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

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.