Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells

Abstract We have previously demonstrated that hypoxia is benefit for maintaining the stemness of cultured human endothelial progenitor cells (EPCs), but the mechanism is still unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of EPCs under hypoxia. We identified the metabolic status of EPCs by using extracellular flux analysis, LC-MS/MS, and 13C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC increased cell stemness under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Although hypoxia decreased mitochondrial adenosine triphosphate (ATP) level, the total ATP level remained unchanging, indicating that energy production does not play a major role in this process. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of EPCs, thereby improving the efficiency of cell expansion in vitro.