MOLECULAR PATHWAYMOLECULAR PATHWAYSS OF OF

PLURPLURIIPOTENCYPOTENCY

Dr. Serdar SivginDr. Serdar Sivgin

February 2010February 2010

KayseriKayseri

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What is What is plurpluriipotencypotency

At the blastocyst At the blastocyst ((day 5 after fertilizationday 5 after fertilization):):

AAn outer layer ofn outer layer of cells, the trophectoderm cells, the trophectoderm (TE)(TE)

AA group of pluripotent group of pluripotent cells, the ICMcells, the ICM (inner (inner cell mass)cell mass). .

TETE will develop into will develop into placentalplacental tissuestissues ICMICM gives rise to gives rise to all cells of the embryoall cells of the embryo proper as proper as

well as well as several extraembryonic tissuesseveral extraembryonic tissues.. ICM ICM andand embryonic stem (ES) cells, possess theembryonic stem (ES) cells, possess the

remarkable property of remarkable property of PLURİPOTENCYPLURİPOTENCY, the , the ability to giveability to give rise to all cells of the organismrise to all cells of the organism..

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KKey transcription factorsey transcription factors in in plurpluriipotencypotency

KKey transcription factors such as ey transcription factors such as Oct4, Sox2Oct4, Sox2 or or NanogNanog::

**affectaffect the the cell cyclecell cycle

**regulate gene expressionregulate gene expression

**modulate themodulate the epigenetic stateepigenetic state

**repair DNA damagerepair DNA damage

Resulting in:Resulting in:

**regulateregulate PLURİPOTENCYPLURİPOTENCY. .

**functionally functionally induceinduce PLURİPOTENCYPLURİPOTENCY

BesidesBesides transcription factors, transcription factors, microRNAsmicroRNAs have recently have recently beenbeen shown to play important roles in gene expressionshown to play important roles in gene expression

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MMolecular mechanisms and key factorsolecular mechanisms and key factors regulating the regulating the specification of ICM and TE lineagesspecification of ICM and TE lineages

At At the morula stage, cells choose their fate depending onthe morula stage, cells choose their fate depending on their their position and polarityposition and polarity. G. Genetic, epigenetic and environmental enetic, epigenetic and environmental factors play anfactors play an important role in important role in early cell-fate early cell-fate

YapYap, the, the co-activatoco-activator for transcription factor r for transcription factor Tead4Tead4 (Yap(Yap localises in localises in the nucleus and increases Tead4 activitythe nucleus and increases Tead4 activity))

Tead4Tead4 subsequently subsequently activatesactivates the trophectoderm the trophectoderm ((TETE)) master master factor factor Cdx2Cdx2

Embryos lacking eitherEmbryos lacking either Tead4 or Cdx2 Tead4 or Cdx2 fail to produce fail to produce functional functional trophectodermaltrophectodermal tissue but ICM cells remain intact and ES cellstissue but ICM cells remain intact and ES cells can becan be derivedderived

TThe counter-activity he counter-activity betweenbetween Oct4 and Cdx2 Oct4 and Cdx2 allows the allows the segregation of the first twosegregation of the first two embryonic embryonic l lineagesineages

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Oct4 (octamer-binding transcription factor)Oct4 (octamer-binding transcription factor) Oocytes, fertilized embryo, embryonic carcinoma cellsOocytes, fertilized embryo, embryonic carcinoma cells

TThe expression of he expression of OOct4 ct4 waswas detected in TE as well as ICM cellsdetected in TE as well as ICM cells

LLoss of Oct4oss of Oct4::

There is iThere is inappropriate differentiation of the inner cell mass and ES cellsnappropriate differentiation of the inner cell mass and ES cells..

