Mechanisms regulating stem cell self-renewal

Abstract

Human embryonic stem cells (hESCs) hold potential in the field of tissue engineering to treat a wide range of diseases, given their capacity for both limitless self-renewal and differentiation to any somatic cell type. However, under standard culture conditions, hESCs have a tendency to spontaneously differentiate. Thus, research is required to understand the mechanisms that regulate stem cell self-renewal and the impact on hESC culture. Human embryonal teratocarcinoma cells (hECCs), the malignant counterparts of hESCs, are also pluripotent, proliferate by self-renewal, and provide a convenient alternative model to study the regulation of pluripotency. Accumulating evidence suggests that glycolysis and hypoxia, through the hypoxia inducible factor HIF-2α, are key regulators of hESC self-renewal, but how changes in metabolism affect gene expression is poorly understood. The aim of this study was to determine how glycolysis affected the epigenetic and metabolic regulation of hESC self-renewal maintenance under hypoxia.

Chromatin immunoprecipitation (ChIP) analysis showed that HIF-2α directly binds to a HRE site in the proximal promoters of the metabolic sensors CtBPs, which link the metabolic state of the cell to changes in gene expression. HIF-2α was also demonstrated to regulate the expression of the chromatin modifiers JMJDs in hESCs under hypoxia, except for JMJD2c. JMJD2c expression peaked within the first 48 hours of exposure to hypoxia and thus was regulated instead by HIF-1α. Inhibiting glycolysis with the addition of glycolytic inhibitors revealed a consequential decrease in JMJD, CtBP and pluripotency marker expression, but intriguingly also HIF-2α, in hESCs maintained under hypoxia by inducing a more heterochromatic state in the proximal promoters of key genes. ChIP analysis revealed that JMJD2a plays a role in hESC self-renewal by inducing a more euchromatic and accessible state around the HREs in the proximal promoters of OCT4, SOX2 and NANOG by removing H3K9me3 histone modifications. CtBPs were also demonstrated to have a role in hESC selfrenewal by acting as a transcriptional coactivator. Furthermore, the mechanisms regulating hESC self-renewal were compared to those in the malignant counterparts, hECCs. All mechanisms analysed were similar between the two cell types, except that pluripotency marker expression was not regulated by environmental oxygen in hECCs. Both HIF-1α and HIF-2α were expressed in hECCs maintained at 20% oxygen, and HIF-α subunit accumulation was caused by high levels of nitric oxide preventing HIF degradation by PHDs.

The data presented in this thesis has identified several mechanisms that enhance self-renewal including hypoxia, metabolic sensors, epigenetics, nitric oxide and most importantly glycolysis. Together, these data have uncovered a potential insight into how hESCs first adapt to hypoxia, but also mechanisms into how that cell identity is maintained and enhanced under long-term hypoxia. However, crucially, glycolysis appears to not be just a feature of pluripotency, but is intrinsic to the acquisition and maintenance of self-renewal.

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Identifiers

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

ORCID for Jeremy Blaydes: orcid.org/0000-0001-8525-0209

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