[In vitro hypoxic culture of human marrow mesenchymal stem cells and their biological features in adults].

OBJECTIVE:To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia. METHODS:The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20% fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group (20% O2, DMEM-LG, Group A), the hypoxic group (1% O2, DMEM-LG, Group B), the normoxic osteoblast induction group (20% O2, conditioned medium, Group C), and the hypoxic osteoblast induction group (1% O2, conditioned medium, Group D). The biological features of the cultured hMSCs under hypoxia were assessed by the cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarin red staining. RESULTS:The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (P < 0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A (P < 0.01). After the induction, hMSCs in the Group B had a decreased number of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increased activity of ALP, which was significantly lower than that in the Group C (P < 0.01). The RT-PCR examination revealed that ALP, osteocalcin, and mRNA expressions of collagen type I and osteonectin in the Group C significantly increased (P < 0.01). By comparison among the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed. CONCLUSION:Hypoxia can promote the proliferation rate of hMSCs, enhance the colony-forming ability and inhibit the differentiation of the osteoblasts.