Embryonic Stem Cell Pluripotency in Mouse
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Embryonic Stem Cell Pluripotency in Mouse

Embryonic stem cells (ESCs) self-renew and are pluripotent, meaning they can differentiate into all cells comprising an adult organism. The extrinsic factors, signaling pathways and transcription factor networks that contribute to maintenance of mouse ESC self-renewal and pluripotency, referred to as the ‘ESC state’, include Leukaemia inhibitory factor (LIF) and Bone morphogenetic protein 4 (BMP4), which are the key cytokines required for maintenance of ESC self-renewal in culture, acting via the Jak-Stat3 and Smad-Id pathways respectively (Ref.1). WNT (Wingless-Type MMTV Integration Site Family Member) signaling is also involved. The cell intrinsic machinery includes the transcription factors Oct4 (Octamer Binding Transcription Factor-4), Nanog (Nanog homeobox) and SOX2 (SRY (Sex Determining Region-Y) Box-2). The Pluripotent state may also be regulated by epigenetic mechanisms involving members of the Polycomb group proteins (Ref.2).

A major signaling pathway contributing to Mouse ESC Pluripotency consists of LIF. LIF belongs to the family of IL-6 (Interleukin-6)-type cytokines, which signals through the common receptor subunit GP130 (GP130 Transducer Chain), along with a ligand-specific receptor subunit (such as the LIFR (LIF Receptor)). GP130 is ubiquitously expressed and plays a role in a variety of cellular processes. Numerous GP130 ligands are produced during early stages of development, contributing not only to the self-renewal of pluripotent cells but also to the development of numerous differentiated cell types. LIF induces heterodimerization of LIFR and GP130. On binding LIF, the intracellular domains of the LIFR¡VGP130 heterodimer recruit the non-receptor JAK (Janus tyrosine Kinase) and the antiphosphotyrosine Immunoreactive Kinase (TYK (Protein-Tyrosine Kinase)) and become phosphorylated. The phosphorylated intracellular domains of the LIFR / VGP130 heterodimer function as docking sites for proteins that contain SH2 domains, which include the transcription factor STAT3 (Signal Transducer and Activator of Transcription-3). Phosphorylation of STAT3 induces nuclear translocation and activation of transcription. Activation of STAT3 is sufficient to maintain Mouse ESC self-renewal in the presence of serum by induction of Oct4 and c-Myc gene. In addition to STAT, LIF stimulation induces other signaling pathways through effector protein, Shp2 (Tyrosine Phosphatase Shp2), which can bind to Y757 of the intracellular domain of GP130 (Y118 in the cytoplasmic domain) and Y969 of LIFR (Y115 in the cytoplasmic domain). When phosphorylated by JAK, Shp2 binds the adaptor protein GRB2 (Growth Factor Receptor-Bound Protein-2) and activate the Ras/ ERK (Extracellular Signal-Regulated Kinase) signaling pathway. However, whereas LIF-induced STAT3 activation promotes self-renewal, LIF-induced ERK activation promotes differentiation, maintaining a balance between STAT3 and ERK signals, thus contributing to the determination of Mouse ES cell fate and suppression of differentiation to Endodermal and Mesodermal lineages. Phosphorylated Shp2 also binds GAB, activating the PI3K/Akt pathway. The PI3K pathway is also activated endogenously by ERas, a novel small GTP binding protein specifically expressed in Mouse ES cells. ERas lacks several amino acids that are conserved among other Ras family proteins and essential for GTPase activity. ERas specifically binds to and activates PI3K. The PI3K pathway inhibits ERK signaling probably through the phosphorylation of Raf by Akt. LIF also activates additional signals including the Ribosomal S-6 kinases and Src kinases (Ref.3, 4 and 5).

