SIGNALLING PATHWAYS GOVERNING SIGNALLING PATHWAYS GOVERNING STEM CELL FATESTEM CELL FATE

Presented By: Huma RehmatInstitute of Biochemistry and Biotechnology,

Punjab University, Lahore, Pakistan

OutlineOutline

What are Stem Cells? Signalling pathwaysSignalling pathways Transcriptional factorsTranscriptional factors Cell cycle ControlCell cycle Control ConclusionConclusion

Stem cellsStem cells

The term Stem Cell was 1st proposed by the Russian scientist Alexander Maksimov in 1908 in Berlin.

Defined by their capacity to self renewal as well as differentiate into one or more mature cell types

PropertiesProperties

Defining properties

Self-renewal,undifferentiated & high potential

Lineage

Plasticity / Transdifferentiation

Signalling pathways

Stem cells specialized in response to external and internal chemical signals

Internal signals: Turn on specific genes causing differential gene expression (transcription factor Oct-4, c Myc etc)

External signals Chemicals secreted by other cells such as growth factors, cytokines, etc. Physical contact with neighboring cells

Potential of Stem CellsPotential of Stem Cells

Totipotent (total): Total potential to differentiate into any adult cell type Total potential to form specialized tissue needed for

embryonic development Pluripotent (plural):

Potential to form most or all 210 differentiated adult cell types

Multipotent (multiple): Limited potential Forms only multiple adult cell types

Oligodendrocytes Neurons

To make multicellular organisms cell must communicate. This communication is mediated by extracellular signal molecules.

Sofisticated mechanisms control which signal molecules are released from a specific type of cell, at what time and concentration they are secreted, and how these signals are interpreted by the target cells

Some signalling molecules act over long distances, some act only on the immediate neighbour cells

Most cells in higher organisms are both emiters and receivers of signals

Cell signallingCell signalling

Signalling pathways

Identified through genetic studies in drosophila.

Highly conserved in evolution and are very important in animal development

signaling molecules are released by signaling cells the signal is called the ligand

the ligand binds to its specific receptor on a target cell

this ligand-receptor interaction induces a conformational or shape-change in the receptorproduces a specific response - called the cellular response

can include a vast array of compoundse.g. transcription factors

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 consist of a of ligands consist of a

heteromericheteromeric complex of type I and type II receptor complex of type I and type II receptor serine/serine/ threonine kinases. threonine kinases.

Binding of BMP to the receptor induces phosphorylation Binding of BMP to the receptor induces phosphorylation of R-Smadsof R-Smads by type I receptors. by type I receptors.

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

In human ES cells, several groups reported that BMP4 In human ES cells, several groups reported that BMP4 induces induces DDIIFFERENTFFERENTIIATATIION.ON.

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

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

LLeukemia eukemia IInhibitory nhibitory FFactoractor ( (LIFLIF))

LIF is a heteromeric complex consistingLIF is a heteromeric complex consisting of of gp130 and the LIF receptorgp130 and the LIF receptor

Upon LIF binding, JAKUpon LIF binding, JAK(Janus kinase) (Janus kinase) kinase kinase phosphorylates tyrosine residues of both gp130 phosphorylates tyrosine residues of both gp130 andand LIFR. LIFR.

These pThese phosphorylation recruits signal hosphorylation recruits signal transducers and activators of transcriptiontransducers and activators of transcription 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 into the nucleus, where they translocate into the nucleus, where they function asfunction as transcription factors transcription factors

LIF and itsLIF and its downstream effector STAT3 are downstream effector STAT3 are essential for maintenance of essential for maintenance of PLURİPOTENCYPLURİPOTENCY inin mouse ES cells. mouse ES cells.

