Effects of fibroblast growth factor on proliferation and differentiation of serially passaged bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The source of bone marrow mesenchymal stem cells (BMSCs) is limited, and the cellular morphology,proliferation and multi-directional differentiation capacities can vary during serial passages in BMSCs in vitro.OBJECTIVE: To study the effects of fibroblast growth factor (FGF) on cellular morphology, proliferation and differentiation of serially passaged BMSCs.METHODS: (1) BMSCs were isolated from Sprague-Dawley rats and cultured. These cells were passaged six times in vitro, and the cellular morphology was observed and photographed. (2) BMSCs at passage 6 were seeded into 96-well plates and randomly divided into control group and FGF treatment group. The proliferation of cells in both groups was detected with cell counting kit-8 kit at days 1, 2, 3, 4, 5, 6, 7 after culture. (3) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cellular morphology was observed and photographed. And then the cells of both groups were treated with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 7 days. The osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN;PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) were detected with real-time PCR. The protein expressions of RUNX2, PPARγ2, SOX9 were detected with western blot assay. (4) BMSCs at passage 6 were seeded into 6-well plates and randomly divided into control group and FGF treatment group. After 7 days treatment with growth medium or growth medium containing FGF, the cells were cultured with osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium for the next 14 days. Then, alizarin red S staining, oil red O staining and alcian blue staining were performed.RESULTS AND CONCLUSION: (1) After in vitro passage for six times, the cellular morphology changed obviously, and FGF treatment recovered the characteristics of primary cells. (2) Compared with the control group, the cell proliferation in the FGF treatment group was significantly increased (P < 0.05). (3) Compared with the control group, the expression of osteogenic, adipogenic and chondrogenic related genes (RUNX2, ALP, OCN; PPARγ2, AP2, ADIPOQ; SOX9, collagen II, aggrecan) was increased significantly in the FGF treatment group (P < 0.05). The protein expressions of RUNX2,PPARγ2, SOX9 were also higher in the FGF treatment group than the control group (P < 0.05). (4) Compared with the control group, the number of extracellular calcium nodules, the number of intracellular lipid droplets, and the expression of acid acidic mucopolysaccharide were significantly increased after FGF pretreatment. To conclude, FGF pretreatment can preserve the stemness of BMSCs serially passaged in vitro.