StemCellBy Wei-Jun Cai Dept. of Histology & EmbryologySchool of Xiangya Medicine, CSU1978- test-tube baby Embryonic Stem Cell HistolyHow does this occur and what are the cellular and molecular mechanisms?How does a single cell develop into a body?Louise BrownSir Martin Evans has recently been honored with the Nobel Prize for Physiology and Medicine (2007) for his contribution towards development of animal models of disease through ESC mediated gene targeting. Sir Martin Evans receiving the Nobel prize1981- Evans and Kaufuman first extract stem cells from mouse embryos Embryonic Stem Cell HistolywNature. 1981 Jul 9;292(5819): 154-6. wDevelop indefinitely in culture, normal karyotype1995- from a non-human primate rhesus monkey embryos 1998 - first extract stem cells from human embryos Embryonic Stem Cell HistolyJames ThomsonIn 1998, James Thomson (University of Wisconsin-Madison) isolated cells from the inner cell mass of the early embryo, and developed the first human embryonic stem cell lines.In 1998, John Gearhart (Johns Hopkins University) derived human embryonic germ cells from cells in fetal gonadal tissue (primordial germ cells).Pluripotent stem cell “lines” were developed from both sourcesWhat Are Embryonic Stem Cell? Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells The Characteristics Of Embryonic Stem Cell ESC are distinguished by two distinctive properties: their ability to replicate indefinitely their pluripotencyable to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult body. The Characteristics Of Embryonic Stem Cell Morphologically umlimited proliferation in vitro and maimtenace of normal diploid karyotype High telomerase activation (telomere) Molecular marker alkaline phosphatase Oct-4（octamer-binding transcription factor 4） SSEAs (Stage-Specific Embryonic antigens) in vitro differntiation potential into cells of three germ layers Capacity to form teratoma, when injected subcutaneouly into Immune-deficient mice wInner cell mass as a nicheES cells can make tumors if in wrong environment嵌合胚嵌合胚 An Overview of Early Development Fertilized eggTotipotentstem cellsTotipotent: Can become any cell in body or placentaFate DecisionPluripotentstem cells(3-5 days old)BlastocystPluripotent: Can become any cell in body except a placentaImplantationFate DecisionGastrulating (day 14) leads toPrimary Germ CellsEndoderm (inner)digestive tract, resp. trackMesoderm (middle)bones, blood cells, heartEctoderm (outer)skin, CNSMultipotent: Can become any cell within a specific germ layer or cell lineageEmbryonic stem cells come from inner cell mass of blastocyst.Gastrulation (day 14) leads toPrimary Germ CellsEndoderm (inner)digestive tract, resp. trackMesoderm (middle)bones, blood cells, heartEctoderm (outer)skin, CNSMultipotent: Can become any cell within a specific germ layer or cell lineageMultipotentTwo kinds of stem cells from animals and humans:TotipotentwIn the 1960s, researchers discovered that the bone marrow contains at least two kinds of stem cellsHematopoietic stem cells, forms all the types of blood cells in the bodyBone marrow stromal cells (bone, cartilage, fat, and fibrous connective tissue)wIn the 1990s - scientists agreed that the adult brain does contain stem cells that are able to generate the brains three major cell types astrocytes and oligodendrocytes (non-neuronal cells)neurons, or nerve cellswAn undifferentiated cell found among differentiated cells in a tissue or organcan renew itselfcan differentiate to yield the major specialized cell types of the tissue or organ wThe primary roles in a living organism are to maintain and repair the tissue wSome scientists now use the term somatic stem cell instead of adult stem cellwUnlike embryonic stem cells, which are defined by their origin, the origin of adult stem cells in mature tissues is unknownwBrain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liverwLabeling the cells in a living tissue with molecular markers and then determining the specialized cell types they generatewRemoving the cells from a living animal, labeling them in cell culture, and transplanting them back into another animal to determine whether the cells repopulate their tissue of originwIsolating the cells, growing them in cell culture, and manipulating