Stem cells (2)

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

STEM CELLS (2)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 3


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Cord blood stem cells• Approx. 130 million babies born yearly – the

umbilical cord blood is the largest potential source of stem cells for regenerative medicine

• In the past 36 yrs 10000 patients were treated for over 80 different diseases

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Cord blood stem cells and fetal stem cells

Cord blood collection from umbilical vein (after

birth)

The cord blood cells are frozen in bag or cryovials

Liquid nitrogen storage tank (-150°)

Analysis of bloodCell separation

Mix and settle Concentrate cells Freeze cells35 min 10 min

Express

Express

Add cordblood

1 2 3

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Cryopreservation• Cryopreservation of primary cells is possible

for long term (so far 20 yrs).• The low-temperature is maintained at -150-

196oC in liquid nitrogen.

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Cord blood processing1. Red cell depletion (using Ficoll, Hetastarch,

Lymphoprep, Prepacyte)2. Depletion of plasma for smaller storage size3. Testing of the final cell pool (infection,

volume, cellularity, stem cell content, CD34+)

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Cord blood processing and cryopreservation• Cord blood is primarily useful in

hematological disorders• Cord blood is collected at birth• Either processed or just simply frozen in

DMSO

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Cord blood banking• Cord blood banks should be set up in every

metropolitan city with HLY specification and linked to an international computer network

• Keeping cord blood for a considerable length of time is costly

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Pluripotenciy of cord blood stem cells

Cord Blood

Stem cellsCBE MSC

Purification

Endodermal Mesodermal EctodermalHepato-Biliary Blood Neural

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Stem cell population in cord blood

Adherent Pre-MSC MSC

Non-adherent

CBE

Lin- CD133+ CD34+

Cord Blood

Bone marrow

Peripheral Blood

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Disorders treatable with cord blood IOncologic disordersAcute lymphoblastic leukemiaAcute myeloid leukemiaAutoimmune lymphoproliferative disordersBurkitt lymphomaChronic myeloid leukemiaCytopenia related to monosomyFamilial hystocytosisHodgkin’s diseaseJuvenile myelomonocytic leukemiaLangerhans cell hystocytosisMyelodysplastic syndromesNon-Hodgkin’s lymphoma

Immune deficienciesAtaxia telangiectasiaCartilage-hair hypoplasiaChronic granulomatous diseaseDiGeorge syndromeHypogammaglobulinaemiaIKK gamma deficiencyImmune dysregulation polyendocrinophatyMucolipidosis type IIMyelokathesisSevere combined immunodeficiencyWiscott-Aldrich syndromeX-linked agammaglobulinaemia, immunodeficiency, lymphoproliferative syndrome

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Disorders treatable with cord blood IIHematological disordersAutoimmune neutropeniaCyclic neutropeniaDiamond Blackfran anemiaEvan’s syndromeRed cell aplasiaRefractory anemiaSevere aplastic anemiaSickle cell diseaseThalassaemiaFanconi’s anemiaGalnzmann’s diseaseCongenital sideroblastic anemiaJuvenile dermatomyositis and xanthogranulomas

Metabolic disordersAdrenoleukodystrophyAlpha mannosidosisType I diabetesGaucher’s diseaseGunther diseaseHermansky-Pudlak syndromeHurler syndromeHurler-Scheie syndromeKrabbe’s diseaseMaroteau-lamy syndromeMetachromatic leukodystrophyMucolipidosis Types II, IIINeimann Pick syndrome, Types A and BSandoff syndromeSanfilippo syndromeTay Sachs disease

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Fat stem cells (ASC)Fat or adipose tissue stem cells (ASC):• Easily obtainable• Consistent immunophenotype• Similar to BMSC• Multipotent• Manipulation by genetic engineering

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Types of adipose tissues

TYPE FUNCTION1 Bone marrow Fills in space no longer used for hematopoiesis

2 Brown adipose tissue (BAT) Protects vital organs in the newborn

3 Ectopic adipose tissueAbnormal fat accumulation in the liver, skeletal or cardiac muscle (e.g. in metabolic syndrome)

4 Mammary adipose tissue Lactation – energy and nutrient source

5 Mechanical adipose tissue Protection from mechanical trauma

6 White adipose tissue (WAT)Insulation, energy storage, reservoir, endocrine organ

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Isolation procedures

Digestion with collagenase at 37oC, 1hr

Stromal vascularfraction (SVF)

Wash in PBS

Aspiration of lipocytes

300g, 5 min

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Immunophenotype of ASCsPositive markers

Marker Name Marker NameCD9 Tetraspan CD55 Decay accelerating factor

CD10Common acute lymphocytic leukemia antigen

CD59 Protectin

CD13 Aminopeptidase CD71 TransferrinCD29 b1-integrin CD73 5’-ectonucleotidaseCD34 Sialomucin CD90 Thy1CD44 Hyaluronate receptor CD105 EndoglinCD49d a4-integrin CD146 Muc-18

