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

Abstract

Background aimsThe 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. 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 BM EPCs 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. We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. On the contrary, inhibiting pyruvate oxidation, TCA or ETC increased cell stemness under normoxia mimicking hypoxic conditions. Although hypoxia decreased mitochondrial ATP level, the total ATP level remained unchanging, indicating that energy production does not play a major role in this process. ConclusionIn summary, hypoxia induced glucose metabolic reprogramming, and such pattern of 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.