them, often by adding growth factors or introducing new genes, to determine what differentiated cells types they can becomewAlso, a single adult stem cell should be able to generate a line of genetically identical cellsknown as a clonewhich then gives rise to all the appropriate differentiated cell types of the tissueCont.wHematopoietic stem cells give rise to all the types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and plateletswBone marrow stromal cells (mesenchymal stem cells) give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendonswNeural stem cells in the brain give rise to its three major cell types: Neurons and two categories of non-neuronal cellsastrocytes and oligodendrocyteswEpithelial stem cells in the lining of the digestive tract occur in deep crypts and give rise to several cell types: Absorptive cells, goblet cells, Paneth cells, and enteroendocrine cellswSkin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layerThe follicular stem cells can give rise to both the hair follicle and to the epidermiswHematopoietic stem cells may differentiate into: Three major types of brain cells (neurons, oligodendrocytes, and astrocytes)Skeletal muscle cellsCardiac muscle cells andLiver cellswBone marrow stromal cells may differentiate into: Cardiac muscle cells and Skeletal muscle cellswBrain stem cells may differentiate into: Blood cells and skeletal muscle cellsPlasticity of adult stem cellsAdult stem cellsEmbryonic stem cellsTypically reside in or near their tissueAre capable of giving rise to functional cells in their tissue (but not other tissue types)Are typically found only in tissues that undergo regular turnover.Decrease in both number and activity as one ages.Created from inner cell mass cells that exist only in very early embryosAre capable of giving rise to all of the cell types in the body including non-regenerativeCan be divided many times (possibly indefinitely) in culture to make many cells.Embryonic or Adult Stem Cells for Cell Replacement Therapy Advantages and Disadvantages Embryonic SC “Pluripotent”Stable. Can undergo many cell divisions.Easy to obtain but blastocyst is destroyed (Ethics)Possibility of immune rejectionHigh potential for tumoursAdult SC“Multipotent”Less Stable. Capacity for self-renewal is limited.No ethical concernsDifficult to isolate in adult tissue.Host rejection minimized or absentLess tumorigenic potentialwFor those who believe that the embryo is a human person from the moment of conception, destruction of the embryo is equivalent to murderwBut the stem cells obtained from human embryos offer great hope for curing diseaseswQuestion: Is there any way of obtaining human embryonic stem cell without destroying or harming human embryos?The other sources of stem cellsTherapeutic CloningClone babyA sexual reproductionHistory of Animal CloningAmphibian (pollywog) (1962)1973年，获第一批carp(鲤鱼)和鲫鱼(crucian)核移植鱼（童第周等，动物学报）；History of Animal Cloning1996年7月5日，乳腺上皮细胞克隆绵羊“Dolly”诞生，哺乳动物体细胞克隆里程碑History of Animal CloningSince then, 13 species of animals including mice (1998), cows (1998), pigs (2000), cats (2001), and rabbits (2002) were successfully cloned.RABBITMOUSECATCattlePIGhorseDolly (sheep)1 live birth out of 29 cloned embryos3%Cloned mice31 live births out of 2468 cloned embryos1%Cloned pigs5 live births out of 335 cloned embryos1%Cloned goats3 live births out of 85 cloned embryos3%Cloned cattle30 live births out of 496 cloned embryos6%Cloned cat1 live birth out of 87 cloned embryos1%Cloned rabbits6 live births out of 371 of cloned embryos1%How Successful Was Animal Cloning? Very low (1-3%) 自1997年“多莉”羊问世之后，全球陆续有几个实验室开展人类体细胞核移 植研究(陆长富等，2000；Cibelli et al., 2001；Lavoir et al., 2005；Heindrychx et al., 2007； Hall et al., 2007；Fan et al., 2009 )。 2000年，中南大学生殖与干细胞工程研究所首次报道人类体细胞核移植胚胎可发育至桑椹胚； 2001年，美国先进细胞公司 Ciblelli 等报道人核移植胚胎可发育至6-c； 2003年，南大学生殖与干细胞工程研究所再次报道人类体细胞核移植胚胎能发育至囊胚； 后来陆续有几个实验室发表文章获得人类核移植囊胚，但获得囊胚数量极但获得囊胚数量极少（少（1-51-5枚）（枚）（Lu et al, 2003, Strojkovic et al., 2005, French et al., 2008, Li et al., 2009, Yu et al., 2009 , Xu et al, 2011）。人类体细胞核移植技术进展？