

Marrow cells offer the advantage of being easily harvested and

cultured from an adult organism , and th e HSC can be isolated and

purified ex vivo. Althou gh m ost of these applications are a long wayfrom any immediate clinical use, these studies raise basic scientificissues that are far from being settled or rationalized, similar, for

example, to the issue of plasticity of postnatal stem cells 10,11. Theyprovide insight on how different the scene of tissue engineering could

be in the relatively near futur e. Beyond theoretical considerations,

and p endin g fur ther experim ental proof where needed, the existenceof heterotopic and pleiotropic stem cells in the bone marrow has

obvious practical implications for th e future of stem cell therap y thatshould no t be missed.

Reconstruction versus correctionDelivery of factors that wou ld stimu late stem cells in situ to initiate a

process leading to regeneration rath er than scar form ation has long

been pursued. But success has been limited owing to problems ofdosage, lack of full activity of recombinant factors, and inability to

sustain a factors presence for an ap pro pr iate length of time. To over-com e these pro blems, gene-activated m atrices are being investigated

that com pr ise plasmid s cod ing for factors in a variety of delivery vehi-cles. These would t ran sduce cells in vivo to bring about appropriate

regeneration, and trials aimed at osteoporotic fractures are beingplanned33. In th ese attem pts, the trad itional principles governing th e

use of carriers merge imaginatively with the frontier of genetic

engineering. No dou bt, if reconstru ction was the initial front ier oftissue engineering, genetic correction is the next. As stem cells are

the critical ingredient in tissue regenerat ion, they are also the criticaltargets of any strategy aimed at correctin g a genetic defect.

Advances in our ability to genetically manipulate cells ex vivo

using viral and no n-viral tran sducing agents has circum vented m anyof the potential hazards of direct gene transfer in hum ans34. Success-

ful stable tran sduction o f holoclone-generating epiderm al cells withretroviral vectors h as paved t he way to th e first trial of gene therapy

for a devastating skin disease, junction al epiderm olysis bullosa, a

recessive disease caused by a misfunctioning laminin molecule35.However, in dominant-negative diseases, a mutant gene must be

silenced, and the techniques for doing this are in their infancy.Homologous recombination and site-directed mutagenesis of stem

cells ex vivo would be the ultimate goal36,37, but antisense RNA, RNAinterference and ham m erhead and h airpin RNAses offer interm edi-

ate strategies to at least prevent expression of a m utan t pro tein38.

With th e prospect of stem cell-mediated gene ther apy, the very def-inition of tissue engineering evolves into engineering of tissue func-

tion . Today, stem cell-based app roach es to tissue reconstru ction op enunpredicted applicative (and market) opportunities. Although this

discussion has been lim ited to systems where extensive preclinical and

clinical studies have already been conducted, more exciting avenuesare in sight in m any areas. But en thusiasm over what un questionably

represents a markedly innovative technique with huge therapeuticpotential m ust be balanced against stringent standards of scientific

and clinical investigation. In addition, we m ust rem ain aware of the

wide range of basic and applied issues associated with each system,with the targeted prob lem s, and with the p redicted solutions. s

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AcknowledgementsThe supp ort of Telethon Fond azione Onlu s Grant ( to P.B.) is gratefully acknowledged.

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