Differentiation Of Bone Marrow Mesenchymal Stem Cells To Cardiomyocytes In Vitro

The scar formation and cardiomyocytes loss result from myocardial infarction(MI) are important causes affecting heart function, Cellular cardiomyoplasty has been proposed as an alternative strategy for cardiac regeneration. A variety of cell types have been proposed as useful candidates. Bone mesenchymal stem cells(MSCs) are a group of cells with highly capability of self-renewal and potentials of multilineage differentiation. MSCs are easy to be got and cultured in vitro and suitable for self-transplantation,which makes them an appropriate source of cells for cell therapy. To investigate MSCs committedly differentiating into cardiomyocytes has great theorial and clinical significance for cell transplantation to treat MI.In our study, we separated rat bone meseachymal stem cells(MSCs) and cultured them in vitro. Then we induced them into cardiomyocytes by means of adding 5-azacytidine(5-Aza) into medium and identified them. We established a practical coculture model of MSCs and cardiomyocytes of rats to study the cardiomyogenic differatiation of MSCs under cardiomyocytesmicroenviroment in vitro.METHODS1. Differentiation with drug : MSCs from adult rats were separatedby percoll separating medium and were cultured in cell culturemedium and medium with 5-aza (1 5 10 20 umol L-1 )for 24 h,and their biologic characteristics were observed. After culture for 4weeks, MSC were identified by means of immunochemistry techniqueand tranmission electronmicroscopy.2.Coculture: MSCs from adult rats were separated and coculturedwith Neonatal ventricular cardiomyocytes which were separated from1-day-old Sprague-Dawley rats. As contrast,MSCs were cultured inthe presence of media conditioned by separate cultures ofcardiomyocytes (conditioned media). Cells were cocultured in anincubator for 2 weeks , Immunostaining against a -actin ,Troponin Iand Connexin43 was performed.RESULTSMSCs isolated by percoll separating medium had better uniform in the process of culture in vitro. Cells adhered to culture plates. MSCs showed fibroblast-like morphology without 5-azacytidine treatment. Four week after treatment with 5-aza. Some cells gradually increased in size and formed a ball-like or stick-like appearance. MSC induced by 5-aza could be identified by the positive staining for Troponin I and Connexin43. The number of cells stained positively in the 10umol L-1 and 20umol L-1 5-aza was visually greater than that of other groups , but more than 50% the cells necrosed in 20umolL -1 5-aza. Electron microscopy revealed sarcomeric organization and gap junction.In coculture experiment , Immunocytochemistry showed that differentiated MSCs from the coculture experiments expressed actin, Troponin I and Connexin43. In contrast, MSCs culture with conditioned media or culture alone presented negative staining with actin, Troponin I and Connexin43. CONCLUSION1. MSCs cultured with 5-aza are differentiated into cardiomyocytes in vitro. The optimal conditions for MSCs to differentiate are 10umol L-1 5-aza.2. MSCs can be transdifferentiated into cardiomyocytes in vitro after coculture with neonatal rat cardiac myocytes. The transdifferentiation of MSCs cultured with conditioned medium into cardiomyocytes can not be observed.3. The microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. Cell-to-cell contact mediates MSCs transdifferentiation.4. MSCs are plastic and can be reprogrammed into a cardiomyogenic lineage,which may be useful for a potential cell therapy to promote cardiac regeneration in patients with ischemic heart disease.