| Although the mortality and morbidity of myocardial infarction (MI) have been greatly decreased with the use of anticoagulants, angiotensin-converting-enzyme inhibitors, effective revascularization and heart transplantation, it still remains one of the major causes of death in heart diseases. Because the limited regeneration of cradiomyocytes, they are mainly replaced by fibrous tissue after MI. The irreversible loss of cardiomyocytes may lead to acute or chronic heart dysfunction, or even severe heart failure. Cellular cardiomyoplasty (CCM) aiming to replace injured myocardium with myogenic cells is regarded as being of great expect.To date, although fetal or neonatal cardiomyocytes, smooth muscle cells, skeletal muscle cells, or their precursors, embryonic stem cells have been employed in the studies of CCM, and some achievements in cardiac structure and function have been made, there still are inherent problems of these cells such as ethical concern and immunological rejection. The multipotentiality of mesenchymal stem cells (MSCs) has been re-recognized for these years. MSCs in various species, including human, can be easily isolated from bone marrow, adapted to ex vivo expansion,and both in vitro and in vivo differentiated into multilineage cells such as bone, cartilage, adipocytes, heptocytes, neurocytes, endothelial cells, marrow stroma cells, smooth muscle, skeletal muscle and cadiomyocytes, disregard of ethical concern and immunological rejection. These advantages enable MSCs to be suitable for cellular autotransplantation and gene therapy. MI occures mainly in the ventricles, so the optimal seed cells for CCM should terminally differentiate into ventricular cardiomyocytes and establish functional connections with host myocardium. Although many reports proved that MSCs tansplantation have this potential, their differentiation rate to ventricular-like cardiomyocytes is quite low. To acquire enough ventricular-like cardiomyocytes for CCM, it is necessary to select them from mingled cells. Unfortunately, no means of specificselection is available. Furthermore, the underlying mechanisms of MSCs differentiation are still unclear, and the feasibility of their differentiation is in question. Therefore, the aim of this study was not only to investigate whether rat bone marrow MSCs could be expanded and differentiated into ventricular-like cardiomyocytes with the inducement of 5-azatytidine (5-Aza) in vitro, but also to verify whether enhanced green fluorent protein (EGFP) under the control of ventricular specific myosin light chain (MLC)-2v promoter could specifically label ventricular-like cardiomyocytes from MSCs. Given it was true, ventricular-like cardiomyocytes could be selected by routine FCAS and used for CCM or related studies.The main methods and results are as follows: 1. Isolation and culture of adult rat bone marrow MSCs.MSCs were isolated from the femora and tibiae of Wistar rats (8-10w, 150-200g) with Percoll gradient density centrifugation and wall-attaching cultivation. The mononuclear cell from bonr marrow were cultured at 37°C in humid air with 5% CO2 in Iscove's modified Dulbecco's medium (IMDM), 10% fine fetal bovine serum ( FBS ), 1% L- glutamine, penicillin (100 μg/ml)/streptomycin (100 ng/ml). The medium was firstly changed 24 h later, after that one time every 3 d. The morphology of MSCs was observed under a phase contrast microscope. Primary fibroblast-like MSCs were found to sparsely attach to the wall of culture dish 4-8 h after incubation, began to proliferate on day 2-4, gradually formed small colonies on day 5-6, and finally reached 80%-90% confluence on day 12-16. After a series of passages, the attached cells became morphological homogeneous without obvious hematopoietic cells, but they gradually became larger in size and flattened-shape cells increased. At the same time, the colony-forming ability of the passaged MSCs was progressively decreased. The growth curves demonstrated that the primary MSCs were in quiescent period for the first 4 d after having...
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