[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.


CONCLUSIONS
Hypoxia can promote the proliferation rate of hMSCs, enhance the colony-forming ability and inhibit the differentiation of the osteoblasts.
Wang
Qingde
Q
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430022, PR China. wqd428@hotmail.com
Yang
Shuhua
S
Yang
Cao
C
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering
D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D015675:Osteocalcin; D000469:Alkaline Phosphatase