Another Extrinsic factor known to support Mouse ESC self-renewal is BMP4 (Bone Morphogenic Protein-4), a TGF-Beta (Transforming Growth Factor-Beta) super family member. In the presence of LIF, BMP4 contributes to the LIF cascade, enhancing the self-renewal and pluripotency of ESCs by activating the gene encoding the transcription factor SMAD4 (Similar to Mothers Against Decapentaplegic homologue-4), which, in turn, activates members of the Id (Inhibitor of Differentiation) gene family. This interaction is facilitated in the presence of serum. The action of BMP is mediated by heterotetrameric Serine/threonine Kinase receptors. Specific receptor subunits that bind to BMP4 include BMPRII (BMP Receptor Type-I) receptor ALK3 (Activin Receptor-Like Kinase-3) and BMPRII (BMP Receptor Type-II) receptor. Upon dimeric BMP4 binding to the receptor complex, BMPRII phosphorylates and activate ALK3. Activated ALK3 causes the phosphorylation of SMAD1 (Similar to Mothers Against Decapentaplegic homologue-1) and SMAD5 (Similar to Mothers Against Decapentaplegic homologue-5) molecules. Upon phosphorylation, these BMP-specific SMADs form a complex with the co-SMAD, SMAD4 and translocate into the nucleus to activate transcription of Id genes, an inhibitor for basic helix-loop-helix transcription factors known to be involved in many cell fate determinations, including neural differentiation. BMP4 thus prevent neural differentiation of Mouse ES cells through the induction of Id expression. Besides activating SMADs, ALK3 also dephosphorylates p38 and ERK and thus inactivates them by unknown mechanisms. This inactivation relieves the negative effects of MAPKs (Mitogen-Activated Protein Kinases) on Pluripotency. As a result of the activation of ALK3, transcription of XIAP (X-Linked Inhibitor of Apoptosis Protein) is up-regulated either by means of the inhibition of p38, ERK or activation of SMAD1, 5, 8 pathways. XIAP protein in the cytoplasm interacts with ALK3, TAB1 (TAK1-Binding Protein-1) to result in the activation of TAK1 (Transforming Growth Factor-Beta-activated Kinase-1) by ALK3, consequently, a recovery of p38 activity. In the absence of ALK3, ALK1 (Activin Receptor-Like Kinase-1), or ALK2 (Activin Receptor-Like Kinase-2) proteins act as BMP4 type I receptor to maintain the pluripotency of ES cells. XIAP does not cause the loss of pluripotency in the absence of ALK3, presumably because of its inability to interact with ALK1 or ALK2. In the presence of LIF, activated STAT3 interacts with T and binds the Nanog promoter, resulting in upregulation of Nanog gene expression. Elevated levels of Nanog, in turn, block the activity of BMPs by interfering with their effector SMAD1, thus limiting mesoderm progression and ultimately maintaining the undifferentiated state of mouse ES cells. BMP proteins are also targets of another ligand cascade that is initiated on binding of WNT to its receptor (Ref.2, 6, 7 and 8).

In addition to LIF and BMP4, WNT pathway also delays the onset of differentiation of Mouse ES cells. However, the role of WNT/ Ctnn-Beta (Catenin-Beta) signaling in ES cell self-renewal is a debatable issue as different models have been proposed. In the absence of WNT activation, Ctnnb1Ctnn-Beta, a cytoplasmic protein which acts as an intracellular signaling molecule of the canonical WNT signaling pathway, is phosphorylated by a complex consisting of APC (Adenomatous Polyposis Coli) gene, AXIN (Axis Inhibitor), and GSK3Beta (Glycogen Synthase Kinase-3-Beta). Phosphorylated Ctnn-Beta is degraded by the Ubiquitin proteasome system, thereby keeping the level of cytoplasmic Ctnn-Beta low. The canonical WNT pathway is activated on binding of the WNT protein to the Frizzled receptor on the cell membrane. Activation of the pathway leads to inhibition of GSK3Beta, subsequent nuclear accumulation of Ctnn-Beta, where it associates with LEF (Lymphoid Enhancer Factor)/ TCF (T-Cell Factor) and CBP and causes expression of target genes like c-Myc, Oct4, Rex1 and STAT5, which are critical for stem cell proliferation. Recently it is found that LEF/ TCF1 complexes with SMAD4, Co-SMAD in the BMP pathway, to cooperatively control gene expression in Mouse ES cells. Activation of the canonical WNT pathway maintains the undifferentiated phenotype in Mouse ESCs, and sustains expression of the pluripotent-state-specific transcription factors Oct4, Rex1 and Nanog in the absence of supplemented LIF. Ctnn-Beta is involved in upregulation of Nanog through binding with Oct3/4. Involvement of c-Myc, a direct target molecule of Ctnn-Beta, in the self-renewal of ES cells has also been demonstrated recently. GSK3Beta, on the other hand, negatively regulates c-Myc activity by phosphorylation following degradation by the Proteasome system. Thus c-Myc is a common target of both LIF and WNT signaling pathways (Ref.5, 9, 10 and 11).

ES cells are not only capable of self-renewing, but also of exhibiting pluripotency, a feature maintained by the core network of transcription factors comprising Oct4, SOX2, and NANOG. The expression levels of Oct4, SOX2, and Nanog are subject to an autoregulatory feedback loop.These regulators have distinct roles but may function in related pathways to maintain the developmental potential of ES cells. Disruption of Oct4 or Nanog results in the inappropriate differentiation of ICM and ES cells to trophectoderm and extra-embryonic endoderm, respectively. In contrast, overexpression of Oct3/4 in ES cells induces differentiation into primitive endoderm and mesoderm lineages. The expression level of Oct3/4 is an important determinant of cell fates in mouse ES cells. The maintenance of pluripotent ES cell self-renewal by Oct4 requires functional LIF/ STAT3 and BMP/ GDF/Id signaling cascades, but the function of LIF/ STAT3 does not seem to be the maintenance of Oct4 expression. Overexpression of Nanog, in turn, circumvents the necessity of either LIF or BMP/ GDF stimulation (Ref.1, 12 and 13).

