luripotencyofEmbryonicStemCells胚胎干细胞的多能性

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1、Jayanti Tokas1,Puneet Tokas2,Shailini Jain3,Hariom Yadav31Department of Biotechnology,JMIT,Radaur,Haryana,India2KITM,Kurukshetra,Haryana,India3NIDDK,National Institute of Health,Bethesda,MD 20892,USAEmail: Stem Cells Stem cells are master cells with two important characteristics Unspecialized cells

2、capable of their own renewal Ability to differentiate into different cell types The stem cells may have various differentiation potentials Totipotent Pluripotent Multipotent UnipotentPluripotent Stem Cells Pluri derived from latin word plures means several or many Most commonly the term is used to d

3、escribe stem cells that give rise to cells derived from all three embryonic germPluripotent Stem Cellsq Embryonic stem cellsq Embryonic carcinoma cellsq Embryonic germ cellsTypes of Pluripotent Stem CellsAbnormal karyotypes,Germline transmission unlikely Undergo spontaneous differentiationUnable to

4、support normal development due to epigenetic modifications during PGCsPluripotent cellsPartially understood Great potential Primordial germ CellsBlastocystFetusAdultZygoteTeratomasInner cell mass(ICM)Embryonic Stem CellsEmbryonic Germ CellsEmbryonic Carcinoma CellsOrigins of pluripotent cells Immort

5、ality Undifferentiation Clonality Broad developmental potentialCriteria for pluripotencyDemonstration of pluripotencyIn vitroa)Differentiation triggered when grown in suspension,Embryoid Body formationb)Different cells obtained spontaneouslyc)Specific growth factors can be used to direct the differe

6、ntiation of ES cells into specific cellsa)Teratoma formation when injected into nude mouseb)When injected into host blastocysts,the ES cells integrate,proliferate and differentiate into all germ lineages including germ cellsIn vivoRegulation of pluripotency in ES cells“Pluripotency is maintained by

7、promotion of proliferation andInhibiting differentiation”Require other processes ES Cells pluripotency Transcription Factors Epigenetic modificationsStage specific functioningTarget genes/receptors required Factors Required Nanog Oct 3/4 Sox 2 LIF c-Myc Klf4 Zic3Core regulatory circuitry in ES cells

8、Activation of target genesNanog Transcription factor containing homeobox domain Downstream effectors of signals of LIF and BMP Elevated levels excludes inclusion of LIF and feeder layer Works with other key factors including Oct4 and Sox2 q POU-domain transcription factorq Maintains pluripotency(ESC

9、s,EGCs,ECCs,GSCs)q Tightly regulated transcription factor,associated with a number of target genes implicated in pluripotency maintenanceq Regulatory elements in target genes are in close vicinity of Sox2-binding sitesq Key factor in the transcriptional framework of self-renewing stem cellsOct3/4 Me

10、mber of HMG-domain DNA-BP family Necessary for embryonal development and to prevent ES cell differentiation Many ES cell pluripotency-associated genes are co-regulated by Sox2 and Oct3/4 A ternary complex formed with Oct4 or Oct1 on enhancer sequence of Fgf4 is must for functioning Cooperate with ot

11、her TFs,e.g.Nanog to activate transcription of pluripotency markersSox2Leukaemia inhibitory factor,LIF Interleukin-6 cytokine family Essential for maintaining pluripotency in vitro in the presence of serum Binds to a heterodimeric receptor comprising of LIF-receptor(LIFR)and gp130 on cell membrane B

12、inding results in the activation of Jak/Stat signal transduction pathway Activated Stat3 maintains pluripotency Boiani and Scholer,2005 Combinatorial signaling pathways(involving LIF and master regulator genes)in maintaining mouse ESC pluripotency Helix-loop-helix/leucine zipper transcription factor

13、Takes part in a variety of cellular functions Downstream effecter of STAT3 in LIF receptor signaling pathway c-Myc is a substrate for GSK3b in Wnt signalling pathway Compensates anti-proliferative effects of Klf4,e.g.in iPS cells c-Myc Member of the quartet,a Krueppel-type zinc finger transciption f

14、actor Can act as an oncogene and as a tumor suppressor protein Over expression inhibits differentiation of ES cells Klf4 upregulates,in concert with Oct3/4,but the role as co-factor for Oct3/4 may be limited to only a few targets Can repress p53,a negative regulator of NanogKlf4llPluripotent lineage

15、s in the mouse embryoPluripotent cells form the ICM of the blastocystAfter giving rise to the primitive endoderm on the surface of the ICM,pluripotent stem cells form the epiblast and start to proliferate rapidly after implantationThey then form the primitive ectoderm,a monolayer epithelium that has

16、 restricted pluripotency which goes on to give rise to the germ cell lineage and to the somatic lineages of the embryoCertain key transcription factors(blue)are required for the differentiation of the various embryonic lineagesDifferentiation of mouse ES cellsES cells differentiate into three cell t

17、ypes q primitive endodermq trophectoderm(TE)q primitive ectoderm mimicking the differentiation potential of pluripotent stem cells in preimplantation embryos In the absence of LIF and in the presence of an excess of Oct3/4,ES cells differentiate into primitive endoderm-like cellsIn the absence of Na

18、nog and in the presence of Gata6,they differentiate into parietal endoderm-like cells Removing Oct3/4 and adding Cdx2 to,ES cell culture induces TE-like differentiation.MEFc,mouse embryonic fibroblast conditioned mediumDifferentiation of mouse ES cellsRegulation of proliferation of mouse ES cellsv P

19、luripotent transcription factors activate the expression of certain effectors that drive ES cell proliferationv Eras and Tcl1 stimulate the phosphoinositide-3-kinase(PI3K)/Akt signaling pathway to promote the cell cycle,whereas b-Myb and c-Myc activate the progression of the cell cycle directlyRegul

20、ation of proliferation of mouse ES cellsTranscriptional regulation of the mouse Oct3/4 geneAB Zic3 contributes to the maintenance of pluripotency by operating downstream of Oct4,Nanog,and Sox2 to inhibit endoderm lineage specification as characterized by endodermal markers Sox17,PDGFRA,Gata4,Gata6,F

21、oxa2,and Sox7 The presence of Zic3 also maintains the expression of the homeodomain protein Nanog,a key regulator of pluripotency in embryonic stem cellsNanogZic3Oct4Sox2Sox17,PDGFRAGata4,Gata6Foxa2,Sox7 EndodermZic3Epigenetic regulations of ES cells pluripotencyThe nucleus shrinks and the distribut

22、ion of electrondense areas(mainly heterochromatin)changes dramatically when ES cells are induced to differentiate into primitive endoderm by the ectopic expression of Gata6 Characteristics of the pluripotent epigenome Small regions of perinuclear heterochromatin exist,but most of the chromatin exist

23、s as euchromatin,bearing histone marks associated with transcriptional activity The hyperdynamics of chromatin proteins(green)might contribute to the maintenance of euchromatin Bivalent domains are also a feature of the pluripotent epigenome,in which active histone marks(such as H3K4me)are flanked by transcriptionally repressive histone marks(such as H3K9me)Epigenetic features of the pluripotent cell nucleus