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&#x2009;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&#x2009;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.
Miskon
Azizi
A
Department of Biomedical Engineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Mahara
Atsushi
A
Uyama
Hiroshi
H
Yamaoka
Tetsuji
T
D000818:Animals; D002454:Cell Differentiation; D016326:Extracellular Matrix Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology; D009206:Myocardium / Q000166:cytology* / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases