MOLECULAR PATHWAY OF PLURIPOTENCY

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• MOLECULAR PATHWAY OF PLURIPOTENCY

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What is pluripotency

• At the blastocyst (day 5 after fertilization)
• An outer layer of cells, the trophectoderm (TE)
• A group of pluripotent cells, the ICM (inner
  cell mass).
• TE will develop into placental tissues
• ICM gives rise to all cells of the embryo proper
  as well as several extraembryonic tissues.
• ICM and embryonic stem (ES) cells, possess the
  remarkable property of PLURIPOTENCY, the ability
  to give rise to all cells of the organism.

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Key transcription factors in pluripotency

• Key transcription factors such as Oct4, Sox2 or
  Nanog
• affect the cell cycle
• regulate gene expression
• modulate the epigenetic state
• repair DNA damage
• Resulting in
• regulate PLURIPOTENCY.
• functionally induce PLURIPOTENCY
• Besides transcription factors, microRNAs have
  recently been shown to play important roles in
  gene expression

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Molecular mechanisms and key factors regulating
the specification of ICM and TE lineages

• At the morula stage, cells choose their fate
  depending on their position and polarity.
  Genetic, epigenetic and environmental factors
  play an important role in early cell-fate
• Yap, the co-activator for transcription factor
  Tead4
• (Yap localises in the nucleus and increases
  Tead4 activity)
• Tead4 subsequently activates the trophectoderm
  (TE) master factor Cdx2
• Embryos lacking either Tead4 or Cdx2 fail to
  produce functional trophectodermal tissue but ICM
  cells remain intact and ES cells can be derived
• The counter-activity between Oct4 and Cdx2
  allows the segregation of the first two embryonic
  lineages

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Oct4 (octamer-binding transcription factor)

• Oocytes, fertilized embryo, embryonic carcinoma
  cells
• The expression of Oct4 was detected in TE as
  well as ICM cells
• Loss of Oct4
• There is inappropriate differentiation of the
  inner cell mass and ES cells.
• So, ES cells cannot be derived of blastocyst
• Overexpression of Oct4
• There is differentiation into primitive endoderm
  and mesoderm
• Oct4 can regulate gene expression by interacting
  with other factors within the nucleus, including
  the high mobility group (HMG)-box transcription
  factor Sox2

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Nanog

• Morula, ICM, germ cells, embryonic carcinoma
  cells
• Required for the germline formation
• Cells lacking Nanog spontaneously differentiate
  into primitive endoderm
• Overexpression of Nanog promotes self-renewal
  independent of the cytokine leukemia inhibitory
  factor (LIF)
• Human and monkey ES cells seem to maintain the
  pluripotency in LIF/STAT3 independent manner

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Sox2 (sex determining region Y)-box 2)

• Oocytes, ICM, epiblast,gut endoderm
• Sox2 plays an important role in the
  maintenance of pluripotency and lineage
  specification.
• may be found in early neural stages.
• One of the earliest expressed genes for
  pluripotency.

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Gata4 and Gata6

• found in extraembryonic endoderm lineages
• work as transcription factors.
• Forced expression of Gata4 or Gata6 in ES cells
  leads to differentiation into primitive endoderm,
  an effect similar to that caused by the loss of
  Nanog function
• Gata4 and Gata6 expression was upregulated in
  the absence of Nanog

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• Bone Morphogenetic Proteins (BMP)
• BMP are members of TGF-ß superfamily
• Receptors of the TGF-ß of ligands
• consist of a heteromeric complex of type I and
  type II receptor serine/ threonine kinases.
• Binding of BMP to the receptor induces
  phosphorylation of R-Smads by type I receptors.
• Phosphorylated R-Smads form complexes with
  Co-Smad and accumulate in the nucleus, where
  together they regulate gene transcription.
• In human ES cells, several groups reported that
  BMP4 induces DIFFERENTIATION.
• In mouse ES cells, BMP4 can induce expression of
  id (inhibitor of diffrerantiation) and suppress
  neural differentiation.
• The self-renewal of mouse ES cells is achieved by
  a delicate balance between the two cytokines, LIF
  and BMP.

