Proliferation Of Mouse Bone Marrow-derived Mesenchymal Stem Cells In Hypoxic Culture

Objective:This study aims to observe the effects of a low oxygen concentration on the proliferation of mouse bone marrow-derived mesenchymal stem cells(MSCs) in vitro, and explore the mechanism of action in order to seek a suitable oxygen concentration for the in vitro expansion of these adult stem cells.Methods:Mouse bone marrow cells were exposed to 1% O2(hypoxia)and 18% O2(normoxia) to calculate the MSCs colony forming rate and the average area. To observe the amplification of MSCs both in normoxia and hypoxia, we use microscopy to observe the MSCs morphology, count cells and detect mitotic index. Also, we adopt the CCK-8 method to detect cell viability, flow cytometry to analysis cell circle and detect the apoptosis rate of cells, ELISA to detect the expression of hypoxia induced factor-1α(HIF-1α), immunocytochemical stainning method to detect the proliferating cell nuclear antigen(PCNA)and expression level of P53 protein.Results:The MSCs colony formation rate in hypoxia group was significantly lower than that in normoxia group, and the average colony area was less as well, but without statistical significance.Compared with normoxia group, the expression level of HIF-1αprotein of MSCs passage cells increased dramatically. After 72 h in hypoxia, the number of MSCs decreased that lead to a significant difference with normoxia. The mitosis index of MSCs in hypoxia was significantly lower, and the absorption value was slightly lower during CCK-8 experiment without significant differences. Compared with normoxia group, both the percentage of G0/G1 and S+G2/M phase of MSCs increased in hypoxia, but there was no significant difference. Regard to the apoptosis of MSCs, the difference was not significant though it shows lower in hypoxia. In hypoxia, PCNA labeling index of MSC cells reduced, while P53 labeling index increased, but no significant difference with the normoxia group as well.Conclusion:The results of this study demonstrate that 1% O2 can inhibit the proliferation of MSCs in vitro. However, the action mechanism is still not clear.