Fynes, K; (2014) Oxygen controlled processing of pluripotent stem cells. Doctoral thesis , UCL (University College London).

Abstract

A major challenge facing the development of effective cell therapies is the efficient differentiation of pluripotent stem cells (PSCs) into pure populations. This is caused, in part, by the heterogeneous presence of functionally distinct subpopulations in undifferentiated PSCs. These can exhibit variable developmental potential, suggesting that there will be a heterogeneous response to differentiation cues, and a low yield of the target cell type. The importance of recapitulating the in vivo stem cell niche during stem cell process development is now widely acknowledged, and thus, manipulation of microenvironmental factors will be invaluable in engineering optimal in vitro conditions for cell culture. Lowering oxygen tension to physiological levels can affect both the expansion and differentiation stages. However, to date, there are no studies investigating the consequences of culturing PSCs under hypoxic conditions on subsequent lineage commitment at ambient oxygen levels. Mouse Embryonic Stem Cells (mESCs) were passaged three times at 2% O2 before allowing cells to spontaneously differentiate as embryoid bodies (EBs) in normoxic (20% O2) conditions. Maintenance of mESCs under hypoxia was associated with a population shift away from a Rex1-positive ICM-like (Inner Cell Mass-like) state, to a primed epiblast-like state exhibiting a significant increase in the expression of early differentiation markers FGF5 and Eomes. Conversely, expression of these committed markers was decreased in human induced pluripotent stem cells (hiPSCs) following the same hypoxic step, and was instead associated with a significant increase in Rex1 expression. Hypoxic preconditioning primed mESCs for their subsequent differentiation into mesodermal and endodermal lineages, as confirmed by increased gene expression of Eomes, Goosecoid, Brachyury, AFP, Sox17, FoxA2, and protein expression of Brachyury, Eomes, Sox17, FoxA2, relative to normoxic cultures. The effects extended to the subsequent formation of more mature mesodermal lineages. A significant upregulation of cardiomyocyte marker Nkx2.5 was also observed, and critically a decrease in the number of contaminant pluripotent cells after 12 days using a directed cardiomyocyte protocol. However, the impact of hypoxic preconditioning was to preferentially prime human cells for ectodermal lineage commitment during subsequent EB differentiation, with significant upregulation of Nestin and β3-tubulin. The research discussed in this thesis demonstrates the importance of oxygen-tension control during cell maintenance on the subsequent differentiation of both mouse and human PSCs, and highlights the differential effects. The reported results have indicated that further to focusing on the differentiation stage itself, it will be critical to consider the prior maintenance of PSCs to fully optimise future stem cell processes. Furthermore, the work has highlighted the importance of considering each stage of bioprocess development individually, to best recapitulate the transient conditions in vivo.

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