A suspension induction for myocardial differentiation of rat mesenchymal stem cells on various extracellular matrix proteins.

The microenvironment of bone marrow-derived mesenchymal stem cells (MSCs) strictly regulates their differentiation. In this study, we have developed a new suspension induction method for myocardial differentiation of bone marrow-derived rat MSCs (rMSCs) in vitro on various extracellular matrix (ECM) proteins. Myocardial differentiation of rMSCs was induced with a conventional monolayer method and our suspension method. In our suspension induction, a cell suspension was treated with the medium in the presence of an inducer, incubated for 2 h under a suspension conditions, and moved to a monolayer culture on gelatin-coated, collagen type I-coated, fibronectin-coated, or polystyrene dishes until the total induction time was 24 h. We evaluated the myocardial differentiation by counting the number of colonies of beating cells, performing immunohistochemical staining, and measuring the expression of cardiac-specific gene mRNA using real-time quantitative polymerase chain reaction. We found that rMSCs induced with the conventional monolayer method did not differentiate efficiently, whereas beating cell colonies were found on ECM-coated dishes of suspension-induced cells, after 3 weeks of culture, especially on gelatin-coated dishes. The beating cells were positively stained with anti-troponin T-C antibody and expressed specific cardiac markers. In conclusion, these results demonstrated that the suspension induction followed by subsequent culture on gelatin ECM substrates is a promising method for differentiating rMSCs into cardiomyocytes in vitro.