So, So, ES cellsES cells cannot be derivedcannot be derived of blastocyst of blastocyst

OOverexpression of Oct4verexpression of Oct4::

There is There is differentiationdifferentiation into primitive endoderm and mesoderminto primitive endoderm and mesoderm

OctOct44 can regulate gene expression can regulate gene expression by interacting with other factors within by interacting with other factors within the nucleus,the nucleus, including the high mobility groupincluding the high mobility group (HMG)-box transcription(HMG)-box transcription factor factor Sox2Sox2

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NanogNanog

Morula, ICM, germ cellsMorula, ICM, germ cells, , embryonic carcinoma cellsembryonic carcinoma cells

RRequired for theequired for the germline formation germline formation

Cells lacking Nanog Cells lacking Nanog spontaneously spontaneously differentiatedifferentiate into into primitive endodermprimitive endoderm

OOverexpressionverexpression of Nanog of Nanog promotespromotes self-renewal self-renewal independent of theindependent of the cytokine leukemia inhibitory factor (LIF)cytokine leukemia inhibitory factor (LIF)

Human and monkeyHuman and monkey ES cells seem to maintain the ES cells seem to maintain the pluripotency in LIF/STAT3pluripotency in LIF/STAT3 independentindependent manner manner

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Sox2 (sex determining region Y)-box 2)Sox2 (sex determining region Y)-box 2) Oocytes, ICM, epiblast,gut endodermOocytes, ICM, epiblast,gut endoderm

Sox2Sox2 plays an important plays an important role in the role in the maintenance of pluripotency maintenance of pluripotency andand lineagelineage specificationspecification..

* may be found in early neural stages.* may be found in early neural stages.

* One of the earliest expressed genes for * One of the earliest expressed genes for pluripotencypluripotency..

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Gata4 and Gata6Gata4 and Gata6

**found in extraembryonic endoderm lineagesfound in extraembryonic endoderm lineages

** work as transcription factors. work as transcription factors.

**Forced expressionForced expression of Gata4 or Gata6 in ES cells of Gata4 or Gata6 in ES cells leads leads to differentiation to differentiation intointo primitive endoderm, an effect primitive endoderm, an effect similar to that caused by thesimilar to that caused by the loss of Nanog functionloss of Nanog function

**Gata4 andGata4 and Gata6 expression was Gata6 expression was upregulatedupregulated in the in the absence ofabsence of NanogNanog

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Bone Bone MMorphogenetic orphogenetic PProteins (BMP)roteins (BMP)

BMPBMP are members of TGF-are members of TGF-ββ superfamily superfamily Receptors of the TGF-Receptors of the TGF-ββ of ligands of ligands consist of a heteromericconsist of a heteromeric complex of type I and complex of type I and

type II receptor type II receptor serine/serine/ threonine kinases. threonine kinases.

Binding of BMP Binding of BMP to the receptor to the receptor inducesinduces phosphorylation of R-Smadsphosphorylation of R-Smads by type I receptors. by type I receptors.

Phosphorylated R-Smads Phosphorylated R-Smads form complexes with form complexes with Co-Smad and accumulate in the nucleus, where Co-Smad and accumulate in the nucleus, where together theytogether they regulate regulate gene transcription. gene transcription.

In human ES cells, several groups reported that In human ES cells, several groups reported that BMP4BMP4 induces induces DDIIFFERENTFFERENTIIATATIION.ON.

In mouse ES cells, BMP4 can induce expression In mouse ES cells, BMP4 can induce expression ofof id (inhibitor of diffrerantiation) id (inhibitor of diffrerantiation) and and suppress suppress neural differentiation.neural differentiation.

TThe self-renewal of mouse ES cells is achieved he self-renewal of mouse ES cells is achieved by aby a delicate balance between the two cytokines, delicate balance between the two cytokines, LIF and BMP.LIF and BMP.

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LLeukemia eukemia IInhibitory nhibitory FFactoractor ( (LIFLIF))

LIF is a heteromeric complex consistingLIF is a heteromeric complex consisting of gp130 and the LIF receptorof gp130 and the LIF receptor

Upon LIF binding, Upon LIF binding, JAKJAK((Janus kinase) Janus kinase) kinase kinase phosphorylatesphosphorylates tyrosine residues tyrosine residues of both gp130 andof both gp130 and LIFR. LIFR.

These pThese phosphorylation hosphorylation recruitsrecruits signal signal transducers and activators of transducers and activators of transcriptiontranscription STAT 1 and STAT3STAT 1 and STAT3

The activated The activated STATSTAT ( (Signal transducer and Signal transducer and activator of transcription 3) activator of transcription 3) proteins proteins translocate translocate into the nucleus, where they function asinto the nucleus, where they function as transcription factors transcription factors

LIFLIF and itsand its downstream effector downstream effector STAT3 STAT3 are essential for are essential for maintenancemaintenance of of PLURİPOTENCYPLURİPOTENCY in in mouse ES cells. mouse ES cells.

