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ESTABLISHMENT IN CULTURE OF MOUSE AND HUMAN STEM CELLS WITH EXPANDED FATE POTENTIAL 10.17863/CAM.22809 Ryan, David John 0000000273272133 Liu, Pentao 0000000157749678 Watts, Colin The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development. University of Cambridge 2018-07-21 Thesis Doctoral Doctor of Philosophy (PhD) en Wellcome Trust Clinical PhD Programme for Academic Clinicians https://www.repository.cam.ac.uk/handle/1810/275567 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/07815edb-31c0-440a-a8eb-2c54b55b2eff/download aca56bcc4a4932bc7e0d7e78e29e326f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/7c109bb9-1ac9-4b13-bbd4-5a25d0970b03/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Expanded Potential Stem Cells Expanded Potential Stem Cell Medium Embryonic Stem Cells Preimplantation Mouse Embryo Blastomeres Totipotency