4In vitro differentiation of ES cells Physiol. Rev., 85, 635, 2005
5Definition of Adult Stem Cells PropertyAssay or experimental identificationHigh proliferative potentialIn vitro expansion and passage(Barrandon Y & Green H, 1987)Relatively undifferentiatedphenotypeLacking differentiation related protein orexpress undifferentiation marker(Coulombe PA, et al., 1989)Slow-CyclingLabel-retaining cells (LRCs) ;almost in G0/G1 phase(Cotsarelis G, et al., 1989)(5/50)
6History of Adult Stem Cell Research Since the 1970’s, bone marrow transplants have been used for treatment of immunodeficiencies and leukemias.Adult stem cell research on humans began in the 1960's, first achieving success in the treatment of a patient with severe combined immunodeficiency disorder in Since the early 1970's, adult stem cells have been successfully used for treatment of immunodeficiencies and leukemias.
7Mammalian epidermal stem cells A hair follicle and a segment of adjacent skin is illustrated. The dermal papilla (DP) signals to matrix stem cells (red) located across a basement membrane (green). Matrix cell daughters (yellow) differentiate into a variety of cell types, including the medulla, cortex and cuticle of the hair shaft (brown), the inner root sheath (IRS) and the outer root sheath (ORS). About two-thirds of the way up an anagen follicle lies the bulge — an expanded region that contains long-term stem cells (red). These cells periodically replenish (arrows) the matrix cells, and also help maintain the sebaceous gland (SG) and the epidermal stem cells (red, top layer) that lie against the basement membrane (not shown) overlying the basal layer in interfollicular regions. b, A mammalian gut crypt is a tube of cells arrayed on a basement membrane (green). Stem cells (red) are located in the basal region along with Paneth cells, but their exact location is variable and both types account for only a fraction of the cells present in the regions shown. A portion of the basement membrane in the stem cell region may be specialized (dark green). Stem cell progeny (yellow) known as transit amplifying cells (TA) move upwards and differentiate. Underlying mesenchymal cells (green) send signals that help regulate stem cell activity.Nature, 414, 98, 2001
8Published Reports on Identification of Human Adult Stem Cells Sources of adult stem cells include bone marrow, blood, the cornea and the retina ofthe eye, brain, skeletal muscle, dental pulp, liver, skin, adipocyte, the lining of the gastrointestinal tract, and pancreas.unipotent?
10PlasticityPlasticity is the ability of an adult stem cell from one tissue to generate the specialized cell type of another tissue.Example: Adult stem cells from bone marrow generated cells that resemble neurons
11Different steps in the transition of adult corneal epithelium into an epidermis Cornea epitheliumEmbryo dermisEpidermisDevelopment127, (2000)(After 21Days)
12Possible Roles of Bone Marrow–Derived and Circulating Stem Cells in the Repair of Solid-Organ Tissue After tissue injury, stem cells that are intrinsic to the tissue replace necrotic cells as a first line of defense. If the pool of endogenous stem cellsis exhausted, exogenous circulating stem cells are signaled to replenish the pool and participate in tissue repair. Thus, circulating stem cellsmay serve as a backup rescue system.N ENGL J MED., 349, 570, 2008
14How Does Cell Therapy Work? Bone marrow transplants are an example of cell therapy in which the stem cells in a donor's marrow are used to replace the blood cells of the victims of leukemia.Cell therapy is also being used in experiments to graft new skin cells to treat serious burn victims, and to grow new corneas for the sight-impaired.In all of these uses, the goal is for the healthy cells to become integrated into the body and begin to function like the patient's own cells.
15What Diseases Can be Cured by Stem Cell Therapies? Any disease in which there is tissue degeneration can be a potential candidate for stem cell therapiesType 1 diabetes mellitus - beta cells of the pancreasParkinson's disease - dopamine-secreting cells of the brainSpinal cord injuries leading to paralysis of the skeletal musclesIschemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvationMultiple sclerosis - loss of myelin sheaths around axonsMyocardium infraction – death of cardiomyocytes
16Stem cell research / Cell therapy Tissue or organSpecific cell typesStem cellsDifferentiationEngraftmentDiseaseDiabetesParkinson’s diseaseSpinal cord injuryBlindness…
17Number of Persons Affected Persons in the United States affected by diseases that may be helped by human pluripotentstem cell researchConditionNumber of Persons AffectedCardiovascular diseases58 MillionAutoimmune diseases30 MillionDiabetes16 MillionOsteoporosis10 MillionCancer8.2 MillionAlzheimer's disease4 MillionParkinson's disease1.5 MillionBurns (severe)0.3 MillionSpinal cord injuries0.25 MillionBirth defects150,000 (per year)Total128.4 MillionData from the Patients' Coalition for Urgent Research, Washington, DC
18Stem cell biology PART2: The Embryonic Stem Cell Jan-Kan Chen College of MedicineChang Gung University
20Early developement in humans Day 0: Fertilization of the oozyte in the oviduct.Zygote – totipotentDay 4-5: (16 cells) – morula, soloid mass of cellsDay 6-7: Blastocyst formation - pluripotent3rd week: Gastrulation, i.e formation of the three germ layers.