Oct4 is known to interact with other transcription factors to activate and repress gene expression in mouse ES cells. Oct4, a member of the POU (PIT/OCT/UNC) class of homeodomain proteins, can heterodimerize with the HMG-box transcription factor, SOX2 and a Forkhead family transcription factor, FoxD3 (Forkhead Box Protein-D3), to affect the expression of several genes in Mouse ES cells. SOX2 is a member of the HMG-domain DNA-binding-protein family that is implicated in the regulation of transcription and chromatin architecture. Although both Oct4 and SOX2 have independent roles in determining other cell types, at least part of their function in pluripotent cells is via a synergistic interaction between the two to drive transcription of target genes. Most common targets of SOX2- Oct4 synergy are FGF4 (Fibroblast Growth Factor-4), UTF1 (Undifferentiated embryonic cell Transcription Factor-1), Fbx15 (F-box protein-15), Nanog, LEFTY1 (Left-right determination factor-1), SOX2 and Pou5f1 (the gene encoding Oct4). Besides SOX2, FOXD3 also plays an important role in pluripotency by interacting with Oct4. Oct4 maintains Nanog activity by directly activating its promoter at sub-steady-state concentration but repressing it at or above steady-state levels. On the other hand, FOXD3 behaves as a positive activator of Nanog to counter the repressive effect of Oct4. The expression of Oct4 is also activated by FOXD3 (Ref.14 and 15).

In addition to Oct4, a homeodomain containing protein Nanog, also sustain pluripotency in ES cells even in the absence of LIF. During development, Nanog function is required at a later point than the initial requirement for Oct4. Nanog expression is regulated by external signal pathways like WNT Pathway. In addition to the external signal pathways, intrinsic transcription factors such as FOXD3, p53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and SOX2 to control a set of target genes that have important functions in ES cell pluripotency. The major downstream target in mouse includes ESRRB (Estrogen Related Receptor-Beta) and RIF1 (Rap1 Interacting Factor-1 homolog (yeast)). ESRRB belongs to the superfamily of nuclear hormone receptors, and is necessary to block the differentiation into mesoderm, ectoderm and neural crest cells but are not required to repress trophectoderm differentiation. RIF1 is an ortholog of a yeast telomeric protein and is upregulated in mouse ES and germ cells. Other genes regulated by Nanog include Tbx3 (T-box 3) and TCL1 (T-Cell Lymphoma breakpoint-1). The function of TCL1 is to repress only a subset of neural crest genes, whereas Tbx3 blocks the mesoderm and ectoderm differentiation. Downregulation of Nanog, SOX2, ESRRB, Tbx3 or TCL1 leads to the immediate induction of Otx2, Pitx2 (Paired-like homeodomain Transcription factor-2), Sox18 and probably additional genes. All three genes are expressed in the epiblast; Otx2 and Pitx2 are important for mesodermal and neuroectodermal development in vivo. Oct4 and Nanog both bind to Mycn (v-Myc Myelocytomatosis viral related oncogene, neuroblastoma derived (avian)), which has recently been reported to be among the key mediators in the self-renewal and proliferation of ES cells. Besides, Nanog and Oct4 directly bind to the promoter regions of Snai1 (snail homolog-1), Kfl6 (Kruppel-like factor 6) and Klf7 genes. Sall4 (Sal-like 4 (Drosophila)), a member of Spalt-like protein family, interacts with Nanog and exists as a complex with Nanog. In addition, Sall4 and Nanog also bind to and regulate the respective regulatory regions of their own genes. Sall4 and Nanog employ autoregulatory feedforward loops to maintain their expression. Nanog might also regulate differentiation through the transcriptional repression of genes that promote differentiation (for example, endodermal GATA-binding protein-6, Gata6). Besides, Nanog also regulate the expression of Oct4 and SOX2. In addition, Nanog also controls important molecular effectors of ES cell fate, as exemplified by FOXD3 and SETDB1 (SET domain, bifurcated-1). Stem cells are capable of producing virtually all cell types in our body, thus possessing great therapeutic potential for many degenerative conditions such as Cancer, Arthritis, and Parkinson diseases. Replacement of aged organs with new ones generated through stem cells and bioengineering would offer enormous hopes for the aging population (Ref.1, 19 and 20).


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  2. Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells.
  3. Epidermal stem cell fate: what can we learn from embryonic stem cells?
  4. Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells.
  5. Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells.
  6. TGFbeta in the differentiation of embryonic stem cells.
  7. BMP signalling inhibits premature neural differentiation in the mouse embryo.
  8. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells.
  9. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency.
  10. Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells.
  11. wnt3a but not wnt11 supports self-renewal of embryonic stem cells.
  12. BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells.
  13. Transcriptional control of pluripotency: decisions in early development.
  14. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.
  15. A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells.
  16. Nanog and transcriptional networks in embryonic stem cell pluripotency.
  17. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression.
  18. The stem cell pluripotency factor NANOG activates transcription with two unusually potent subdomains at its C terminus.
  19. A heterogeneous expression pattern for Nanog in embryonic stem cells.
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