Cardiomyocyte-like cell differentiation by FGF-2 transfection and induction of rat bone marrow mesenchymal stem cells

Abstract

Objective(s)To investigate and test the hypotheses that FGF-2 enhanced myocardial differentiation with rat bone marrow mesenchymal stem cells (BMSCs). Materials and methodsLentiviral vectors carrying the FGF-2 gene were transfected into rat BMSCs firstly. According to the different inducing agents, they were divided into the following four groups: group A (BMSCs blank control group), group B (FGF-2 induction group), group C (Lenti-FGF-2-GFP lentivirus transfection group), and the group D (Lenti-control-GFP lentiviral transfer). Then several kinds of experimental methods such as real-time PCR, immunocytochemical staining, immunofluorescence staining, Western blot, and transmission electron microscopy were used to elucidate the effects by which FGF-2 adjusts myocardial differentiation in rat BMSCs. ResultsThe results of real-time PCR showed that GATA-4 and Nkx2.5 were expressed in all groups of cells. Compared with the experimental control group, the expression of GATA-4 and Nkx2.5 genes was the strongest after induction of 2 weeks in each induction group, and gradually decreased after induction of 4 weeks. Among them, the relative expression levels of GATA-4 and Nkx2.5 genes in Lenti-FGF-2-GFP were highest at all time points. The expressions of cTnI, cTnT, Cx43, and Desmin were detected by immunocytochemical staining and immunofluorescence staining. After 4 weeks of induction, cTnI, cTnT, Cx43, and Desmin were positively expressed in the cytoplasm of cells. Statistical analysis showed that the integrated optical density (IOD) values of the markers in the Lenti-FGF-2-GFP were the strongest. Cx43 and cTnI were weakly positive or negative in the experimental control group. There was a significant difference in the positive expression of each marker in each induction group and the experimental control group. Western blot analysis showed that Tromyosin (Tm) and Desmin were expressed in the blank group, FGF-2 drug-induced group, Lenti-FGF-2-GFP, and empty virus control transfection group after 4 weeks of induction, among which FGF-2 lentivirus transfected. The expression levels of Tm and Desmin were the highest in the staining induction group. Statistical analysis showed that the positive expressions of Tm and Desmin in each experimental group were statistically significant. Transmission electron microscopy showed that the nucleus of the cells transfected and induced by FGF-2 was located at the center of the cells. Myofilaments, rough endoplasmic reticulum, and mitochondria, and ribosomes were seen in the cytoplasm. ConclusionThese results indicate that FGF-2 can transfect and induce differentiation of BMSCs into cardiomyocyte-like cells. Lentivirus-mediated FGF-2 transfection induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells better than FGF-2 direct induction.