Reproduction Abstracts

Human embryonic stem cells (hESCs) proliferate by self-renewal and hold much promise for regenerative medicine since they have the potential to develop into all cells of the body. In culture, hESCs are difficult to maintain as they have a propensity to spontaneously differentiate. A low, 5% oxygen concentration (hypoxia) promotes hESC maintenance but the mechanisms which regulate this effect are unknown. We hypothesise that changes in environmental oxygen alter the expression of microRNAs (miRNAs) to regulate hESC pluripotency. TaqMan human miRNA arrays (Card A, Applied Biosystems) were performed on hESCs cultured at either 5%, or 20% oxygen and 40 miRNAs were found to be differentially expressed. Bioinformatic analyses of a subset of miRNAs that were down-regulated under hypoxic compared to atmospheric oxygen tensions were predicted to target NANOG, a key transcription factor regulating hESC pluripotency. RT-qPCR was used to confirm a significant down-regulation of these miRNAs. To determine whether these miRNAs regulate NANOG expression, specific pre-miRNAs or negative control pre-miRNAs were transfected into hESCs cultured at 5% oxygen. Using Western Blotting, NANOG protein expression was found to be significantly reduced. Dual-luciferase reporter assays are currently being performed to determine whether these miRNAs bind directly to the NANOG 3′UTR. These data suggest that miRNA expression is altered by environmental oxygen and regulate hESC self-renewal. Funded by the Gerald Kerkut Charitable Trust, the University of Southampton and the Society for Reproduction and Fertility.