Human and monkeyHuman and monkey ES cells seem to ES cells seem to maintain the pluripotency in LIF/STAT3maintain the pluripotency in LIF/STAT3 independentindependent manner manner 11

Wnt/Wnt/ ββ-catenin pathway-catenin pathway

The The winglesswingless gene had originally been identified as a recessive mutation gene had originally been identified as a recessive mutation

affecting wing and haltere development in affecting wing and haltere development in DrosophilaDrosophila melanogastermelanogaster[3[3]] ΒΒetaeta-catenin -catenin is a is a cytoplasmic protein cytoplasmic protein that functions in cellthat functions in cell--cellcell adhesion adhesion

by linking cadherins to the actin cytoskeletonby linking cadherins to the actin cytoskeleton..

In the absence In the absence of of WntWnt((combination of combination of WgWg (wingless (wingless) ) and and Int)Int) activationactivation, , bbetaeta-catenin -catenin is is phosphorylatedphosphorylated by a complex by a complex consisting of consisting of APC APC genegene, , Axin, and glycogen synthase kinase (GSK) 3b.Axin, and glycogen synthase kinase (GSK) 3b.

Phosphorylated bPhosphorylated betaeta-catenin -catenin is is degradeddegraded by the by the ubiquitinubiquitin proteasomeproteasome system, thereby keeping the level of cytoplasmicsystem, thereby keeping the level of cytoplasmic bbetaeta-catenin low.-catenin low.

NNeural differentiation of mouseeural differentiation of mouse ES cells ES cells was was attenuated attenuated by the by the activation of Wnt signalingactivation of Wnt signaling by overexpression of Wnt1 or treatment with by overexpression of Wnt1 or treatment with lithiumlithium

chloridechloride,, an inhibitor of GSK3b an inhibitor of GSK3b

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Wnt/Wnt/ ββ-catenin pathway may -catenin pathway may promote promote SELF-RENEWALSELF-RENEWAL (i(in n mmouse and human ouse and human ES cellsES cells))

Wnt Wnt binds to its receptorbinds to its receptor ( (Frizzled, LRP5 or LRP6Frizzled, LRP5 or LRP6)) Activated DishevelledActivated Dishevelled inactivates inactivates the the APC/APC/ Axin/Axin/ GSK3b GSK3b complex. complex. Since this complex Since this complex induces degradation of induces degradation of ββ-catenin -catenin in the absence of Wnt in the absence of Wnt

ligand, its inactivationligand, its inactivation results in the stabilization and accumulation of results in the stabilization and accumulation of ββ--catenin protein in the nucleus. catenin protein in the nucleus.

ΒΒ-catenin binds to and -catenin binds to and activatesactivates LEF/TCFLEF/TCF transcription factors.transcription factors.

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PI3 kinases PI3 kinases are are lipid kinases lipid kinases that catalyze thethat catalyze the phosphorylation of phosphorylation of inositol phospholipids inositol phospholipids

PI3 kinase pathway PI3 kinase pathway is likely to be a crucialis likely to be a crucial regulator of regulator of ES cell ES cell proliferation. proliferation.

PI3 kinasePI3 kinase pathway pathway may be involved may be involved in the maintenance of in the maintenance of pluripotencypluripotency in both mouse and human ES cellsin both mouse and human ES cells

PI3 kinase inhibitorPI3 kinase inhibitor, suppressed progression of cells from, suppressed progression of cells from the G1 the G1 to S phase and to S phase and decreased cell proliferationdecreased cell proliferation

PTEN is PTEN is a negative regulator a negative regulator of the PI3 kinase pathway. of the PI3 kinase pathway. In lIn loss of oss of negative regulatnegative regulationsions of PTEN of PTEN promotespromotes ES cell proliferation and ES cell proliferation and tumorigenicity tumorigenicity

Phosphatidyl inositol 3 (Phosphatidyl inositol 3 (PI3PI3)) kinase kinase

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

. .

The PI3 kinase pathway The PI3 kinase pathway can be activated via can be activated via different different routes. routes.

