Hypoxia inducible factors regulate pluripotency and proliferation in human proliferation in human embryonic stem cells cultured at reduced oxygen tensions

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and OCT4 mRNA as well as OCT4 protein compared to hypoxic conditions. HIF-1alpha protein was not expressed at 20% oxygen and displayed only a transient, nuclear localization at 5% oxygen. HIF-2alpha and HIF-3alpha displayed a cytoplasmic localization during initial hypoxic culture but translocated to the nucleus following long term culture at 5% oxygen and were significantly upregulated compared to cells cultured at 20% oxygen. Silencing of HIF-2alpha resulted in a significant decrease in both hES cell proliferation and OCT4, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1 was concomitantly increased. HIF-3alpha up-regulated HIF-2alpha and prevented HIF-1alpha expression with knock-down of HIF-3alpha resulting in the reappearance of HIF-1alpha protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF-3alpha was found to regulate the expression of both HIF-1alpha and HIF-2alpha, it is HIF-2alpha which regulates hES cell pluripotency as well as proliferation under hypoxic condition