JOURNAL ARTICLE

[Time differences of bone marrow mesenchymal stem cells and nucleus pulposus-like cells in a non contact co-culture system]

Lei Chen, Xiunian Hu, Lengxi Fu, Junjian Ye
Chinese Journal of Reparative and Reconstructive Surgery 2012, 26 (11): 1369-74
23230675

OBJECTIVE: To investigate time differences in directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells (NPCs) in a non contact co-culture system so as to search for the best time for transplantation in vivo.

METHODS: Six New Zealand white rabbits (aged 6 weeks, weighing 1.5-2.0 kg) were selected. BMSCs were collected and cultured for immunocytochemistry identification of CD34, CD44, CD45, and CD90; NPCs were isolated and identified immunocytochemically by RT-PCR. The 2nd passage BMSCs and the primary NPCs were co-cultured in a non contact co-culture system. The cell morphological changes were observed and the cell growth curves were made at 1, 3, and 5 passages after co-culture. The expressions of the aggrecan and collagen type II genes were detected by RT-PCR in BMSCs at 5, 10, and 15 days after co-culture; the expressions of the aggrecan and collagen type II proteins were detected by Western blot at 5, 10, 15, 20, 25, and 30 days after co-culture.

RESULTS: The expressions of CD44 and CD90 were positive, CD34 and CD45 were negative in BMSCs. The expressions of the collagen type II and aggrecan were positive in NPCs. At 2 weeks after co-culture, the morphology of BMSCs changed obviously, the cells were polygonal and irregular shape. The cell growth rate showed no difference within 3 passages, but decreased obviously after 3 passages. RT-PCR showed that the expressions of collagen type II and aggrecan genes at 10 and 15 days were significantly higher than those at 5 days (P < 0.05), no significant difference was found between at 10 days and at 15 days (P > 0.05). Western blot showed that the expressions of collagen type II and aggrecan proteins gradually increased with time, and there was significant difference within 5, 10, and 15 days (P < 0.05), but no significant difference was found after 15 days of co-culture (P > 0.05).

CONCLUSION: In a non contact co-culture system, BMSCs can differentiate into the NPCs. The expression of collagen type II and aggrecan can reach a stable level at 15 days after co-culture, and it is the suitable time for transplantation in vivo.

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