Jayanti Tokas 1, Puneet Tokas 2, Shailini Jain 3, Hariom Yadav 3 1 Department of Biotechnology, JMIT, Radaur, Haryana, India 2 KITM, Kurukshetra, Haryana, India 3 NIDDK, National Institute of Health, Bethesda, MD 20892, USA

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

Stem Cells Fate Replenish old/damaged tissue by proliferation and maturation Differentiation - senescence and apoptosis Fail to differentiate or undergo apoptosis - Neoplasia Stem Cell

Stem cell niche Niche signals regulate stem cell  Self-renewal  Survival  Maintenance Niche cells adhere to stem cells by cell-cell adhesion Stem/niche organization provides cues to organize spindles and self-renewal signals Niches exist in many adult tissues: hematopoietic system, neural tissues, skin, gut epithelium, etc

Cytokines Prime molecules modulating cellular growth and differentiation of embryonic, hematopoietic and other stem cell types Act in low concentrations ( M) Complex molecules Two important characteristics  Redundancy  Pleiotropism

STAT signaling Principal mediator of the biological responses to ligands that activate gp130 Post-translationally regulated by tyrosine and serine phosphorylation Structure: 1. Tetramer, a leucine zipper like domain located at the amino terminus 2. DNA binding domain 3. Src homology domain 3- like region 4. Src homology domain 2 (SH2), a critical site of tyrosine phosphorylation 5. Carboxy-terminal transactivation domain

Y58 0 P Y N- SH2 PTPase C- SH2 Y542 PTPase N-SH2C-SH2 BP pY Y +PO 4 PTPase S Y +PO 4 p Y542 pY580 C-SH2 N- SH2 Activation by External phosphotyrosines Activation through binding of SH2 domains to C-terminal phosphotyrosines of SH2 Inactive Active Abbreviations : S, substrate BP, bisphosphorylated SH2 domain binding protein SH 2 Two alternate pathways SHP-2 signaling

P Cytokine R JAK Cytokin e JAK P P P P STA T SH 2 P P P STA T SH 2 P STA T SH 2 STA T SH 2 P P Nucleus Cell membrane Cytoplasm Gene expression STA T SH 2 P P Cytokine Receptor Signaling (Constantinescu, 2000)

Oct 3/4 Marker for undifferentiated cells Master regulator of initiation, maintenance and differentiation of pluripotent cells Exerts effects via certain target genes like genes encoding, the transcriptional co-factor UTf-1, Zfp42/Rex-1, PDGF, otx-2, etc Requires several cofactors to execute its functions of maintaining pluripotency

Relationship between Oct-3/4 expression and stem cell fate

Embryonic Stem cells self renewal LIF maintains ES cells self renewal Signal transduction by LIF and other IL-6 family members occurs via a common signal transducer gp130 Dimerization of the receptor associated subunit activates receptor associated kinases like Src and the Janus family of kinases e.g. Jak1, Jak2 and Tyk2 Homo-dimerization (IL-6/IL-6R and IL-11/IL-11R) or hetero-dimerization (LIF/LIFR) Embryonic stem cell renewal factor (ESRF) : a LIF independent signaling factor

IL-6 family cytokines STAT3 Jak1/2/Tyk2 Self-renewal - P ERK1/2 -GTP -P Raf Ras SHP2 Gab1 Grb2 -P MEK Differentiation - phosphorylation -dimerization gp130 Box3 Box2 Bo x1 P-Y126 P-Y173 P-Y265 P-Y 275 Y118-P STAT3-P Signaling pathways induced by IL-6 family cytokines Essentials of Stem Cell Biology, pg no. 32

Hematopoietic stem cells (HSC) self renewal Expression of cell surface molecules TpoR+Flk2+KIT HOXB4 EpoR and t EpoR (tEpoR EpoR ) SCF and IL-6 : expression of receptor CXCR4 of stromal derived factor Self renewal blockade : IL-3 and TNF

Hematopoietic stem cells (HSC) self renewal

Keratinocyte Stem Cells Present in hair follicle bulge area Cell adhesion, cell migration, and clonogenicity Stem cells express more β1 integrin and adhere more rapidly to collagen IV Misu :  Direct target gene of Myc  Up-regulated in many cancers  Depletion might result in increased self renewal capacity

Role of Myc in regulating epidermal stem cell fate Adhesion and cytoskeletonGrowth promoting target genes

New Players in Stem cell self renewal and Future directions Nanog – marker for pluripotent cell lines Induced pluripotent stem cells from human fibroblasts Maintenance of self renewal property in ES via Nanog is independent of LIF/STAT3, and also Oct-3/4 Understanding the molecular mechanism of stem cell renewal and commitment - unparalleled progress for tissue replacement, transplantation and other stem cell based therapies Expression of genes AML1 or RUNX1 (human cord stem cells) Stem cell research in livestock a long way to go……

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