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• Leukemia Inhibitory Factor (LIF)
• LIF is a heteromeric complex consisting of gp130
  and the LIF receptor
• Upon LIF binding, JAK(Janus kinase) kinase
  phosphorylates tyrosine residues of both gp130
  and LIFR.
• These phosphorylation recruits signal transducers
  and activators of transcription STAT 1 and STAT3
• The activated STAT (Signal transducer and
  activator of transcription 3) proteins
  translocate into the nucleus, where they function
  as transcription factors
• LIF and its downstream effector STAT3 are
  essential for maintenance of PLURIPOTENCY in
  mouse ES cells.
• Human and monkey ES cells seem to maintain the
  pluripotency in LIF/STAT3 independent manner

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Wnt/ ß-catenin pathway

• The wingless gene had originally been identified
  as a recessive mutation affecting wing and
  haltere development in Drosophila melanogaster3
  
• ?eta-catenin is a cytoplasmic protein that
  functions in cell-cell adhesion by linking
  cadherins to the actin cytoskeleton.
• In the absence of Wnt(combination of Wg
  (wingless) and Int) activation, beta-catenin is
  phosphorylated by a complex consisting of APC
  gene, Axin, and glycogen synthase kinase (GSK)
  3b.
• Phosphorylated beta-catenin is degraded by the
  ubiquitin proteasome system, thereby keeping the
  level of cytoplasmic beta-catenin low.
• Neural differentiation of mouse ES cells was
  attenuated by the activation of Wnt signaling by
  overexpression of Wnt1 or treatment with lithium
• chloride, an inhibitor of GSK3b

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• Wnt/ ß-catenin pathway may promote SELF-RENEWAL
  (in mouse and human ES cells)
• Wnt binds to its receptor (Frizzled, LRP5 or
  LRP6)
• Activated Dishevelled inactivates the APC/ Axin/
  GSK3b complex.
• Since this complex induces degradation of
  ß-catenin in the absence of Wnt ligand, its
  inactivation results in the stabilization and
  accumulation of ß-catenin protein in the nucleus.
  
• ?-catenin binds to and activates LEF/TCF
  transcription factors.

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Phosphatidyl inositol 3 (PI3) kinase

• PI3 kinases are lipid kinases that catalyze the
  phosphorylation of inositol phospholipids
• PI3 kinase pathway is likely to be a crucial
  regulator of ES cell proliferation.
• PI3 kinase pathway may be involved in the
  maintenance of pluripotency in both mouse and
  human ES cells
• PI3 kinase inhibitor, suppressed progression of
  cells from the G1 to S phase and decreased cell
  proliferation
• PTEN is a negative regulator of the PI3 kinase
  pathway. In loss of negative regulations of PTEN
  promotes ES cell proliferation and tumorigenicity
  

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Activation of the Ras/ERK pathway and PI3 kinase
pathway by growth factors

• .
• The PI3 kinase pathway can be activated via
  different routes.
• Gab1 can bind to Grb2, resulting in tyrosine
  phosphorylation and activation of the PI3 kinase
  pathway.
• The PI3 kinase-regulatory subunit p85 can bind
  to a phosphorylated tyrosine residue of the
  receptor.
• Activated Ras can induce membrane localization
  and activation of the p110 catalytic subunit of
  PI3 kinase.
• The PI3 kinase pathway is constitutively
  activated by ERas in mouse ES cells.
• The PI3 kinase pathway can promote self-renewal
  of mouse and human ES cells, possibly by
  suppression of the ERK pathway

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Phosphatidyl inositol 3 (PI3) kinase

• Activation of PI3 kinases is induced by many
  different receptor tyrosine kinases for growth
  factors, such as FGF, EGF, and PDGF, and leads to
  PIP3
• Akt1 is a serine/threonine kinase. Akt1 binds to
  PIP3 and is translocated to the inner cell
  membrane, where it is phosphorylated and
  activated by another serine/threonine kinase PDK1
• Activated Akt1 modulates the function of
  numerous substrates, such as Mdm2, IKK, and mTOR,
  and elicits various cellular responses, including
  proliferation and suppression of cell death.
• (everolimus, sirolimus mTOR inhibitors in
  RCC and AML)

