Research of HIF-1 Mediated SDF-1/CXCR4 Axis on the Directional Migration and Neural Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. At present, the mechanisms of targeted therapy of MSCs are known to relate with the hypoxia-inducible factor-1 (HIF-1) and its regulated biological axis stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4). SDF-1 and CXCR4 have a wide distribution in various cells and tissues, which plays an important role in the development of immune system, circulatory system and central nervous system. We thus inferred that SDF-1/CXCR4 may participate in the differentiation as well as the migration of stem cells. Our research aims to explore the effect of HIF-1α and its mediated SDF-1/CXCR4 axis on the directional migration and neural differentiation of MSCs, which may lead to a break in the efficiency and target distribution of MSCs therapy.

(1) Influence of hypoxia, CXCR4 antagonist (AMD3100) and SDF-1α on the proliferation of rat Mesenchymal Stem Cells (rMSCs): rMSCs were isolated from bone marrow of rats, and expanded in vitro. The growth feature of rMSCs exposed to hypoxia (PO₂=1%) or normoxia was identified by growth curve, while the effect of AMD3100 (5ug/ml) and SDF-1α (10ng/ml and 100ng/ml) on the proliferation ability of rMSCs was detected by cell counting kit-8. (2) Effect of hypoxia on the expression of HIF-1α, CXCR4 and SDF-1α: Firstly, we used RT-PCR, western blotting and flow cytometry to detect the expression of HIF-1α and CXCR4 mRNA and protein levels in rMSCs which treated with hypoxia for 0h, 6h, 12h, 24h, 48h and 72h. Secondly, HIF-1α, SDF-1α mRNA and protein level in the hippocampus of rats which suffered hypoxia- ischemia for 1d, 3d, 5d, 7d, 14d and 21d were also detected by the same assays. (3) Research of HIF-1α and SDF-1/CXCR4 axis on the migration of rMSCs: We first detected the change of CXCR4 mRNA and protein levels in rMSCs treated with AMD3100 (5ug/ml) and SDF-1α (10ng/ml) by RT-PCR, western blotting and flow cytometry, and then studied SDF-1/CXCR4 axis on the migration of rMSCs using Transwell assay. (4) Effect of HIF-1α and SDF-1/CXCR4 axis on the differentiation of rMSCs: protein level of NSE and GFAP as well as positive rate of neural-induced rMSCs which have been pretreated with AMD3100 (5ug/ml) were detected by western blotting and immunocytochemistry.

Persistent hypoxia promoted the proliferation of rMSCs, while AMD3100 and SDF-1α at the concentration mentioned above had no effect. Compared to normal control, the protein expression of HIF-1α in rMSCs increased in hypoxic condition while the mRNA of HIF-1α did not change. Furthermore, the mRNA and protein level of CXCR4 both increased in rMSCs exposed to hypoxia for 6h and 12h, and the results confirmed by flow cytometry. We found HIF-1α mRNA was stably expressed in hippocampus, and increased significantly in hypoxia-ischemia brain damaged (HIBD) rats in a time dependent manner, which reached the peak on 7d. As expected, SDF-1α mRNA in hippocampus of HIBD rats was higher than that of normal control group, which reached the peak on 7d (P<0.01) and stably expressed till 21d, while the protein level is mainly in concordance. Moreover, CXCR4 mRNA was extremely up-regulated in rMSCs treated with SDF-1α (10ng/ml), however, in 5 ug/ml AMD3100 treated rMSCs, which decreased markedly (P<0.01), and the results were confirmed by western blotting and flow cytometry assays (P<0.05). Transwell assay manifested that SDF-1α had obvious chemotaxis to rMSCs. Protein level and positive cell number of NSE and GFAP were extremely down-regulated in rMSCs which pretreated with 5ug/ml AMD3100.

Increased expression of HIF-1α led to the up-regulation of SDF-1/CXCR4 axis, and rMSCs displayed chemotaxis migration ascribed to the receptor-ligand interactions of SDF-1α and CXCR4, suggesting that HIF-1 and its mediated SDF-1/CXCR4 axis are of great significant on the directional migration of rMSCs. We also showed that CXCR4 antagonisation reduced the neural differentiation capabilities of rMSCs, thus suggested that SDF-1/CXCR4 axis may deeply involve in the neural differentiation of rMSCs.

No relevant conflicts of interest to declare.