CD54 Intracellular adhesion molecule CD166 Activated leukocyte cell

adhesion molecule

HLA-ABC Histocompatibility locus antigen-ABC a-SMA a-smooth muscle actin

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Immunophenotype of ASCsNegative markersMarker Name Marker NameCD11b ab-integrin CD50 Intracellular adhesion

molecule-3

CD14 LPS receptor CD56 Neural cell adhesion molecule

CD16 Fc receptor CD62 E-selectionCD18 b2-integrin CD104 B4-integrin

CD45 Common leukocyte antigen HLA-DR Histocompatibility locus

antigen-DR

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Cytokine profile of ASCsCYOTOKINE FUNCTION GROUP

• Adiponectin• Leptin• Plasminogen activator inhibitor-1

Adipokines

• Hepatocyte growth factor• Pigment epithelial derived factor• Vascular endothelial growth factor

Angiogenic

• Flt-3 ligand• GCSF• Leukemia inhibitory factor• IL-7• MCSF

Hematopoietic

• IL-6, IL-8, IL-11• Leukemia inhibitory factor• TNFa

Pro-inflammatory

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Immunogenecity of ASCs• Lack of immunogenicity is linked to the

absence of the major histocompatibility class II antigens (HLA-DR) on their surface.

• Their immunosuppressive properties are linked to prostaglandin E2 production.

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Differentiation potential of ASCs• Adipocyte• Cardiac myocytes• Chondrocyte• Endodermal and ectodermal lineages• Endothelial and smooth muscle cells• Hematopoietic support• Neuronal lineage• Osteoblast• Skeletal myocytes

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Differentiation into adipocytes

ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION

WAT etc. Forskolin (AMP agonist) Neutral lipids Functional fat pads

Methylisobutylxanthine (AMP agonist)

Adiponectin Plastic surgery

Glucocorticoid receptor ligands (dexamethasone)

CAAT/enhancer binding protein-a fatty acid binding protein (aP2)

Cosmetic and reconstructive surgery

PPAR-g2 ligands (thiazolidinediones)

Leptin

Insulin Lipoprotein lipase

bFGF PPAR-g2

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Differentiation into cardiac myocytes


BAT 5-azacytadine Sarcomeric actininRepair injured cardiac tissue after ischemic injury

WAT Cardiomyocyte extract Connexin-43

Desmin

troponin-I

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Differentiation into chondrocytes


Bone marrow adipose tissue

Ascorbate Aggrecan Knee, hip chondroid tissue

BAT Dexamethasone Chondroitin sulfate

WAT TGF-b Collagen type II

3D structure Collagen type IV

BMP-6Chondrocyte specific proteoglycans

FGF

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Differentiation into osteocytes


WAT Ascorbate Osteocalcin Bone implantation

Bone marrow adipose tissue

Dexamethasone DMP-1 Bone fracture repair

1,25-dihydroxy vitamin D3 Osteoadherin

B-glycerophosphate

BMP-2

BMP-7

Runx2

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Differentiation into skeletal myocytes


Low concentration FBS myoD

Horse serum myogenin

Myosin light chain kinase

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Differentiation into neuronal cells


IndomethacinGlial fibrillary acidic protein (GFAP)

Central nervous system injury

Insulin Nestin

methylisobutylxanthine Intermediate filament

Glutamate receptor subunits

S-100

B-III tubulin

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Differentiation into endodermal and ectodermal lineages


Bone marrow adipoid tissue

Hepatocyte growth factor Albumin Liver

Oncostatin M A-fetoprotein

DMSO together with HGF, bFGF, nicotinamide

Urea

ATRA Cytokeratin-18Epithelial tissue repair (Crohn’s disease)

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Differentiation into endothelial and smooth muscle cells


WAT CD31 Vascular trauma

calponin Urogenital trauma

A-smooth muscle actin

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Hematopoietic support


ASCs secrete:IL6,IL7, IL8,IL11 SCF, TNFa, MCSF, GMCSF

CD34+ into T, NK, B markers

For patients requireng hematopoietic stem cells reconstruction following high-dose chemotherapy

STEM CELLS (3)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 4


TÁMOP-4.1.2-08/1/A-2009-0011

Application of ESCs and ASCs

Vascular lumen

Vasculogenic zone Vascular disorders

Gene therapiesGenetically modified

stem cell-based deliveryIntravenous

injection

Mesoderm

Cardiac diseases

CSCsAtria nicheApex niche

HSCEPC MSC

OsteoblastsChondrocytes

AdipocytesMyoblasts

Common lymphoidprecursor

NK-cellsT-lymphocytes

Dendritic cellsB-lymphocytes

Common myeloidprecursor

Macrophages

PlateletsErythrocytes

MonocytesGranulocytes

HSCsEndosteum surface nicheMicrovasculature nicheMSCsPerivascular surface niche

Bone

Hemopoietic and Immunesystem disorders

Endoderm

BASCsBronchioalveolarduct junction niche

Lung disorders

PSCsPancreatic ductputative niche

PancreasInsulin-secreting b-cells

Endocrine isletsof Langerhans

Exocrineacini

Diabetes

LiverHepatocytes

HDCsBile duct(canal of Hering)

