Abstract
Transforming growth factor β1 (TGF-β1) and salvianolic acid B (Sal-B) are key signaling factors for stem cell differentiation into cardiomyocytes (CMs). The present study compared the biological effect of TGF-β1 and Sal-B, alone or in combination, on bone marrow mesenchymal stromal cells (BMSCs) that differentiate into myocardial-like cells in a simulated myocardial microenvironment in vitro. BMSCs were isolated from bones of limbs of 10 male Sprague Dawley rats and cultured. The 2nd-generation BMSCs were co-incubated with TGF-β1 and Sal-B, alone or in combination, for 72 h. The control group was BMSCs cultured without any inductive substance. The levels GATA binding protein 4 (GATA4) and homeobox protein NKx2.5 were determined by reverse-transcription quantitative polymerase chain reaction and immunofluorescence staining was used to evaluate α-sarcomeric actin and cardiac troponin I (cTNI) as cardiomyogenic differentiation markers. The ultrastructure of BMSCs in each group was also observed. BMSCs were initially spindle-shaped with irregular processes. The cells gradually increased in number 24 h post-inoculation and proliferated 7 days later. Compared with the control group, BMSCs in the treatment groups had fusiform shapes, orientating with one accord and were connected with adjoining cells forming myotube-like structures on day 28. The morphology and architecture/myotubes of BMSCs was similar among the treatment groups, but the amount of cells in the combined group was comparatively higher. The results of immunofluorescence staining revealed the expression of the CM-specific proteins α-sarcomeric actin and cTNI in these cells. The expression of these cardiac-specific markers in the combined group was significantly higher than that in the other groups (P<0.01 or P<0.05). In addition, the transcriptional expression of GATA4 and NKx2.5 in the treatment groups was stable and significantly higher than that in the control group on day 7. Transmission electron microscopy showed that BMSCs in the treatment groups all had myofilaments, rough endoplasmic reticulum and mitochondria in the cytoplasm when compared with the control group. Taken together, these results indicated that the combination of TGF-β1 and Sal-B effectively promotes cardiomyogenic differentiation of BMSCs in vitro and their application may represent a therapeutic strategy for the treatment of ischemic heart disease.