Bone Mesenchymal Stem Cells' Cardiomyogenic Differentiation In Vitro Cardiomyocytes Microenviroment

Objectives: Bone mesenchymal stem cells (BMSCs) are thought to be multipotent difFeratiation cells. Resent studies in vivo have suggested the differentiation depended on the specific microenviroment. It has been confirmed the bone mesenchymal stem cells can differentiate into cardiomyogenic cells in vivo myocardial microenviroment, but the mechanism of which is unknown. In this study, we cultured the cardiomyocytes and the endothelial cells to establish the cell microenviroment in vitro and incubated bone mesenchymal stem cells, studied the morphological changes of the mesenchymal stem cells, their expressed phenotypes, and the effects of seeding density on the differentiation and provide theoretic support for their transplantation to heart in vivo.Method: We harvested SD rat's bone marrow and isolated it using density gradient centrifugation (Mark's mehtod) . After labeled by PK.H-26, the mesenchymal stem cells were co-cultured with cardiomyocytes(CMC) of SD rat , autologous endothelial cells(EC) at 1, 4 and 8X 105cells/well respectively. In blank control group, mesenchymal stem cells were cultured alone at the same density. Studied the morphological changes of these cells continuously and identified cardiomyogenic cells with special structural protein of cardiomyocyte,Actin, after co-culture one week and four weeks-Results: The labled cells can be identified throughout this study. In vitro, the bone mesenchymal stem cells present the fibroblast shape. After co-cultured with CMC ,the volum of the cells turn into a larger one gradually. Seeded at 1 X 105cells/well. the cells extended the langer and liked a spindle,ten percent of which were resemble the myotube in shape and presented positive staining with Anti-Actin after one week .Seeded at 4 and 8X 105cells/well,the cells' shape did not change markly but divided vigorousely. After two weeks, seeded at 1 X 105cells/well ,more mesenchymal stem cells' shape largened and conflicted with the adjacent cells ,which was more similar to myotube. Structure like intercalated diskesinitially formed with the adjacent cardiomyocytes. When seeded at 4,8 X 105cells/well ,the cultures reached 90%of confluence but the shape did not change stilly. Up to the three week, seeded at 1 X 105cells/well ,some cells appeared similar to the cardiomyocytes. The closer stem cells to cardiomyocytes , the more obvious changes were found.Also,the volum of the cells established intercalated-disk-like with cardiomyocytes changed incording to the contraction of the latter. Whereas, seeded at 4and 8X 105cells/well ,the cells needed to process passage so as to the ratio of stem cells to cardiomyocytes was very low and the langened or myotube-like shape did not be found. After four weeks, seeded at 1 X 105cells/well , being stained with Anti-Actin ,the cells turned on positive staining.However,seeded at 4 and 8 X 105cells/well ,the number of stained cells did not up to l%.Also, those mesenchymal stem cells incubetes with endothelial cells and those cultured alone presented negative staining with Anti-Actin.Conclusions:This study showed bone mesenchymal stem cells can differentiated into cardiomyolic cells under cardiomyocytes microenviroment in vitro.In the differentiation, the proper density of seeding play a crutial role.Cardiomyogenic cells can form intercalated diskes with adjaecent cardiomyocytes .