21Factors associated with early embryogenesis Inner cell mass: FGF-4 (embryogenesis and differentiation of trophectoderm)Trophectoderm: leptin and STAT3 (implantation)Trophoblast (mouse): Mash 2 (placenta formation)Epiblast: goosecoid, T, Evx-1, follistatin (primitive streak formation)
22Regulation of body pattern and differentiation GATA-4, -6: Early differentiationHox: Anterior-posterior polarityNodal and Lefty: Left-right symmetryHex: Anterior-posterior developmentMrg1: Heart formationBMP-4: Differentiation of mesenchymal cell, primitive streak migration, CNS developmentWnt3: Formation of the primitive streak and the nodeHNF-4, STAT-3: Visceral endoderm differentiation
23Culture of human embryonic stem cells Thomson et al., (1998) Science 282 :
24How Many Human Embryonic Stem Cell Lines are There? The actual number of human embryonic stem cell lines is a matter of some debate.To date, more than 100 human embryonic stem cell lines have been derived worldwide.However, most of those lines have not adequately characterized yet.Only 22 cell lines are eligible for federal funding in the USA.
25Comparison of some properties of mouse and human embryonic stem cells Mouse ES CellsHuman ES CellsTelomerase activity+Regulation ofself-renewalVia gp 130 receptors,MEF feeder layer,Nanog, BMP-4Feeder cells (MEF or human cells), serum, bFGF, MatrigelGrowth characteristicsin vitroTight, rounded, multilayer clustersFlat, loose aggregatesEB formationSimple and cystic EBsCystic EBsTeratoma formationin vivoMEF, mouse embryonic fibroblasts; EB, embryoid body.
26Maintaining mouse embryonic stem cells in their undifferentiated state LIF, either produced by feeder cells or added exogenously, allows mouse ES cells to proliferate without differentiation in vitroLIFR and gp130 are required for LIF binding, which in turn activates STAT3, which is necessary for continued proliferation of ES cellsSTAT3 and Oct-4 may interact and perhaps affect the function of a common set of target genesActivation of ERK and SHP-2 inhibit self-renewal of ES cellsIn mouse ES cells, Oct-4 expression and Gab-1 activation suppress Ras-ERK signalling pathway, and suppress induction of differentiation
27Leukemia inhibitory factor (LIF) Early blastocyst development and implantationSurvival for primordial germ cellMaintenance of mouse embryonic stem (ES) cell but not human ES cell
28Effect of LIF on self-renewal of mouse embryonic stem cells +LIF 24h+LIF 48h-LIF 24h-LIF 48hNature 336, (1988)
30JAK/STAT3 signaling pathways Regulation ofself-renewal in mouse ES cells by Oct3/4, Nanog, BMP-dependent SMAD, and LIF-dependentJAK/STAT3 signaling pathwaysPhysiol. Rev. 85: , 2005
31Examples demonstrating the developmental potential of human ES cells in vitro Cell Types DevelopedEctoderm, endoderm, mesoderm, and neural precursorsCardiomyocytesCardiomyocytes, endodermal, hematopoietic, and neuronal cellsNeuronal, epithelial, pancreatic, urogenital, hematopoietic, muscle, bone, kidney, and heart cellsNeural epithelium, embryonic ganglia, stratified squamous epithelium, gut epithelium, cartilage, bone, smooth and striated muscle cellsCells with properties of pancreatic -like cellsCardiomyocytes, pigmented and nonpigmented epithelial cells, neural cells, mesenchymal cells, erythroid, macrophage, granulocyte, and megakaryocyte cellsMyeloid, erythroid, megakaryocyte colony-forming cellsNeural precursors, glial and neuronal cells: incorporation into the brain (H1, H9, H9.2 lines)Neural precursors, glial and neuronal cells: incorporation into the brain (HES-1 line)Neural progenitor, dopaminergic, GABAergic, glutamatergic, glycinergic neurons, astrocytesNeural progenitor, neuronal cellsTrophoblastHepatocytes
32Directed differentiation of human ES cells in vitro Human ES cells differentiate spontaneously if removed from feeder cells and grown in suspension culturebFGF: Epidermal epithelial cells (keratin)Activin A:Muscle cell-like syncytium (enolase)Retinoid acid: Neuron (neurofilament H)Mouse BM stromal cell: Hematopoietic precursor cell (CD34)
33Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cellsNone of the growth factors directs differentiation exclusively to one cell typeActivin A and TGF-1 mainly induce mesodermal cellsRA, EGF, BMP-4 and bFGF activate ectodermal and mesodermal markersNGF and HGF allow differentiation into the three germ layersMost of the factors inhibit differentiation of specific cell types, and this inhibitory effect is more pronounced than an induction effect(PNAS 97: , 2000)
34An ES colony + HGF + activin A + RA + bFGF + BMP-4 Schuldiner et al PNAS 97: , 2000
35Schuldiner et al PNAS 97:11307-12, 2000 DS: differentiated embryonic cells; ES:embryonic stem cell:Schuldiner et al PNAS 97: , 2000
36Schuldiner et al PNAS 97:11307-12, 2000 Thick arrow and large fonts indicate an induction compared with the control(no GF)Dashed arrow indicates a decrease in expression.Schuldiner et al PNAS 97: , 2000
37Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell Nature Biotechnology 25, (2007)Nature Biotechnology 25, (2007)
41Self-organizing transcription faactors network for ES cells self-renew Cdx2Oct4Gata6NanogPrecusor(totopotent)Inner cell mass(pluripotent)Trophectoderm(multipotent)Primitive endodermEpiblastOct4: Loss of Oct4 causes differentiation of ES cells into trophectoderm.Overexpression of Oct4 results in differentiation into primitive endoderm and mesoderm.Sox2: One of the target genes of Oct4 and is required in ES cells with pluripotent sustenance.Nanog: Nanog can activate Oct4 promoter and also as transcription repressor for cell differentiation genes.
