Role of microRNAs in the hypoxic regulation of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) derived from the inner cell mass of the blastocyst, are pluripotent, capable of indefinite self-renewal and have the capacity to differentiate into all cells of the three germ layers. Thus, hESCs hold great potential for a wide range of applications such as regenerative medicine and drug development. However, improved propagation and use of hESCs in medical applications requires a better knowledge of the underlying mechanisms that regulate hESC pluripotency.

Hypoxia (5% oxygen) has been shown to be beneficial for maintaining pluripotent stem cells. Hypoxia inducible factors are crucial for this process, being stabilised under low oxygen conditions and promoting the expression of several pluripotency associated genes. However, microRNAs (miRNAs) as multi-purpose gene regulators might also have an important role in regulating these processes. Currently, little is known about specific miRNAs that may regulate pluripotency in hESCs in response to changes in environmental oxygen. Thus, this thesis aims to determine the involvement of miRNAs in the hypoxic regulation of hESCs.

Using miRNA arrays, differentially expressed miRNAs were found in hESCs cultured at 5% oxygen compared to those maintained at atmospheric, 20% oxygen. Validation of the arrays using RT-qPCR showed that hESCs cultured at 5% oxygen displayed increased levels of the hypoxamir miR-210 and decreased levels of miR-122-5p and miR-223-3p. Using bioinformatic analysis miR-122-5p and miR-223-3p were found to target the NANOG 3’UTR.

Using synthetic pre-miRs to transiently up-regulate either miR-122-5p or miR-223-3p in hESCs cultured at 5% oxygen significantly reduced NANOG protein expression. DualLuciferase-Reporter Assays and site-directed mutagenesis confirmed the location of a novel binding-site for miR-223-3p in the NANOG 3’UTR. The data presented in this thesis show that miRNA-223-3p directly regulates NANOG expression and that miRNAs have a physiological role in regulating the response of hESCs to environmental oxygen.

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Identifiers

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

ORCID for Tilman Sanchez-Elsner: orcid.org/0000-0003-1915-2410

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