Characterisation of a novel culture condition for the establishment and maintenance of mouse embryonic stem cells and implications for the mechanisms of self-renewal

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Pluripotency is defined as the ability of a cell to give rise to all the cell types of the \nadult organism. In vivo this property is possessed transiently by the cells of the \nepiblast in the developing embryo but it can be maintained indefinitely by deriving \nembryonic stem (ES) cells. How the pluripotent state is established in the cells of the \nearly embryo and how it is ‘captured’ and maintained in the form of ES cells is a \nfascinating question for biology with practical implications. It is hoped that ES cells \nwill be of use in biomedical research and cell replacement therapy. Our \nunderstanding of their biology and our ability to manipulate the cells in vitro will be \nof great importance if these hopes are to be realised. \nThe starting point for the work presented in this thesis was the development of a \nnovel culture condition for the derivation and maintenance of mouse ES cells (Q-L. \nYing and J. Nichols). The media is formed by the addition of three small molecule \ninhibitors to a previously described serum-free media, N2B27, and is termed 3i \n(three inhibitors).The inhibitors are SU5402, PD184352 and CHIRON99021, and \nthey inhibit the FGF receptor, mitogen activated protein/extracellular signalregulated \nkinase (ERK) kinase (MEK), and glycogen synthase kinase 3 (GSK3) \nrespectively. \nI attempt to further our understanding of pluripotency and self-renewal in ES cells by \ngenetic and biochemical examination of ES cells cultured in 3i. Analysis of \nintracellular signalling pathways together with descriptions of genetic mutants for the \ntargets of the inhibitors validates the mode of action and the specificity of the three \ninhibitors. Self-renewal of mouse ES cells is considered dependent on activation of \nSTAT3 through provision of the cytokine leukaemia inhibitory factor (LIF). I \ndemonstrate unequivocally that this pathway is not required for self-renewal in 3i by \ncharacterising Stat3-null ES cells. Further experiments reveal that preventing \nactivation of ERK downstream of the growth factor FGF4, produced by the ES cells \nthemselves, is key to preventing differentiation. Pleiotropic effects of GSK3 \ninhibition are observed and candidate GSK3 targets with known or predicted effects \non self-renewal are investigated as potential downstream effectors. I propose that \nactivation of canonical Wnt signalling, together with a global derepression of \nbiosynthetic capacity, mediate the pro-self-renewal effects of GSK3 inhibition. \nThe description of culture conditions that function independently of signalling \npathways previously thought essential for self-renewal provides fresh insight into the \nnature of ES cell self-renewal and the relationship of ES cells to the pluripotent cells \nof the developing embryo. There are practical implications for ES cell biology as \nthere is reason to hope that the new conditions will translate more readily to other \nmammalian species to facilitate the derivation of ES cells and will provide an \noptimal platform for differentiation of ES cells into somatic cell types of interest.