Hepatic disorders

LungEctoderm

NSCsSubventricularzone nicheHippocampus niche(dentate gyrus region)

Neuron Astrocyte Oligodendrocyte

Brain and spinal cord disorders

Eye disorders

RSCsCiliary epitheliumniche

CESCsLimbus nicheRetina

Cornea

KSCsBasal layer nichebESCseNCSCsBulge nicheSKPsDermal papilla

Skin disorders

HeartCardiomyocytes

Embryonic developmentInner cell mass

ESCsBlastocyst

Pluripotent ESC

Mesodermal stem cellEndodermal stem cell

Ectodermalstem cell

Hemangioblast

HSC EPC

Vascular wall-resident stem cellsEPCs and MSCs

New endothelial cell

BloodstreamMacrophagePlatelets

Erythrocyte MonocyteEosinophil

NK-cell

T-lymphocyteDendritic cell B-lymphocyteBasophil

Neutrophil

Media

Brain

Eye

Skin

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Genetic engineering and gene delivery using ASCs• Lentiviral vectors can transduce ASCs• Other recombinant viral vectors• Nucleofection

TÁMOP-4.1.2-08/1/A-2009-0011Approaches and methods for controlling stem cell growth and differentiation

Bioreactors come in many sizes and designs and include stirred, rotary and perfused systems. All serve to improve exchange efficiency of nutrients and waste products and delivery of growth factors to enable longer term culture, helping to scale-up cell numbers or to grow larger pieces of tissue.

Scaffold can provide physical (e.g. surface roughness, porosity, etc.) and also biochemical (e.g. controlled release of doped growth factors) cues to promote attachment, recruitment, differentiation and delivery of cells.

Co-culture with the cells or tissues of interest (i.e. the target for tissue repair) can help to encourage differentiation. This can include direct physical contact and/or indirect biochemical signaling

Biochem factors added to culture medium (including serum) stimulate differentiation. Requires knowledge of factors likely to induce differentiation but is rarely, if ever, 100% effective.

Differential adhesion assays using specific ECM proteins or receptor ligands can help in encouraging selection of specific cell types based on affinity and kinetics of cell-substratum interactions. Related to this is the colony forming unit (CFU) assay.

Cell sorting techniques like MACS or FACS can positively select (or negatively deplete unwanted cell types) using cell surface antibodies or fluorescent transduced markers like GFP.

FACS MACS

Transduction with lineage specific genes can help to drive differentiation. Reporter tags like GFP aid selection (i.e. FACS) and reveal when and where genes are activated

GeneReporter(e.g. GFP)

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Reprogramming

Virus carriesreprogramming factors

into somatic cell’s nucleusPluripotent iPSC line

Culture asper hESCs

Somatic cell isreprogrammed

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Differentiation of Cells IPrecursor cell

Regulatoryprotein 2

Regulatoryprotein 3

Regulatoryprotein 1

Cell A Cell B Cell C Cell D Cell E Cell F Cell G Cell H

Regulatoryprotein 3

Regulatoryprotein 3

Regulatoryprotein 3

Regulatoryprotein 2

Cell division

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Differentiation of Cells II

Blastocyst

Zygote

Gastrula

Germ cells

Sperm Egg

Mesoderm (Middle layer)

Smooth muscleCardiac muscle Skeletal musclecells

Red blood cells Tubule cellof the kidney

Endoderm (Internal layer)

Lung cell(Alveolar cell)

Thyroid cell Pancreatic cell

Ectoderm (External layer)

Skin cells ofepidermis

Neuron of brain Pigment cell

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Mature, organ specific primary cells I

Biopsy PurificationCell culture

Cells forengineering

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Mature, organ specific primary cells II

Biopsy

Purification

Cells forengineering

Differentiated tissue cells

Tissue specific resident stem cell Cell cultures

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Differentiation of epidermis

Filaggrin, loricin, trichohyalin, involucrin, SPRRs, S100 proteins

Stratum corneum

Granular layer

Basal layer

Basal membrane

Keratin-2e

Keratin-1, -10

Keratin-5, -14

Epid

erm

al d

iffer

entia

tion

Spinous layer

Lipid envelopeCornified envelopeExtrusion of lipids from lamellar granules

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Mature tissue specific cells in tissue engineering• Biopsy or resection• Purification• Regaining proliferation capacity in cell culture• Re-differentiation

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Regulatory issues ICells• GLP• GMP• Permit to work on ES

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Regulatory issues IIAnimals• Permission to work on animals• UK: Home Office Licence 1986• EC 1394/2007

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Regulatory issues IIIHuman Embryonic Stem CellsEthical issues of using human embryos as sources of stem cells

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Regenerative medicine• Organ regeneration by inducing self-

regenerative biochemical and cellular processes

• Organ regeneration by addition of in vitro generated full organs or specific tissues of an organ

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Organ failure• Organ failure due to disease, accident or

aging requires full organ replacement or regeneration

• Ideally, one’s own tissues (autologue) should provide the necessary biomaterial for generation of such organs

Stem cells (2)

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