Effects of hypoxic preconditioning on the migration of bone marrow derived mesenchymal stem cells

Abstract

To explore the effects and probable mechanism of CoCl2-induced hypoxic preconditioning on the migration of bone marrow derived mesenchymal stem cells (BMSC). BMSC were cultured by whole bone marrow adherence and identified by surface markers (CD29, CD90 and CD45) with flow cytometry (FCM). The methods of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and FCM were applied to establish the model of CoCl2-induced hypoxic preconditioning. The migratory capacity of BMSC with hypoxic preconditioning was analyzed by the assays of scratch wound healing and transwell migration. The protein and mRNA expressions of HIF-1α and CXCR4 of BMSC were detected by Western blot and real-time polymerase chain reaction (PCR). After silencing HIF-1α by siRNA technique and blocking CXCR4 by its antagonist AMD3100, the changes of migratory capacity of BMSC were also tested. Cultured BMSC were uniformly positive for CD29 and CD90 and negative for CD 45. According to the results of MTT and FCM, 200 µmol/L CoCl2 and 24 h culture time was the ideal hypoxic preconditioning model of BMSC. The migratory capacity of BMSC in hypoxic preconditioning group was higher than the one in control group (scratch wound healing assay: (0.396 ± 0.018) mm vs (0.200 ± 0.011) mm, transwell migration assay: 21.0 ± 4.5 vs 8.5 ± 1.7, both P < 0.05). The protein and mRNA levels of HIF-1α and CXCR4 of BMSC in hypoxic preconditioning group were significantly higher than in control group. After silencing HIF-1α or blocking CXCR4 by AMD3100, the migratory capacity of BMSC in hypoxic preconditioning group decreased and had no difference with the control group. Hypoxic preconditioning may enhance the migratory capacity of BMSC in vitro. And it is partially attributable to the up-regulation of HIF-1α/CXCR4 axis after preconditioning.