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Title: Investigation of phosphoinositide 3-kinase dependent signalling in the regulation of embryonic stem cell fate
Author: Kingham, Emmajayne
ISNI:       0000 0004 2674 8260
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2009
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In order to harness the therapeutic potential of stem cells, a clear understanding of the factors and mechanisms governing their fate is required. Self-renewal, pluripotency and proliferation are important cellular functions for maintaining the stem cell state. The class IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses including cell migration, proliferation and survival. Moreover, this class of PI3Ks were previously reported to play a role in proliferation and maintenance of self-renewal in murine embryonic stem cells (mESCs). Here the activation of PI3Ks, the intracellular signalling, including cross-talk between other important pathways, and the roles of specific catalytic subunits of the PI3K class I family have been investigated. Despite inhibition of PI3Ks giving rise to differentiated cell types, early lineage commitment of mESCs was shown to be regulated by pathways not involving PI3Ks. Differentiation towards mesoderm, endoderm and ectoderm were detected upon broad selectivity inhibition of PI3Ks with LY294002. Cross-talk between PI3K and MAPK pathway signalling was highlighted as a possible mechanism for PI3Ks to regulate self-renewal. Inhibition of PI3Ks with LY294002 led to an enhancement in MAPK pathway activation. On further investigation, activation of MAPK pathway signalling by inducible expression of constitutively active Mek brought forth a minimal reduction in self-renewal. Furthermore, inhibition of p110β induced an enhancement in Erk phosphorylation akin to that induced by LY294002, implicating this isoform in regulating MAPK signalling under normal mESC culture conditions. Insulin was shown to activate PI3Ks in mESCs and could be inhibited by treatment with pharmacological inhibitors of the p110α catalytic subunit isoform. Further investigation into the role of p110α in mESCs revealed a role in cell proliferation and metabolic activity. However, pharmacological or siRNA-mediated interference of this isoform did not perturb self-renewal. In contrast, p110β was identified as having a predominant role in the maintenance of self-renewal of mESCs. Both specific pharmacological inhibition and siRNA targeted knockdown of p110β led to a marked loss in alkaline phosphatase staining and a reduction in Nanog and Rex1 expression, indicating a loss of self-renewal. Thus, independent roles for p110α and p110β in regulating mESC proliferation and self-renewal were found to be the result of coupling to different PI3K catalytic subunit isoforms. Interestingly, reducing proliferation by inhibition of p110α or mTOR led to a greater decline in mESC self-renewal when induced by inhibition of p110β. This demonstration of cross talk between the pathways that regulate proliferation and self-renewal suggests a priming effect where the rate of proliferation could sensitise mESCs to levels of p110β activation in order to regulate self-renewal and ultimately cell fate.
Supervisor: Welham, Melanie Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available