To further investigate the role of PKM2 in the regulation of hESC metabolism and pluripotency under hypoxic conditions, siRNA was used to silence PKM2 expression. a glycolytic enzyme. PKM2 expression was increased in hESCs SB 204990 cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two individual mechanisms by which genes regulating glucose uptake and SB 204990 metabolism are involved in the hypoxic support of pluripotency in hESCs. Human embryonic stem cells (hESCs) are derived from the inner cell Rabbit polyclonal to PLK1 mass of the blastocyst and are pluripotent; they have the capacity to differentiate into all cell-types in the human body1,2,3,4. Thus hESCs have great potential to provide cellular therapy for a range of diseases. For this hope to be realised with security and efficiency, hESCs need to be managed as highly pluripotent populations in the absence of spontaneous differentiation. Much data suggests that environmental culture conditions and specifically the oxygen tension have an impact around the maintenance of pluripotency. Use of low environmental oxygen tensions has been shown to reduce the amount of spontaneous differentiation, as well as being beneficial for hESC maintenance in terms of increased expression of important pluripotency markers and decreased incidence of chromosomal abnormalities5,6,7,8,9,10. Maintenance of hESCs at atmospheric oxygen has also been found to decrease hESC proliferation and glycolytic and amino acid metabolism of hESCs9,10,11. Higher rates of glucose uptake and lactate production were measured in hESCs cultured at 5% oxygen than in those managed at 20% oxygen, which was mirrored by the increased expression of the pluripotency markers OCT4, SOX2, and NANOG9. Interestingly, this association between glycolytic metabolism and pluripotency was also exhibited in hESCs cultured at 5% oxygen in the absence of FGF2, where a reduction of SOX2 expression, glucose uptake and lactate production was observed when compared with hESCs cultured in the presence of FGF29. These findings suggest that a high rate of glucose uptake and lactate production is characteristic of highly pluripotent stem cells and that hypoxia might be beneficial for the maintenance of hESCs at least partially by supporting glycolytic metabolism. Importantly, expression of many glycolytic genes has been shown to be promoted under hypoxia in other cell-types, providing a mechanism by which hypoxic conditions might regulate metabolism in hESCs12,13,14,15,16. How glucose metabolism is regulated in hESCs is not known, but access into the cell via glucose transporters is likely to be important. However, which glucose transporter is responsible for glucose uptake SB 204990 in hESCs is not known. The glucose transporter GLUT1 has been found in many cell types, and its expression has been found to be regulated by hypoxia in mouse ESCs (mESCs)17,18,19,20. Expression of mRNA was also found to be increased in hESCs cultured at 5% oxygen compared with those at atmospheric oxygen, suggesting that its regulation may drive changes in rates of glucose consumption with changing environmental oxygen tension9. This hypoxic promotion SB 204990 of GLUT1 expression was demonstrated to be regulated by HIF-29. GLUT3 had been considered to be a neuron-specific glucose transporter, but a much wider tissue distribution has since been exhibited in humans21,22,23. GLUT3 has a higher affinity for glucose than GLUT1 and has a high turnover, which makes it an efficient transporter24,25. Silencing GLUT3 expression in murine blastocysts led to a SB 204990 greater decrease in glucose uptake than silencing GLUT1 expression, suggesting that GLUT3 might be more important for glucose uptake, at least in preimplantation development26. Expression of both transporters, GLUT1 and GLUT3, is regulated by hypoxia in mouse blastocysts27. Glucose utilisation may also be regulated through the activity of glycolytic enzymes. Pyruvate kinase catalyses the breakdown of phosphoenolpyruvate to produce pyruvate and ATP. As this reaction is the final rate-limiting step of glycolysis, it is possible that the rate of glucose uptake and lactate production is controlled through regulation of this step. PKM1 and PKM2 are two splice variations from the gene that differ by just 23 proteins due to on the other hand spliced exons 9 or 10, respectively28. PKM2 continues to be found to market the Warburg impact in tumor cells, which details an elevated reliance on glycolysis when plenty of air can be designed for oxidative phosphorylation29 actually,30,31. Knockdown of PKM2 in tumor cell lines led to decreased prices of glycolytic rate of metabolism and decreased cell viability, but, oddly enough, cell viability had not been decreased after PKM2 knockdown in human being adult pores and skin fibroblasts or human being umbilical vein endothelial cells29,32..