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Pluripotency of Embryonic Stem Cells

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Title: Pluripotency of Embryonic Stem Cells Author: Jayanti Tokas, Puneet Tokas, Shailini Jain, Hariom Yadav Last modified by: Eugene Shubnikov Created Date – PowerPoint PPT presentation

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Title: Pluripotency of Embryonic Stem Cells


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_at_gmail.com
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

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

4
Pluripotent Stem Cells
5
Types of Pluripotent Stem Cells
  • Embryonic stem cells
  • Embryonic carcinoma cells
  • Embryonic germ cells

6
Origins of pluripotent cells
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
7
Criteria for pluripotency
  • Immortality
  • Undifferentiation
  • Clonality
  • Broad developmental potential

8
Demonstration of pluripotency
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

10
In vivo
  • 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

11
Regulation of pluripotency in ES cells
Pluripotency is maintained by promotion of
proliferation and Inhibiting differentiation
12
Stage specific functioning
Target genes/ receptors required
Require other processes
13
Factors Required
  • Nanog
  • Oct 3/4
  • Sox 2
  • LIF
  • c-Myc
  • Klf4
  • Zic3

14
Core regulatory circuitry in ES cells
Activation of target genes
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

16
Oct3/4
  • 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

17
Sox2
  • 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

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

19
Combinatorial signaling pathways (involving LIF
and master regulator genes) in maintaining mouse
ESC pluripotency
Boiani and Scholer, 2005
20
c-Myc
  • 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

21
Klf4
  • 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

22
Pluripotent lineages in the mouse embryo
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

24
Differentiation of mouse ES cells
25
Differentiation of mouse ES cells
  • 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

26
Regulation of proliferation of mouse ES cells
27
Regulation of proliferation of mouse ES cells
  • Pluripotent transcription factors activate the
    expression of certain effectors that drive ES
    cell proliferation
  • Eras and Tcl1 stimulate the phosphoinositide-3- k
    inase (PI3K)/Akt signaling pathway to promote
    the cell cycle, whereas b-Myb and c-Myc activate
    the progression of the cell cycle directly

28
Transcriptional regulation of the mouse Oct3/4
gene
A
B
29
Zic3
  • 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

30
Epigenetic regulations of ES cells pluripotency
31
Characteristics of the pluripotent epigenome
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
32
Epigenetic features of the pluripotent cell
nucleus
  • 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)

33
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