42Maintaining pluripotency Autoinductive FGF4/Erk signaling poises ESCs for lineage entry and must be resisted to allow self-renewal. A.Oct4 and Sox2 direct expression of fgf4 and poise ES cell from lineage commitment, Elevated Erk activity provides a signal rendering pluripotent cells susceptible to lineage inductive cues.B. Self-renewal of the pluripotent ES cell state requires overcoming the fgf4/Erk signal. The actions of FGF can be 1) blocked by inhibitors; 2)reversed by constitutive Nanog expression; 3) counteracted by LIF and BMP4.Cell, 132:532,2008
43Signaling Transduction Pathways Involved in Maintaining Mouse ESC LIF-STAT3 pathway:LIF (leukemia inhibitory factor) stimulates mESC through the gp130, which works as a heterodimer together with LIFR.Activation of gp130 leads to the activation of the JAK and STAT.Wnt pathway:Wnt/b-catenin signaling involved in the maintenance of pluripotency of ESC. Wnt signaling activation can upregulate c-Myc and STAT3 expression.BMP4 pathway:BMP4 phosphorylates Smad1/5 in mouse ES cells.Smad1/5 activation results in the expression of inhibitor of differentiation (ld) protein, which blocks the neural differentiation.
44Induced pluripotent stem cells, iPS cells Man-made pluripotency can be achieved through induced reprogramming of somatic cellsCell stem cell 2,2,151-9,2888
45Startrgies for the generation of pluripotent stem cells from somatic cells
46Mouse gene combinations for iPS induction The relation of ES cellAnd iPS cell is unclear, they may beSimilar but not identical.Nature review molecular biology 9,725,2008
47Klf4: Serves as upstream regulator of Oct4, Sox2, Nanog, and c-Myc. C-Myc: A major downstream target for the LIF/STAT3 and the Wnt signalling pathways that support maintenance of pluripotency.Lin28: RNA binding protein. Play a central role in blocking miRNA mediated differentiation in stem cells.
48Putative Role of the Four Factors in the Induction of iPS Cells Pluripotent stem cells are immortal and have open and active chromatin structure. Myc induces these two properties.Myc also induces apoptosis and senescence , which are suppressed by KLF4.Oct3/4 change the cell fate from tumor cells to ES cells.Forced expression of c-Myc and KLF4 alone would result in the generation of tumor cells, but not pluripotent stem cells.
49Putative Role of the Four Factors in the Induction of iPS Cells .Oct-3/4 and Sox2 activate multiple target genes synergistically.KLF4 may also function as cofactor of Oct-3/4 and Sox2.KLF4 : Kruppel-like factors, are zinc-finger proteins.
50Following injection into blastocysts, iPS cells contributed to mouse embryonic development
51Induction of Pluripotent Stem Cells from Fibroblast Cultures Kazutoshi Takahashi , Keisuke Okita , Masato Nakagawa & Shinya YamanakaNature Protocols 2:3081-9, 2007Cell 131: , 2007Generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4 and c-Myc.
52Generation of iPS cells from adult somatic cells with four factors: Science 318, 1917, 2007Generation of iPS cells from adult somatic cells with four factors:Oct4, Sox2, nanog and Lin28
57Summary of Policies Defined Around the World CountriesHuman Embryo Cloning (=creating embryo)Use of Stem Cell LinesUse of Superfluous EmbryosFrance, SpainProhibitedAuthorizedItaly, Austria, IrelandU.K. DenmarkIsrael, Sweden, Belgium, IndiaGermany(imported)U.S.A.Prohibited (public)Free (private)Authorized underrestricted condition (public)(in most states)CanadaUnder considerationJapan, Netherlands, KoreaTaiwan