Role of environmental oxygen in regulating the proteome and fatty acid composition of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) are pluripotent, self-renewing cells capable of giving rise to derivatives of all three germ layers. However, hESCs have a propensity for spontaneous differentiation. Compared to a standard culture in normoxia (20% oxygen), hESCs maintained under hypoxic conditions (5% oxygen) exhibit a lower rate of spontaneous differentiation and upregulation of key genes regulating pluripotency. Little is known about the effect of low oxygen level on global protein expression and fatty acid content and synthesis in hESCs and mechanisms regulating its beneficial effect. This study aimed to determine the proteome of hESCs cultured at 5% or 20% oxygen and investigate changes in fatty acid composition and metabolism. Using mass spectrometry, 129 out of the 2674 detected proteins were differentially expressed in hESCs maintained at 5% oxygen compared to those cultured at 20% oxygen; 38 were upregulated (Log2FoldChange > 0.5) and 31 were downregulated (Log2FoldChange < -0.5) in cells maintained in hypoxia. Proteins involved in the cellular response to oxygen and autophagy were upregulated in hESCs cultured in hypoxic conditions. Pathway analysis revealed a strong association with functions such as cell death and survival. PARP-1 and caveolin-1, proteins regulating DNA repair mechanisms and cell membrane functions respectively, were significantly increased in hESCs cultured at 5% oxygen compared to 20% oxygen. Hypoxia was found to significantly alter the composition of fatty acids in hESCs compared to 20% oxygen. In particular, the concentration of eicosapentaenoic acid (EPA) was significantly increased in hESCs cultured at 5% oxygen. The expression of Δ5-desaturase (D5D), the enzyme mediating EPA synthesis, was also significantly higher in hESCs cultured in hypoxia. The expression of D5D in hypoxic conditions was found to be directly regulated by HIF2α which binds to a predicted hypoxia-response element within the FADS1 (gene encoding D5D) proximal promoter. HIF2α binding was significantly enriched in hESCs cultured at 5% oxygen. EPA was supplemented to the hESC culture to investigate whether EPA might be beneficial for cells maintained at 20% oxygen. Supplementation of EPA to the medium had no effect on the self-renewal, proliferation, reactive oxygen species generation or glucose metabolism but, was found to regulate the expression of D5D and caveolin-1. These data suggest that environmental oxygen regulates the phenotype of hESCs through changes in global protein expression. The regulation of D5D by HIF2α and increased EPA content suggests that fatty acid biosynthesis may have an important role in the maintenance of hESCs under hypoxic conditions. The better understanding of changes in hESCs maintained under hypoxic conditions could improve cell culture for future use in regenerative medicine and cell therapies.

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Identifiers

ORCID for Franchesca Houghton: orcid.org/0000-0002-5167-1694

ORCID for Philip Calder: orcid.org/0000-0002-6038-710X

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