Characterisation of a novel culture condition for the establishment and maintenance of mouse embryonic stem cells and implications for the mechanisms of self-renewal.
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Date
2009Author
Wray, Jason Patrick
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Abstract
Pluripotency is defined as the ability of a cell to give rise to all the cell types of the
adult organism. In vivo this property is possessed transiently by the cells of the
epiblast in the developing embryo but it can be maintained indefinitely by deriving
embryonic stem (ES) cells. How the pluripotent state is established in the cells of the
early embryo and how it is ‘captured’ and maintained in the form of ES cells is a
fascinating question for biology with practical implications. It is hoped that ES cells
will be of use in biomedical research and cell replacement therapy. Our
understanding of their biology and our ability to manipulate the cells in vitro will be
of great importance if these hopes are to be realised.
The starting point for the work presented in this thesis was the development of a
novel culture condition for the derivation and maintenance of mouse ES cells (Q-L.
Ying and J. Nichols). The media is formed by the addition of three small molecule
inhibitors to a previously described serum-free media, N2B27, and is termed 3i
(three inhibitors).The inhibitors are SU5402, PD184352 and CHIRON99021, and
they inhibit the FGF receptor, mitogen activated protein/extracellular signalregulated
kinase (ERK) kinase (MEK), and glycogen synthase kinase 3 (GSK3)
respectively.
I attempt to further our understanding of pluripotency and self-renewal in ES cells by
genetic and biochemical examination of ES cells cultured in 3i. Analysis of
intracellular signalling pathways together with descriptions of genetic mutants for the
targets of the inhibitors validates the mode of action and the specificity of the three
inhibitors. Self-renewal of mouse ES cells is considered dependent on activation of
STAT3 through provision of the cytokine leukaemia inhibitory factor (LIF). I
demonstrate unequivocally that this pathway is not required for self-renewal in 3i by
characterising Stat3-null ES cells. Further experiments reveal that preventing
activation of ERK downstream of the growth factor FGF4, produced by the ES cells
themselves, is key to preventing differentiation. Pleiotropic effects of GSK3
inhibition are observed and candidate GSK3 targets with known or predicted effects
on self-renewal are investigated as potential downstream effectors. I propose that
activation of canonical Wnt signalling, together with a global derepression of
biosynthetic capacity, mediate the pro-self-renewal effects of GSK3 inhibition.
The description of culture conditions that function independently of signalling
pathways previously thought essential for self-renewal provides fresh insight into the
nature of ES cell self-renewal and the relationship of ES cells to the pluripotent cells
of the developing embryo. There are practical implications for ES cell biology as
there is reason to hope that the new conditions will translate more readily to other
mammalian species to facilitate the derivation of ES cells and will provide an
optimal platform for differentiation of ES cells into somatic cell types of interest.