Transcript 1387273989

MOLECULAR PATHWAYS OF
PLURIPOTENCY
Dr. Serdar Sivgin
February 2010
Kayseri
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What is pluripotency
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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 PLURİPOTENCY, 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 PLURİPOTENCY.
*functionally induce PLURİPOTENCY
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)
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BMP are members of TGF-β superfamily
Receptors of the TGF-β 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)
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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 PLURİPOTENCY
in mouse ES cells.
Human and monkey ES cells seem to maintain
the pluripotency in LIF/STAT3 independent
manner
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Wnt/ β-catenin pathway
The wingless gene had originally been identified as a recessive mutation
affecting wing and haltere development in Drosophila melanogaster[3]
Βeta-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/ β-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 β-catenin in the absence of Wnt ligand, its
inactivation results in the stabilization and accumulation of β-catenin protein in the
nucleus.
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Β-catenin binds to and activates LEF/TCF transcription factors.
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
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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
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 and
human 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 PI3K–Akt
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?
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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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Thank you…