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Akt signaling pathway
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Activation of the Ras/ERK pathway and PI3 kinase
pathway by growth factors

• Binding of growth factors to their receptors
  induces autophosphorylation of receptors and/or
  phosphorylation of receptor-associated proteins.
• The adaptor protein Grb2 binds to the
  phosphorylated tyrosines through its SH2 domains
  and activates the Ras/ERK pathway through the
  GTP-GDP exchange factor SOS.
• Activation of the Ras/ERK pathway induces
  differentiation in mouse ES cells.

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Molecular mechanisms of reprogramming

• Re-establishing pluripotency in a somatic cell
  is a complicated process.
• The most important changes include the
  activation of an ES-cell-specific transcription
  network
• re-setting the epigenetic landscape
• alteration of the cell cycle signature
• overcoming the DNA damage response triggered
  by these drastic changes

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Induced pluripotency with key factors

• ES cell factors such as Oct4, Sox2, cMyc, and
  Klf4 in fibroblast cells can reprogram them to a
  pluripotent state.
• The most efficient method to make iPS cells is
  through viral transduction.
• Failure of silencing indicates incomplete
  reprogramming and raises the danger of
  carcinogenesis by the oncogene cMyc.
• To avoid insertional mutagenesis and transgene
  reactivation, other methods that do not alter the
  genome have been developed, such as
  non-integrating
• episomal vectors,
• minicircle vectors and
• PiggyBac transposon system

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Differences between mouse and human ES cell

• The stem cells of teratocarcinoma are embryonal
  carcinoma (EC) cells, which express
  characteristics, similar to those of the inner
  cell mass (ICM)
• There are significant differences between mouse
  and human cells (EC and ES)
• Cell surface antigens of mouse EC and ES cells
• SSEA1()/SSEA3(-)/SSEA4(-)
• Cell surface antigens of Human EC cells
• SSEA1(-)/SSEA3()/SSEA4()
• (these phenotype is similar to that of human
  ES cells and human ICM cells)
• Human EC and ES cells have capacity to generate
  trophoblastic cells.
• This does not usually occur in mouse EC and
  ES cells.

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Similarities and differences between mouse
andhuman ES cell genomic targets

• Heart and neural crest derivatives expressed 1
  (Hand1) and Myst3 genes were identified as
  targets of Oct4 and Nanog in human ES cells,
• whereas others such as Estrogen-related
  receptor b (Esrrb) were observed only in mouse
  cells
• Rif1 has been implicated in regulating telomere
  length and might be important for self-renewal
• Esrrb has been shown to be important for
  placental development and germ cell
  proliferation.
• Tcl1 is highly expressed in mouse ES cells,
  enhances cell proliferation and survival through
  augmentation of phosphoinositide-3 kinase
  PI3KAkt signaling

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Core transcriptional regulatory circuitry in
pluripotent mouse and human ES cells.
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Epigenetic control of pluripotency

• What is epigenetic?
• Each of the cells within our body contains the
  same genetic material, yet these cells can look
  and behave very differently
• Each cell contains the same genes but some are
  switched on (expressed) and some are switched off
  (not expressed).
• The specific complement of genes expressed and
  not expressed in a cell determines its
  characteristics and this is controlled by
  epigenetics.
• ES cell chromatin characteristics
• abundance of acetylated histone modifications
• increased accessibility to nucleases
• .

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Epigenetic characteristics of pluripotent and
lineage committed cells

• ES cells lacking Eed can contribute to most cell
  lineages, suggesting that
• PcG proteins are not necessary for maintaining
  pluripotency
• Eed mutant ES cells spontaneously differentiate
• PcG proteins are necessary for ES cell identity
• Gene expression is influenced by enzymatic
  activities that can induce both global and local
  changes in chromatin structure

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Epigenetic characteristics of pluripotent and
lineage committed cells.
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