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

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CHAPTER 1: Introduction Xu et al (Xu et al., 2001) introduced a feeder free culture system for the first time for hESC culture. This consists of plating the cells on Matrigel, with MEF conditioned KSR medium supplemented with FGF-2 (Amit et al., 2004) (Figure 1.2). Matrigel TM Basement Membrane Matrix (BD Biosciences) is a solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, a tumour rich in extracellular matrix proteins. It contains collagen IV, laminin, heparin sulphate proteolycan and entactin, as well as several growth factors such as FGF-2, insulin-like growth factor-1, nerve growth factor (NGF), transformed growth factor β1 (TGFβ1), epidermal growth factor and platelet-derived growth factor (PDGF). Similarly, hESC can be cultured on Laminin (purified from EHS mouse tumours or from human placenta) (Xu et al., 2001) or Fibronectin (extracted from mouse, bovine or human plasma and human foreskin fibroblasts) (Amit et al., 2004). One of the biggest goals of hESC research is to culture the cells in the absence of animal products in order to minimize the risk of contamination with animal pathogens. Another goal is the development of a chemically defined media that could be used to test factors that modulate selfrenewal and differentiation, because serum can contain antagonist of those factors. Ludwig and colleagues have reported the development of a feeder-independent hESC culture composed of protein components uniquely derived from recombinant sources or purified from human material which they called TeSR1 (Ludwig et al., 2006b). This is a DMEM/F12 based medium supplemented with human serum albumin, FGF-2, LiCl, g-aminobutyric acid (GABA), pipecolic acid and TGFβ. They also derived two new cell lines in this defined media, but they developed chromosomal abnormalities. Because this media was highly costly, the same group have developed a modified version, the mTeSR1, which includes animal sources of protein (bovine serum albumin and Matrigel) and cloned zebrafish FGF-2 (Ludwig et al., 2006a). There are also studies currently being conducted that aim to identify components that support hESC self-renewal, especially the ones secreted by MEF, which will be described later. - 8 -
CHAPTER 1: Introduction Standard method Serum-free method on feeder layer Serum-free method on Matrigel •6x10 4 cells/cm 2 Feeders •Serum containing medium •Mechanical passaging •2x10 4 cells/cm 2 Feeders •Serum replacement medium plus FGF-2 •Non-mechanical passaging (enzymatic or non-enzymatic) •Matrices and no Feeders •MEF conditioned medium plus FGF-2 •Passaging (enzymatic or non-enzymatic) Overview of plate (day 7) Colony detail (at passaging) Overview of plate (day 1 after passaging) FIGURE 1.2- Different methods of culturing of hESC (adapted from Grandela and Wolvetang, 2007). 1.1.4- Maintenance factors of self-renewal and pluripotency In order to have a continuous source of hESC for research and therapeutic purposes, it is essential to maintain them for extended periods in their undifferentiated state. ESC self-renewal can be maintained in vitro by culturing cells in the presence of specific factors. 1.1.4.1- LIF LIF, a cytokine that belongs to the IL-6 family, is sufficient to maintain mESC without the presence of feeders or serum containing medium. LIF acts via heterodimerization of two members of class I cytokines, the low affinity LIF receptor (LIFR) and the signal transducer gp130 (Davis et al., 1993; Gearing et al., 1992) and ultimately activates the Jak/STAT pathway that is sufficient for self-renewal in these cells (Boeuf et al., 1997; Matsuda et al., 1999; Nakamura et al., 1998; Niwa et al., 1998). Although the LIFR and gp130 are present in hESC, and there is activation of STAT members in response to recombinant human LIF or mouse LIF (Daheron et al., 2004; Humphrey et al., 2004), this is not sufficient to maintain hESC in an undifferentiated state. - 9 -
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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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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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