Gab1Gab1 can can bindbind to to Grb2Grb2, resulting in tyrosine , resulting in tyrosine phosphorylation and activation of the PI3 kinase phosphorylation and activation of the PI3 kinase pathway.pathway.

TThe PI3he PI3 kinase-regulatory subunit p85 can bind kinase-regulatory subunit p85 can bind to a to a phosphorylated tyrosine phosphorylated tyrosine residue of the residue of the receptor. receptor.

AActivated ctivated RasRas can induce can induce membrane membrane localization and activation of the p110 catalytic localization and activation of the p110 catalytic subunit of PI3 kinase.subunit of PI3 kinase.

TThe PI3 kinase pathway ishe PI3 kinase pathway is constitutively constitutively activated by activated by ERasERas in mouse ES cells. in mouse ES cells.

The PI3 kinase pathwayThe PI3 kinase pathway can can promotepromote self-self-renewal renewal of mouse and human ES cells, possibly of mouse and human ES cells, possibly by by suppressionsuppression of the ERK pathway of the ERK pathway

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Phosphatidyl inositol 3 (Phosphatidyl inositol 3 (PI3PI3)) kinase kinase

Activation of PI3 kinases Activation of PI3 kinases is is inducedinduced by by many different many different receptor tyrosine kinases receptor tyrosine kinases forfor growth factors, such as FGF, EGF, and growth factors, such as FGF, EGF, and PDGF, and leads toPDGF, and leads to PIP3PIP3

Akt1 Akt1 is a serine/threonine kinase. Akt1 is a serine/threonine kinase. Akt1 binds to binds to PIP3PIP3 and is translocated to the and is translocated to the inner cell membrane, where it isinner cell membrane, where it is phosphorylated and activated by another phosphorylated and activated by another serine/threonineserine/threonine kinase kinase PDK1PDK1

Activated Akt1 modulates the function ofActivated Akt1 modulates the function of numerous substrates, such as numerous substrates, such as Mdm2, IKK, Mdm2, IKK, and mTORand mTOR, and elicits various cellular, and elicits various cellular responses, including responses, including proliferationproliferation and and suppression of cellsuppression of cell deathdeath. .

(everolimus, sirolimus mTOR inhibitors in (everolimus, sirolimus mTOR inhibitors in RCC and AML)RCC and AML)

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Akt signaling pathwayAkt signaling pathway18

Activation of the Ras/ERK pathway and PI3 kinase pathway by growth factorsActivation of the Ras/ERK pathway and PI3 kinase pathway by growth factorsBinding of Binding of growth factors growth factors to their receptorsto their receptors induces induces autophosphorylationautophosphorylation of receptors of receptors and/or phosphorylation of receptor-and/or phosphorylation of receptor-associated proteins. associated proteins.

The adaptor protein The adaptor protein Grb2Grb2 binds to binds to the the phosphorylated tyrosines through its SH2 phosphorylated tyrosines through its SH2 domains and domains and activatesactivates the the Ras/ERK pathway Ras/ERK pathway through the GTP-GDP exchange factorthrough the GTP-GDP exchange factor SOS. SOS.

Activation of the Activation of the Ras/ERKRas/ERK pathway pathway inducesinduces differentiationdifferentiation in mouse ES cells. in mouse ES cells.

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Molecular mechanisms of reprogrammingMolecular mechanisms of reprogramming

Re-establishing pluripotency in a somatic cell is aRe-establishing pluripotency in a somatic cell is a complicated complicated process. process.

The most important changesThe most important changes include the activation of an ES-include the activation of an ES-cell-specific transcriptioncell-specific transcription networknetwork; ;

**re-setting the epigenetic landscapere-setting the epigenetic landscape

**alterationalteration of the cell cycle signatureof the cell cycle signature

**overcoming the DNAovercoming the DNA damage response triggered by damage response triggered by these drastic these drastic changeschanges

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Induced pluripotency with key factorsInduced pluripotency with key factors

ES cell factors such as ES cell factors such as Oct4, Sox2, cMyc, and Klf4 in fibroblastOct4, Sox2, cMyc, and Klf4 in fibroblast cells can cells can reprogram them to a pluripotent state.reprogram them to a pluripotent state.

TheThe most efficient method to make iPS cells is through most efficient method to make iPS cells is through viralviral transductiontransduction. .