The other sources of stem cells Alternative sources of pluripotent stem cells from amnitotic fluidThe process of deriving stem cell lines from amniotic fluidProposed by Landry and Zucker Landry, D. W. and H. A. Zucker, “Embryonic death and the creation of human embryonic stem cells,” The Journal of Clinical Investigation 114, 1184-1186 (2004).w“organismic death for the early-stage human embryo: the irreversible loss of the capacity for continued and integrated cellular division, growth and differentiation.” wExtracting stem cells from an embryo that is organismically dead would not harm the embryo, since it would already be dead.Howard ZuckerThe other sources of embryonic stem cells Dead embryosSuch entities in fact sometimes occur in naturethey are know as teratomasand they can never develop into full human beings even though the teratomas may show evidence of certain human characteristics such as fingernails.Typically these are viewed as freakish and some critics have maintained that this solution is morally and aesthetically repulsive. TeratomasThe other sources of embryonic stem cellswCurrently, to perform preimplantation genetic diagnosis (PGD), doctors extract a single cell from the early embryo.wCan we do the same in order to then use the cell to develop hESCs? Removing a single CellThe other sources of embryonic stem cells The Induced Pluripotent Stem (iPS) CelliPS : Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells. iPS cells are generated from somatic cells.iPS cells generation in patient fibroblastswParkinsons disease (Wernig and Jaenisch, 2008, Maehr and Melton PNAS 2009). wAmyopathic Lateral Sclerosis, (Dimos and Eggan Science 2008)wType I diabetes (Maehr and Melton PNAS 2009)wADA-SCID, SBDS, Gaucher disease, Duchenne and Becker Muscular dystrophi, Huntington disease, JDM, Down syndrome, Lesch-Nyhan syndrome. (Park and Daley Cell 2008).iPS cells generation from other cell typesBlood cells (Loh and Daley 2009). B-cells (Hanna and Jaenisch Cell 2008)Blood stem cells (Emiinli and Hochedlinger Nat Genet 2009)Pancreatic b-cells (Stadtfeld and Hochedlinger Cell Stem Cell2008)Hepatic and gastric endoderm (Aoi and Yamanaka Science 2008)Neural stem cells (Kim and Scholar, Nature 2008)iPS cell reprogramming: Problems Use of viral vectors for inductionUse of viral vectors for induction Low efficiency of reprogrammingLow efficiency of reprogramming Risk of Risk of tumourtumour formation formation Efficient differentiation protocols requiredEfficient differentiation protocols required wTo test new drugs. Tested for safety on differentiated cells generated from human pluripotent cell lineswCancer cell lines, for example, are used to screen potential anti-tumor drugswThe generation of cells and tissues which can be used for cell-based therapieswReplacement cells and tissues to treat diseasesParkinsons and Alzheimers diseasesSpinal cord injuryStroke, burnsHeart disease, DiabetesOsteoarthritis, and Rheumatoid arthritisApproximately one billion heart cells are lost during a serious heart attack. If one survives the initial event, a progressive chronic heart failure often occurs due to the replacement of functional beating heart cells with a tough fibrous scarSubstantia Nigra and Parkinson DiseaseDAParkinson disease vsnormalAlzheimers Disease & the BrainLoss of nerve cells within brain “shrunken, shriveled” Hippocampus, basal forebrain and cortex are affectedADNormalBarriers to bringing hESCs to clinicChanges in their epigenetic profilesChromosomal aberrations during their establishment and maintenancePost transplantation challenges like risk of tumorsImmune-rejections Fate decision How do stem cells develop into a functional population of specialized cells ?50THANK YOU!THANK YOU!wEssentially involves tricking a human egg into thinking it had been fertilized when it had not wThe egg would then develop to the 50-100 cell stage, at which point hESCs could be extractedwAre these really embryos? Could they actually develop as human beings? There is no way to answer that question without implanting the embryo, and this is itself a morally dangerous stepThe other sources of embryonic stem cells PaethenogenosiswCan we know that the IVF embryos are really dead?wWill the screening to find dead embryos itself harm some embryos?Will this be an incentive to produce even more embryos than necessary for IVF?Will this in fact yield stem cells of sufficient quality?wWill this harm the embryo?wAre the removed cells not themselves