Lewis, S.M.; (2012) Localisation of mTOR complex proteins in embryonic stem cells: relationship with cell division and survival. Doctoral thesis , UCL (University College London).

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Abstract

The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation in response to nutrients and growth factors. The role of mTOR in the biology of embryonic stem cells has not been extensively characterised. The principle aim of this research was to assess the feasibility of manipulating the mTOR pathway to provide bioprocessing improvements for the expansion and differentiation of embryonic stem cells. In order to achieve this aim, the cellular distribution and localisation of mTOR pathway phosphoproteins was assessed and the effect of pathway inhibition on ES cell proliferation and viability was characterised. The key findings of this study revealed that in mouse ES cells, mTOR pathway proteins were phosphorylated during mitosis. Secondly, inhibition of mTOR in growth factor-stimulated pluripotent stem cells impaired proliferation but not viability. Upon withdrawal of growth factors, cells lost their compact shape and exhibited a spread morphology. Under these conditions mTOR inhibition reduced the viability and proliferation of ‘compact’ cells but not ‘spread’ cells. Thus, during the expansion of ES cells the mTOR pathway may be stimulated. During early differentiation, inhibition of the pathway may improve the purity of the final cell preparation and reduce the prevalence of parent stem cells. In conclusion, the experimental findings discussed here show that temporal manipulation of the mTOR pathway in embryonic stem cells may provide bioprocessing advantages resulting in an increased yield of parent cell populations during expansion and increased purity of differentiated cells. The sensitivity of the mTOR pathway to the bioavailability of nutrients may offer a cost-effective route to optimising cell production in an industrial scale process. Further work to elucidate the role of the mTOR complex 2 in ES cell proliferation may provide an additional level of control to independently modulate cell proliferation and growth during expansion and differentiation.

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