Regulation of Apoptosis and Differentiation by p53 in Human ...

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CHAPTER 1: Introduction 1.1.4.2- FGF-2 FGF-2, also known as basic fibroblast growth factor (bFGF), is a member of a family of over 20 polypeptide growth factors (Ornitz and Itoh, 2001) FGFs are found in different species ranging from nematodes to humans. The recombinant FGF-2 is a 16.5-kDa peptide with 144 amino acids (Okada-Ban et al., 2000). The biologic activity of FGF-2 is mediated by four related transmembrane tyrosine kinase receptors: FGFR-1, -2, -3, and -4, and possibly other transmembrane receptors (Powers et al., 2000; Khurana and Simons, 2003). Ligand binding results in homodimerization of these receptors, trans-phosphorylation and signal transduction. Numerous splice variants of multiple genes generate a wide diversity of FGFRs. FGF-2 has a pleiotropic effect on different types of cells and its cellular response can be affected by type of receptor binding, rate of intracellular growth factor uptake and interacting molecules inside the cell (Goldfarb, 2001). FGF-2 can act as a survival factor by blocking apoptosis (Fisher, 1997; Stachowiak et al., 1997), it also stimulates angiogenesis (Slavin, 1995; Bikfalvi, 1995) and cell proliferation via activation of the Ras/Raf-MAPK pathway by enabling adaptor proteins Grb2, Shc, and Nck to participate in signal transduction cascades (Klein and Schneider, 1997). It can also promote tumor progression (Bikfalvi, 1995; Vacca et al., 2001). FGF-2 was been used in hESC culture (Xu et al., 2001; Amit et al., 2004) in serum- and feeder-free culture methods because it inhibits hESC differentiation. Two studies were published that show that higher doses of FGF-2 (100 and 40 ng/ml) override the requirement for MEF feeders or its conditioned medium on maintenance of hESC and this effect is mediated by the inhibition of BMP signaling (Xu et al., 2005a; Xu et al., 2005b; Levenstein et al., 2006). Bendall and colleagues have found that under feeder-free culture conditions, the hESC organize themselves in order to create a niche in which derived hESC derived fibroblast-like cells are dependent on FGF-2 and in response to this factor express IGF-1 reported to promote selfrenewal of undifferentiated hESC (Bendall et al., 2007). 1.1.4.3- Transforming growth factor β (TGFβ) pathway TGFβ superfamily members play an important role in a wide range of cellular processes including tissue differentiation, morphogenesis, proliferation, migration and apoptosis in embryonic development, as well in adult homeostasis and response to disease and injury (Massague, 1990). The TGFβ superfamily of ligands include TGFβ, Activin, Nodal, Bone morphogenetic proteins (BMP) and Growth/Differentiation factors (GDF), among others. All of these have been associated with ESC (Valdimarsdottir and Mummery, 2005). These ligands bind to heteromeric complex of serine/threonine kinase receptors called TGFβ type I and type II receptors. The type I receptor acts downstream of the type II receptor and transduces the signal through the phosphorylation of Smads (Massague, 2000; Piek et al., 1999), wich play theirs role in the nucleus acting as transcription factors or target genes. It has been shown that maintenance of hESC in an undifferentiated state requires the action of the TGFβ/Nodal/Activin branch of the TGFβ superfamily (Amit et al., 2004; Ludwig et al., 2006b; Vallier et al., 2005; Vallier et al., 2004). It has been suggested that TGFβ1 prevents differentiation - 10 -
CHAPTER 1: Introduction along the primitive endoderm lineage (Poon et al., 2006). On the other hand, BMP signaling needs to be repressed to prevent differentiation into to primitive endoderm or along the trophoblast lineage (Pera et al., 2004; Xu et al., 2002c). While TGF, Activin, and Nodal bind to a set of receptors that stimulates the activation of SMAD 2/3, BMP4 binds to different receptors and activates the SMAD 1/5/8 signaling (Valdimarsdottir and Mummery, 2005). Culture medium supplemented with Activin A can maintain hESC in the undifferentiated state for over 20 passages without the need for feeder layers or conditioned medium from MEF (Beattie et al., 2005; Xiao et al., 2006). The bone morphogenetic protein antagonist, Noggin, in combination with FGF-2, was shown to maintain the pluripotency of hESC in the absence of feeder layers (Wang et al., 2005). These three growth factors are all expressed in MEF, suggesting that they may be important factors secreted by MEF for maintaining undifferentiated hESC. 1.1.4.4- MEF secreted factors A great deal of research is focusing on the identification of factors that are secreted by MEF. Conditioned medium from MEF is sufficient for the maintenance of hESC on Matrigel. This suggests that secreted factors produced by MEFs can activate the molecular programs required for maintaining hESC in an undifferentiated state. The identification of these factors, signaling pathways and downstream target genes is fundamental for the future of hESC research, especially because the ultimate goal is to produce chemically defined and xeno-free media to culture these cells. A genome-wide expression profiling performed by Greber and colleagues revealed that FGF-2 acts on MEF to regulates the expression of key members of the TGFβ pathway (upregulates Inhba, Tgfb1, Grem1, and downregulates Bmp4) that are most likely to influence hESC self-renewal (Greber et al., 2007). They also showed that FGF-2 has similar effect on hESC, suggesting that TGFβ ligands act in an autocrine manner. 1.1.4.5- Other factors Another study claimed that activation of the canonical Wnt pathway is sufficient to maintain selfrenewal of both hESC and mESC and this was achieved by using a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), 6-bromoindirubin-3'-oxime (BIO) (Sato et al., 2004). However, data reported by others (Dravid et al., 2005) and in our laboratory (Kathryn Davidson, personal communication) do not support the findings claimed by Sato and colleagues. Wnts seem to promote proliferation and inhibition of apoptosis of hESC rather than promoting self-renewal. Pebay and colleagues (Pebay et al., 2005) have also reported a system for the maintenance of stem cells. They have shown that sphingosine-1-phosphate and PDGF, two known mitogens components of serum, can maintain hESC self-renewal in a serum-free culture medium but still in the presence of feeder layers. They suggested that this is mediated by activation of the extracellular signal-regulated kinases (ERKs). In summary, different pathways seem to be required for ESC maintenance, proliferation and repression of differentiation. - 11 -
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CHAPTER 3: Characterization of hESC
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CHAPTER 6: Bibliography - 128 -
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CHAPTER 6: Bibliography Benard, J.,
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CHAPTER 6: Bibliography Duensing, A
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CHAPTER 6: Bibliography Itskovitz-E
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CHAPTER 6: Bibliography Melino, G.,
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CHAPTER 6: Bibliography Wei, C. L.,
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APPENDIX I - 144 -
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APPENDIX I 4% Paraformaldehyde (PFA
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TABLE A 2.1- List of genes upregula
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PMP22 1.76 0.70 NM_153322 PDE6G 1.8
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TABLE A 2.2- List of functions of u
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Protein Synthesis Ophthalmic Diseas
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1.73 0.897 CN292254 LOC651029 1.73
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Reproductive System Disease Cellula
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