equivalent to embryos?wCan one research this issue without harming embryos?I.Pluripotent Stem Cells Derived from Organismically Dead Embryos (Landry-Zucker Proposal) II.Pluripotent Stem Cells via Blastomere Extraction from Living Embryos III.Pluripotent Stem Cells Derived from Biological Artifacts IV.Pluripotent Stem Cells via “Parthenogenesis.” V.Amniotic Fluid that contains embryotic stem cellsVI.Pluripotent Stem Cells Derived from “cloning”Six Possible Sources for ESCTakahashi and Yamanaka, Cell, Aug 25, 2006therapeutic cloningwGoal is to make genetically matched ES cellswProof-of-concept in micewallows somatic (not germline) gene/cell therapyRideout WM et al. 2002. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109: 17-27. CELL TYPE DEVELOPEDANIMAL MODEL REFERENCEOligodenrocyte progenitorSpinal cord injury induced mouseKeirstead et al., 2005 Nakamura et al., 2005 CardiomyocytesRat, Swine, MiceLaflamme et al., 2007 ; Leor et al., 1996 ; Kehat et al., 2004 ; Caspi et al., 2007 HepatocyteCCl4-injured SCID mouse modelSeo et al., 2005 ChondrocyteCanine Spinal Fusion modelMuschler et al., 2003 Endothelial cellsSurgical induction of hind limb ischemia in athymic mouseCho et al., 2006 Neural precursorsQuinolinic acid (QA)-induced Huntingtons disease (HD) model in ratsSong et al., 2007 Pancreatic cells Streptozotocin-treated diabetic miceShim et al., 2007 Skeletal myoblastsSCID/Beige miceBarberi et al., 2007 Neuroepithelial precursors and Dopaminergic neuronsParkinsons disease rodent modelSonntag et al., 2007 Ben-Hur et al., 2004 hESCsOpen neural tube defect (ONTD) model in chick embryosLee et al., 2006 T lymphoid lineageEngraftment into human thymic tissues in immunodeficient miceGalic et al., 2006 A list of animal injury and disease models where hESCs have been shown to be effective Risk of tumorsTransplantation of hES cell based therapies involves the risk of tumor formation arising from undifferentiated population of the transplanted cells.Studies with both ESCs and ES derived differentiated cells have shown that they can form teratocarcinomas in adult mice if injected subcutaneously, intramuscularly or into the testis. Presence of even one undifferentiated cell may potentially lead to teratomas, a cancerous tumor which is derived from germ cells and can from all the three germ layers. Genetic instabilityQuestions on the suitability of ESCs for transplantation purpose is raised because of the observed genetic instability of cloned cells and extreme inefficiency of the process. Cloned animals like Dolly give the outward appearance of full health, but the probability of their having numerous genetic defects is very high.Hochedlinger and Rudolf Jaenisch (2002) showed that in mice, the reprogramming of the inserted genetic material by the embryonic cells proceeded in a very unregulated way . Transplant rejectionThe immune system tends to reject the transplanted ESCs as foreign. This rejection can be inhibited by the use of immunosuppressive drugs which can have serious side effects.Alternate approaches using homolologous recombination techniques can allow the host immune system to recognize and mark the ESCs as self. Elimination of MHC class I and II gene loci is also proposed, though this would be technically challenging and would be clinically problematic Epigenetic reprogramming and culture adaptationTwo major causes for epigenetic changes in hESCs have been identified. The epigenetic changes in preimplantation embryos used for derivation of the hES cell lines Epigenetic changes during their maintenance in the culture over time Chromosomal abnormalities during prolonged cultureSeveral reports also indicate that these cells acquire chromosomal abnormalities or karyotype aberrations during prolonged culture in parallel with epigenetic changes.Such adaptations may result in enhanced cloning efficiencies after plating single cells . A reduced tendency for apoptosis and is expected to have a reduced capacity for differentiation which is difficult to assess quantitatively.A recent report by Baker et al., (2007) demonstrates accumulation of specific chromosomal aberrations within several well-established hESC lines over time.Different ways of self renewalAsymmetricCell divisionSpecialized cellSpecialized cellb