Bone Marrow Mesenchymal Stem Cells Of Adult Mice Differentiate Into Cardiomyocytes-like In Vitro

Objective: A number of cardiovascular diseases, such as myocardial infarction, lead to cardiomyocyte loss and consequently deterioration of cardiac function. Because cardiomyocytes have a severely limited capacity for dividing new cardiomyocytes and had to replace damaged tissue. The necrosis in ischemic myocardium is repaired by fibrous scar tissues. This cause left ventricle remodeling, and ultimately lead to heart failurethat is a final stage of the development of many chronic heart disease. The use of cell transplantation therapy is a promising option to regenerate cardiac tissue. A numbe of cell types including skeletal myoblasts, fetal cardiomyocytes, smooth muscle cells, embryonic stem cells and bone marrow mesenchymal stem cells (MSCS), have been employed for transplantation study, and show benefits on heart functions. MSCS are nonhematopoitic multipotent stem cells that exist in bone marrow over the whole lifespan of mammals. MSCS could differentiate to myogenic cells and be obtained by a simple routine of bone marrow aspiration. Autologous bone marrow cell transplantation have not immuno-rejective reation. So they are ideal dornor cells for seeding to myocardial infarction area. The studies were designed to investigated the factors governing the growth and the possibility that induced MSCS of adult mice differentiate into cardiomyocytes-like in vitro.Methods: Bone marrow mesenchymal stem cells were aspirated from adult SD mice,s femal bone and isolated by graditent centrifugation method. MSCS were purified , expand in vitro culture . The MSCS (primary,one and three passage) were respectively treated with 5-aza for 24h. The MSCS differentiation was observed by phase-contrast microscope, cardiac-specific antigen of the diffrentiated cells was detected using immunohistochemistry, the expression of cardiomyocytes-specific gene was evaluated by reverse transcriptase-polymerase chain reaction(RT-PCR).Results: The MSCS attached to culture dish sparsely and the majority of the cells displayed a spindle-like shape and took the apperance of fibroblasts. These cells began to proliferate and increase in size at about day 4 in the culture condition, and gradually grew to form small colonies. About 2 weeks growing, colonies gradually grew expanded in size with the adjacent ones interconnected with each other and the cultures reached confluence 80%-90%. Passaged MSCS were larger in size and more heterogenous in morphology and in growth properties , and took the apperance of spindle-like or triangle-like shape. The primary and the first passaged MSCS treated with 10μmol/L 5-aza were observed under the same culture conditions for control cells . The growth properties of MSCS in both primary and first passaged did not obviously change treated with 10μmol/L 5-aza. While the MSCS of the third passage by 5-aza for 4 week gradually increased in size and formed a ball-like or stick-like appearance, and connected each other with adjoining cells, and began to form myotube-like structures. The MSCS of primary, one passage induced by 5-aza for 4 weeks were not the positive staining for Desmin and Troponin T, but the MSCS of the third passage induced by 5-aza for 4 weeks showed postive staining for Desmin and Troponin T. Desmin positive cells were 20% and Troponin T positive cells were neatrly 8%. The MSCSof the third passage induced by 5-aza for 4 weeks expressed cardiac-special genes, including ANP, GATA-4. Conclusions: In summary, the study demonstrated that the MSCS cultured with 5-aza can differentiate into cardiomyocytes-like in vitro. The MSCS are ideal dornor cells as seeding cellular cardiomyocytes to myocardial infarction area. But the differentiated mechanism is incompletely understood.