Abstract:

Objective To investigate the effect of indirect contact co-culture with cardiac myocytes induction in the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in order to find the optimal induction conditions. Methods 2-3 weeks old 24 SD rats and newborn 1-3 day old 96 SD rats were used. BMSCs were derived by adhesive screening, and cardiac myocytes（CMs）were isolated by differential adhesion. According to the induction conditions, the cells were divided into groups A, B, and C. In group A［the 5-azacytidine（5-Aza-CR）induction group］, 10 μmol/L 5-Aza-CR was used to induce for 24 hours under a light-free environment. In group B (the co-cultured CMs induction group), at the beginning of the experiment, the CMs and BMSCs were cultured separately; when each had attached, and they were co-cultured. In group C (the 5-Aza-CR and co-cultured CMs group), CMs were seeded in the upper side of the transwell chamber, and 5-Aza-CR-induced BMSCs were seeded in the lower side. The phase contrast microscope was used to continuously observe morphological changes of BMSCs in each group; the cells were collected in 2-4 weeks. Expression of α-sarcomeric actin and cTnT in induced BMSCs was inspected by immunohistochemistry and immunofluorescence. Results 1.Cells of group B had a trend of aggregate growth. Shedding or degrading cells of group C were significantly less than in group A, but fatty vacuolization appeared in a part of the cells. 2.Resultsof immunohistochemistry and immunofluorescence: The expression of cTnT in group A, B, and C was negative or at low levels at 2 weeks of induction, while the expression of α-actin was weak. The expression of cTnT and α-actin in each group was positive, respectively, at 4 weeks of induction. The positive expression rates of cTnT and α-actin were (20.22±2.30)% and (28.05±2.45)% in group A, (21.18±1.30)% and (29.06±1.86)% in group A, and (26.28±2.89)% and (33.91±2.18)% in group C. The difference of group A and C was statistically significant (P<0.05), but the difference of group A and B was not (P＞0.05). Conclusion CMs indirect contact co-culture can induce BMSCs to differentiate into cardiomyocyte-like cells, and induction effects could be improved by being induced with 5-Aza-CR together.