| Objective To establish the experimental methodology of isolating, culturing, proliferating and inducing rat bone marrow mesenchymal stem cells (MSCs) in vitro;observe whether or not MSCs induced by 5-azacytidine (5-aza) in vitro can differentiate into cardiomyocyte-like cells;and investigate the feasibility of acute myocardial infarction (AMI) treatment by allogeneic transplantation of MSCs.Methods Fifty-five healthy female Wistar rats aged two months were selected. Five were donors for MSCs. The others would be used as AMI animal models randomly separated into two groups including the transplanted group (n-25) and the control group (n=25).Bone marrow taken out from bilateral femora, tibiae and humeri was cultured with DMEM culture solution in culture bottles. MSCs were isolated and purified by the wall-adhering method. MSCs were digested and transferred to the next generation once they achieved 80% fusion. The third generation of MSCs began to be induced by 5-aza (10 μmol/L), and the following generations were all induced in the same way. The inducement started every time the new generation occupied 50% of the bottle bottom, and every inducement persisted for 24 hours. The morphology and growth of every generation were observed under fluorescence phase-contrast microscope. The fifteenth generation with 80% fusion was prepared for transplantation. These transplant cells had been induced 13 times by 5-aza and cultured for four months. Few transplant cells were observed under electron microscope and identified by immunohistochemistry. The other cells were labeled by 5-bromodeoxyuridine (BrdU) and transplanted into the AMI models.Myocardial infarctions were made by liquid nitrogen freezing. The prepared cellslabeled by BrdU were injected into the infarcted parts of the transplanted group. The same volume of DMEM solution was injected into the infarcted parts of the control group. Tissue Doppler echocardiography (TDE) was applied one day preoperation, one week postoperation and four weeks postoperation. Cardiac function was evaluated by systolic peak velocity (Vs), left ventricle end diastolic volume (LVEDV) and left ventricle ejection fraction (LVEF). The transplanted rats were sacrificed four weeks postoperation and the transplanted parts were harvested for BrdU immunofluorescent stain.Results The transplant cells (i.e. the fifteenth generation of MSCs) were same as cardiomyocytes in morphology, and expressed the cardiac-specific proteins, a-actin and troponin T, showing that MSCs induced by 5-azacytidine (5-aza) in vitro could differentiate into cardiomyocyte-like cells. Ten rats of the transplanted group died with 40% mortality. Twelve rats of the control group died with 48% mortality. The difference of the mortality between both groups had no statistical significance (P>0.05). In the control group (?=13), Vs, LVEDV, LVEF was respectively 13.97 + 2.06 cm/s, 0.31 ±0.06 ml, (77.50+4.81)% one day preoperation, 6.79+1.41 cm/s, 1.13 + 0.14 ml, (40.33 + 10.51)% one week postoperation, and 8.81 + 1.83 cm/s, 1.10 + 0.15 ml, (45.77 + 7.56)% four weeks postoperation. In the control group, the difference of the cardiac function among preoperation, one week postoperation and four weeks postoperation had statistical significance (.PO.05). The cardiac function four weeks postoperation was better than one week postoperation, but worse than preoperation. In the transplanted group (?=15), Vs, LVEDV, LVEF was respectively 14.01 + 1.03 cm/s, 0.30±0.05 ml, (76.83+4.52)% one day preoperation, 7.02 ±2.01 cm/s, 1.16 ±0.13 ml, (42.56 ±9.58)% one week postoperation, and 11.15 ± 1.78 cm/s, 0.67 + 0.11 ml, (65.38±9.57)% four weeks postoperation. In the transplanted group, the difference of the cardiac function among preoperation, one week postoperation and four weeks postoperation had statistical significance (PO.05). The cardiac function four weeks postoperation was better than one week postoperation, but worse than preoperation. The differences of the cardiac function preoperation and one week postoperation between both groups had no statistical significance (P>0.05), while the difference of the cardiac function four weeks postoperation between both groups hadstatistical significance (P<0.05). The transplanted group had better cardiac function than the control four weeks postoperation. The BrdU-labeled cells were found in the histological sections of the transplanted parts under fluorescent microscope, which suggested the transplant cells survived in the host cardiac muscles.Conclusion Rat MSCs cultured generation by generation and repeatedly induced by 5-aza in vitro can differentiate into cardiomyocyte-like cells, which if transplanted into the rat infarcted cardiac muscles will survive and help improve the host's cardiac function. Our study provides experimental support for clinical treatment of AMI by MSCs transplantation.
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