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Slide 1 - Pluripotency of Embryonic Stem Cells Jayanti Tokas1, Puneet Tokas2, Shailini Jain3, Hariom Yadav3 1Department of Biotechnology, JMIT, Radaur, Haryana, India 2KITM, Kurukshetra, Haryana, India 3NIDDK, National Institute of Health, Bethesda, MD 20892, USA Email: yadavhariom@gmail.com
Slide 2 - Stem Cells Stem cells are master cells with two important characteristics Unspecialized cells capable of their own renewal Ability to differentiate into different cell types The stem cells may have various differentiation potentials Totipotent Pluripotent Multipotent Unipotent
Slide 3 - Pluripotent Stem Cells Pluri derived from latin word plures – means several or many Most commonly the term is used to describe stem cells that give rise to cells derived from all three embryonic germ
Slide 4 - Pluripotent Stem Cells
Slide 5 - Embryonic stem cells Embryonic carcinoma cells Embryonic germ cells Types of Pluripotent Stem Cells
Slide 6 - Abnormal karyotypes, Germline transmission unlikely Undergo spontaneous differentiation Unable to support normal development due to epigenetic modifications during PGCs Pluripotent cells Partially understood Great potential Origins of pluripotent cells
Slide 7 - Immortality Undifferentiation Clonality Broad developmental potential Criteria for pluripotency
Slide 8 - Demonstration of pluripotency
Slide 9 - In vitro Differentiation triggered when grown in suspension, Embryoid Body formation b) Different cells obtained spontaneously c) Specific growth factors can be used to direct the differentiation of ES cells into specific cells
Slide 10 - a) Teratoma formation when injected into nude mouse b) When injected into host blastocysts, the ES cells integrate, proliferate and differentiate into all germ lineages including germ cells In vivo
Slide 11 - Regulation of pluripotency in ES cells “Pluripotency is maintained by promotion of proliferation and Inhibiting differentiation”
Slide 12 - Require other processes Stage specific functioning Target genes/ receptors required
Slide 13 - Factors Required Nanog Oct 3/4 Sox 2 LIF c-Myc Klf4 Zic3
Slide 14 - Core regulatory circuitry in ES cells Activation of target genes
Slide 15 - Nanog 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
Slide 16 - POU-domain transcription factor Maintains pluripotency (ESCs, EGCs, ECCs, GSCs) Tightly regulated transcription factor, associated with a number of target genes implicated in pluripotency maintenance Regulatory elements in target genes are in close vicinity of Sox2- binding sites Key factor in the transcriptional framework of self-renewing stem cells Oct3/4
Slide 17 - Member 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 other TFs, e.g. Nanog to activate transcription of pluripotency markers Sox2
Slide 18 - Leukaemia 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 Binding results in the activation of Jak/ Stat signal transduction pathway Activated Stat3 maintains pluripotency
Slide 19 - Boiani and Scholer, 2005 Combinatorial signaling pathways (involving LIF and master regulator genes) in maintaining mouse ESC pluripotency
Slide 20 - Helix-loop-helix/leucine zipper transcription factor 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
Slide 21 - Member of the quartet, a Krueppel-type zinc finger transciption factor 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 Nanog Klf4
Slide 22 - ll Pluripotent lineages in the mouse embryo
Slide 23 - Pluripotent cells form the ICM of the blastocyst After giving rise to the primitive endoderm on the surface of the ICM, pluripotent stem cells form the epiblast and start to proliferate rapidly after implantation They then form the primitive ectoderm, a monolayer epithelium that has restricted pluripotency which goes on to give rise to the germ cell lineage and to the somatic lineages of the embryo Certain key transcription factors (blue) are required for the differentiation of the various embryonic lineages
Slide 24 - Differentiation of mouse ES cells
Slide 25 - ES cells differentiate into three cell types – primitive endoderm trophectoderm (TE) 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 cells In the absence of Nanog 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 medium Differentiation of mouse ES cells
Slide 26 - Regulation of proliferation of mouse ES cells
Slide 27 - Pluripotent transcription factors activate the expression of certain effectors that drive ES cell proliferation 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 directly Regulation of proliferation of mouse ES cells
Slide 28 - Transcriptional regulation of the mouse Oct3/4 gene A B
Slide 29 - 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, Foxa2, and Sox7 The presence of Zic3 also maintains the expression of the homeodomain protein Nanog, a key regulator of pluripotency in embryonic stem cells Zic3
Slide 30 - Epigenetic regulations of ES cells pluripotency
Slide 31 - The nucleus shrinks and the distribution 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
Slide 32 - Small regions of perinuclear heterochromatin exist, but most of the chromatin exists 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
Slide 33 - THANK YOU