Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis.

This study aimed to examine the proliferative behavior and molecular mechanisms of rat bone marrow-derived MSCs (rBMSCs) cultured under three different oxygen concentrations. Passaged rBMSCs exhibited significantly greater proliferation rates at 1% O2 and 5% O2 than those at 18% O2 and the cells exposed to 1% O2 showed the highest proliferative potential, which was evidenced by the growth curves, colony-forming efficiencies, and CCK-8 absorbance values. The rBMSCs grown under hypoxic culture conditions (1% O2 and 5% O2) had the increased percentage of cells in S + G2/M-phase and the decreased apoptotic index, compared with normoxia (18% O2). It was revealed for the first time that there were more phosphohistone H3 (PHH3)-positive cells and higher expressions of proliferating cell nuclear antigen (PCNA) in the hypoxic cultures of rBMSCs than in the normoxic culture. Hypoxia upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic proteins Bax and the cleaved caspase-3 in cultured rBMSCs. The levels of hypoxia-inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were increased in the hypoxic-cultured rBMSCs. Nevertheless, no significant difference was observed in p53 level of rBMSCs between different oxygen concentrations. In conclusion, the hypoxia exerts a promoting effect on the in vitro expansion of rBMSCs via several signaling and molecular pathways involved in the control of cell cycle and apoptosis.