Failure of silencing indicates incompleteFailure of silencing indicates incomplete reprogramming and raises the reprogramming and raises the danger of danger of ccarcinogenesisarcinogenesis by the oncogene cMycby the oncogene cMyc..

To avoid insertionalTo avoid insertional mutagenesis and transgene reactivation, other mutagenesis and transgene reactivation, other methods that do not alter themethods that do not alter the genome have been developed, such as genome have been developed, such as non-integratingnon-integrating

episomal vectorsepisomal vectors, ,

minicircle vectors minicircle vectors and and

PiggyBac transposon systemPiggyBac transposon system

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DDifferences between mouse andifferences between mouse and human ES cellhuman ES cell

The stem cells of tThe stem cells of teratocarcinomaeratocarcinoma are embryonal are embryonal carcinoma carcinoma (EC) (EC) cellscells, , whichwhich express characteristics,express characteristics, similar to those of thesimilar to those of the inner inner cell mass cell mass (ICM)(ICM)

There are There are significant differences between mouse andsignificant differences between mouse and human cells human cells (EC and ES)(EC and ES)

Cell surface antigens of mCell surface antigens of mouse EC and ES cellsouse EC and ES cells::

SSEA1(+)/SSEA3(-)/SSEA4(-)SSEA1(+)/SSEA3(-)/SSEA4(-)

Cell surface antigens of HCell surface antigens of Human ECuman EC cellscells::

SSEA1(-)/SSEA3(+)/SSEA4(+)SSEA1(-)/SSEA3(+)/SSEA4(+)(these(these phenotype is similar to that of phenotype is similar to that of human ES cells and human human ES cells and human

ICM cellsICM cells))

HHuman EC and ES cells uman EC and ES cells have capacity tohave capacity to generate trophoblastic generate trophoblastic cells. cells.

This does not usually occur in mouseThis does not usually occur in mouse EC and ES cells. EC and ES cells.

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Similarities and differences between mouse andSimilarities and differences between mouse andhuman ES cell genomic targetshuman ES cell genomic targets

HHeart and neural crest derivativeseart and neural crest derivatives expressed 1 (expressed 1 (HHandand11) and ) and MMystyst3 3 genes genes were identified aswere identified as targets of Otargets of Octct4 and N4 and Nanoganog in human ESin human ES cellscells,,

whereas others such as Estrogen-related receptor bwhereas others such as Estrogen-related receptor b ((EsrrbEsrrb) were ) were observed only observed only in mouse cellsin mouse cells

Rif1Rif1 has been implicated in has been implicated in regulating telomereregulating telomere length length and might and might be be important for self-renewalimportant for self-renewal

EsrrbEsrrb has been shown to be important has been shown to be important for for placental development placental development and and germgerm cell proliferationcell proliferation..

Tcl1Tcl1 is highly expressed in is highly expressed in mouse mouse ES cellsES cells, , enhances cell enhances cell proliferation proliferation and survival through augmentationand survival through augmentation of of phosphoinositide-3 kinase phosphoinositide-3 kinase PI3K–Akt signalingPI3K–Akt signaling

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Core transcriptional regulatory circuitry in pluripotent mouse and human ES cells.

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Epigenetic control of pluripotencyEpigenetic 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 characteristicsES cell chromatin characteristics::

abundance of abundance of acetylated histone modifications acetylated histone modifications

increased accessibility to increased accessibility to nnucleasesucleases

..

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ES cells lackingES cells lacking Eed Eed can contribute to most cell lineages, can contribute to most cell lineages, suggesting thatsuggesting that

PcG proteins are PcG proteins are not necessary not necessary for for maintaining pluripotencymaintaining pluripotency

Eed mutant ESEed mutant ES cells spontaneously differentiatecells spontaneously differentiate

PcG proteins arePcG proteins are necessary for ES cell identitynecessary for ES cell identity

Gene expression is Gene expression is influencedinfluenced by enzymatic activities by enzymatic activities that can that can induce both global and local induce both global and local changes in chromatinchanges in chromatin structurestructure

Epigenetic characteristics of pluripotent and lineage committed cellsEpigenetic characteristics of pluripotent and lineage committed cells

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Epigenetic characteristics of pluripotent and lineage committed cells.

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Thank you…Thank you…