Objective: To investigate the feasibility of human bone marrow mesenchymal stem cells (hBMSCs) in vitro differentiation into vascular smooth muscle cells with induction of platelet-derived growth Factor BB (PDGF-BB).
Methods: Bone marrow mesenchymal stem cells of adult healthy donors were separated from iliac crest aspiration and expanded in DMEM-LG medium. Cells at passage 1 were transferred to EGM-2 medium containing PDGF-BB (20 ng/ml) and cultured for 14 days. The expression of SM alpha-actin, SM calponin, SMMHC and SM 22alpha were detected by immunofluorescence and observed with fluorescence microscope. mRNA expression of SMalpha-actin, SM calponin, SMMHC as well as SM 22alpha was analyzed by RT-PCR. The method of Western-Blot was applied to determine protein expression of SM 22alpha. Cells with induction were observed for the expression of SM alpha-actin,SM calponin,SMMHC by FACs analysis.
Results: With the induction of PDGF-BB, the morphology of cells changed to a spindle fibroblastic appearance. By fluorescence microscope observation, expression of SM alpha-actin, SM calponin and SMMHC was found intracellularly in PDGF-BB treated hBMSCs at 14 days. Western-Blot detection confirmed SM 22alpha expression by 14 days induction. RT-PCR of characteristic vascular smooth muscle cells related genes, such as SM alpha-actin, SM calponin, SMMHC and SM 22alpha revealed differentiation of vascular smooth muscle cells phenotype in monolayer culture upon stimulation with PDGF-BB for 14 days. The positive expression of SM alpha-actin, SM calponin and SMMHC in induced cells was significantly higher than that in non-induced cells (P < 0.05, n=3).
Conclusion: These results suggested hBMSCs could be differentiated into vascular smooth muscle cell phenotype with PDGF-BB induction in vitro.