Human and monkeyHuman and monkey ES cells seem to maintain ES cells seem to maintain the pluripotency in LIF/STAT3the pluripotency in LIF/STAT3 independent independent mannermanner

Wnt/Wnt/ ββ-catenin pathway-catenin pathway

TThe he 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 Drosophila melanogaster

ΒΒetaeta-catenin is a cytoplasmic protein that functions in cell-catenin is a cytoplasmic protein that functions in cell--cellcell adhesion adhesion by linking cadherins to the actin cytoskeletonby linking cadherins to the actin cytoskeleton..

In the absence of WntIn the absence of Wnt(combination of (combination of WgWg (wingless) and (wingless) and Int)Int) activation, activation, bbetaeta-catenin is phosphorylated by a complex-catenin is phosphorylated 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 is degraded by the ubiquitin-catenin is degraded by the ubiquitin 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 was attenuated by the activation ES cells was attenuated by the activation of Wnt signalingof Wnt signaling by overexpression of Wnt1. by overexpression of Wnt1.

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

Wnt binds to its receptorWnt binds to its receptor ( (Frizzled, LRP5 or LRP6Frizzled, LRP5 or LRP6)) Activated Dishevelled inactivatesActivated Dishevelled inactivates the APC/the APC/ Axin/Axin/ GSK3b complex. GSK3b complex. Since this complex induces degradation of Since this complex induces degradation of ββ-catenin in the absence of Wnt -catenin 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 activates LEF/TCF-catenin binds to and activates LEF/TCF transcription factors.transcription factors.

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

Phosphatidyl inositol 3 (Phosphatidyl inositol 3 (PI3PI3)) kinasekinase

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

. .

TThe PI3 kinase pathway can be activated via he PI3 kinase pathway can be activated via different different routes. routes.

Gab1 can bind to Grb2, resulting in tyrosine Gab1 can bind to Grb2, 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 phosphorylated tyrosine residue of the to a phosphorylated tyrosine residue of the receptor. receptor.

AActivated Ras can inducectivated Ras 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 ERas in mouse ES cells. activated by ERas in mouse ES cells.

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

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

Activation of PI3 kinases Activation of PI3 kinases is induced by is induced by many different receptor tyrosine kinases many different 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 PIP3 PIP3

Akt1 is a serine/threonine kinase. Akt1 Akt1 is a serine/threonine kinase. Akt1 binds to PIP3 and is translocated to the binds to PIP3 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 PDK1kinase PDK1

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

KKey transcription factorsey transcription factors in in plurpluriipotencypotency

KKey transcription factors such as c Myc, Oct4, Sox2ey transcription factors such as c Myc, Oct4, Sox2 or or NanogNanog::

*affect*affect 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 induce functionally induce PLURİPOTENCYPLURİPOTENCY

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

Cell cycle control in differentiated cells and Cell cycle control in differentiated cells and embryonic stem cellsembryonic stem cells

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

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

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

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Induced Pluripotent Stem CellsInduced Pluripotent Stem Cells

• In 2006, researchers in Japan successfully inducted mouse somatic cells to become pluripotent stem cellso pluripotency depends on transcription factors o retroviruses used for introduction of these TF's

• First successfully performed in human cells by same group at University of Wisconsin who first isolated hESC in 2007 and the same group in Japan in 2007

• Possible problems with this cell is retrovirus that randomly inserts could interrupt tumor suppressor genes

• Current research focusing on using protein only induction• Promising field as avoids the controversy of embryonic SC

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 viralmost efficient method to make iPS cells is through viral 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 vectors, episomal vectors,

minicircle vectors and minicircle vectors and

PiggyBac transposon systemPiggyBac transposon system

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References

Burdon, T., Smith, A., & Savatier, P. (2002). Signalling, cell cycle and pluripotency in embryonic

stem cells. Trends in Cell Biology , 12(9), 432–438. doi:10.1016/S0962-8924(02)02352-8

Blank, U., Karlsson, G., & Karlsson, S. (2012). Signaling pathways governing stem-cell fate.

Blood, 111(2), 492–503.

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