Department of Biomedical Engineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Tissue Eng Part C Methods. 2010 Oct;16(5):979-87. doi: 10.1089/ten.TEC.2009.0218.
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.
骨髓间充质干细胞（MSCs）的微环境严格调节其分化。在这项研究中，我们开发了一种新的悬浮诱导方法，用于体外诱导骨髓源性大鼠 MSCs（rMSCs）向心肌分化，使用各种细胞外基质（ECM）蛋白。用传统的单层方法和我们的悬浮方法诱导 rMSCs 的心肌分化。在我们的悬浮诱导中，将细胞悬浮液用含有诱导剂的培养基处理，在悬浮条件下孵育 2 小时，然后转移到涂有明胶、I 型胶原、纤连蛋白或聚苯乙烯的培养皿上进行单层培养，直到总诱导时间达到 24 小时。我们通过计算搏动细胞集落的数量、进行免疫组织化学染色以及使用实时定量聚合酶链反应测量心脏特异性基因 mRNA 的表达来评估心肌分化。我们发现，用传统的单层方法诱导的 rMSCs 不能有效地分化，而悬浮诱导细胞在 ECM 包被的培养皿上培养 3 周后，特别是在明胶包被的培养皿上，可以发现搏动细胞集落。搏动细胞被抗肌钙蛋白 T-C 抗体阳性染色，并表达特定的心脏标志物。总之，这些结果表明，悬浮诱导后在明胶 ECM 底物上进行后续培养是体外诱导 rMSCs 分化为心肌细胞的一种有前途的方法。
