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Defining The Role Of Oxygen Tension In Human Pluripotent Stem Cell Fate Decisions

Mohammed, Reema; (2019) Defining The Role Of Oxygen Tension In Human Pluripotent Stem Cell Fate Decisions. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Early embryo development occurs in a relatively low oxygen microenvironment in the reproductive tract (1.5- 5.3% O2). Human Pluripotent Stem Cells (hPSCs) are routinely cultured in atmospheric condition (20% O2). The majority of in vitro protocols compare 20% O2 with one or two 'hypoxic‘ conditions (range from 1%-5% O2). Therefore, there is a lack of information on how intermediate oxygen tensions might affect hPSCs behaviour. In this project, we aimed to define the role of oxygen tension in regulating self-renewal and early differentiation properties of hPSCs. Using short-term monolayer protocol, hPSCs were cultured in parallel under a full spectrum of oxygen levels (0%, 2%, 5%, 8%, 12% and 20% O2). Cells were examined for morphological changes, growth kinetics, and expression of genes associated with pluripotency, embryonic germ layers, metabolism and hypoxia using qualitative RT-PCR, Immunostaining and Flow Cytometry. Our results revealed that culturing within a threshold of 2 to 5% O2, was more beneficial for maintaining the self-renewal capacity of hPSCs based on morphology, cell growth and OCT4 and NANOG expression. Although cells under 2% and 5% O2 conditions exhibited more uniform phenotypic profile which was associated with slow mitotic division, some signs of differentiation were observed under 5% O2. Spontaneous differentiation of hPSCs under mild hypoxia (8% and 12% O2) revealed striking morphological changes indicating the acquisition of a mesenchymal-like population that displayed positive expression of BRACHYURY, α-SMA, S100A4 and Vimentin. RT-qPCR results demonstrated cadherin switch that was coincided with SNAIL up-regulation. Which indicate the acquisition of EMT-like event during mesodermal commitment similar to that observed in vivo during early gastrulation. Interestingly, when mild hypoxia combined with directed mesoderm differentiation medium, a noticeable increase in mesoderm- and EMT- associated markers observed at faster kinetics. This study provides evidence for the importance of oxygen condition in regulating stem cells fate.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Defining The Role Of Oxygen Tension In Human Pluripotent Stem Cell Fate Decisions
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10081128
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