The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible remain 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 BM EPCs under hypoxia. We identified the metabolic status of BM 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 promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.

BackgroundThe benefits of hypoxia for maintaining the stemness of cultured human endothelial progenitor cells (EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. There is growing evidence to suggest a role for cellular metabolism in the regulation of stem cell fate and self-renewal. This study aimed at exploring changes in glucose metabolism and roles in maintaining EPC stemness under hypoxia.MethodsExtracellular flux analysis, LC-MS/MS and 13C tracing HPLC-QE-MS were used to establish EPC metabolic status. Then inhibitors of glucose metabolism were used to assess the impact of the dependent pathways on cell stemness. The key enzymes of glycolysis, tricarboxylic acid cycle (TCA), pentose phosphate pathway (PPP) and mitochondrial respiration were inhibited, and the cell survival rate, clone formation rate, mRNA expression of stemness markers, Nanog, Oct4, Klf4 and Sox2, and adenosine triphosphate (ATP) level were compared.ResultsReprogramming of pathways concerned with glucose metabolism was found under hypoxic conditions, including increased rates of flux through glycolysis and the pentose phosphate pathway (PPP), together with decreased flux through the tricarboxylic acid (TCA) cycle and mitochondrial respiration. Use of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and the mitochondrial electron transport chain (ETC) revealed that inhibition of glycolysis or the PPP impaired cell proliferation under both normoxic and hypoxic conditions. By contrast, inhibition of pyruvate oxidation, via targeting of the TCA cycle or ETC, increased cell stemness under normoxic conditions. Moreover, promotion of pyruvate oxidation reversed the effect of hypoxia in maintaining cell stemness. Although hypoxia decreased mitochondrial ATP level, total cellular ATP level was unchanged, indicating that energy production does not play a major role in hypoxic effects. ConclusionIn summary, hypoxia-induced reprogramming of pathways involved in glucose metabolism maintained EPC stemness. Artificial manipulation of cell metabolism may be a way to regulate EPC stemness, thereby facilitating in vitro cell expansion.

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.