The Effect Of M-CSF On Proliferation And Differentiation Of BMSCs In Vitro And BMSCs In Myocardial Ischemia

Background:Although several repair mechanisms have been described in the animals and human heart,all fall too limit to prevent clinical heart disease in most acute or chronic pathological cardiac conditions.Moreover,despite many breakthroughs in cardiovascular medicine,the complications of myocardial infarction remain a serious problem.Bone marrow mesenchymal stem cells could provide a promising strategy against myocardial infarctions and postinfarct congestive heart failure,based on that bone marrow mesenchymal stem cells can generate new cardiomyocytes in animals and human beings.However,due to its poor replicative capacity and short proliferative longevity,it is difficult to isolate enough MSCs from the bone marrow required for large areas of injured tissues.Thus,the expansion of MSCs in vitro is essential for cell transplantation.The purpose of this study was to identify macrophage colony-stimulating factor(M-CSF) contributing to production of MSCs and maintenance of their cardiomyocyte differentiation.PartⅠIsolation,Characterization and Cardiomyocyte Differentiative Potential of Rat BMSCs in vitroObjective:To isolate and expand a highly purified population of adult rat BMSCs, and to analyze its biological and electrophysiological characteristics.Methods:BMSCs were isolated from rat bone marrow with density gradient centrifugation and cultured in low-glucose DMEM supplemented with 10%fetal calf serum.Trypan blue staining and cell counting from day 1-7 were used for the cell concentration.Flow cytometry(FCM) was used to analyze the expression of CD44,CD29,CD45,CD11,and the cell cycle.To induce differentiation,BMSCs were treated with 5-azacytidine(5-aza) for 24h and then cultured with DMEM without 5-aza. Immunohistochemistry analysis were used to test the expression of MHC and cTnI. Patch-clamp recording technique was used to analyze electrophysiological characteristics of BMSCs.Result:FCM analysis showed that the BMSCs were CD44,CD29 positive,and CD45,CD11 negative.S phase of the passage 2 BMSCs is 23%.The passage 3 BMSCs that were induced by 5-aza could express MHC and cTnI.Before BMSCs were induced by 5-aza,only the outward currents of I_K+ were recorded by Patch-clamp.The inward currents of I_Ca2+ and I_Na+ were be recorded after BMSCs induced by 5-aza for three weeks.Conclusion:BMSCs have cardiomyocyte differentiation potential and can be differentiated into excitability cells from unexcitability cells by 5-azacytidine.PartⅡThe effect of M-CSF on Proliferation and Cardiomyocyte Differentiation Potential of BMSCs in VitroObjective:To investigate the effect of M-CSF on proliferation of rat BMSCs,and to analyze the cardiomyocyte differentiation potential of the BMSCs expanded by M-CSF.Methods:Trypan blue staining and cell counting were used to determine the effect of the different concentration of M-CSF on proliferation of rat BMSCs.FCM was used to analyze the expression of CD44,CD29,CD45,CD11,and the cell cycle of the BMSCs expanded by M-CSF.To analyze the cardiomyocyte differentiation potential of the BMSCs expanded by M-CSF,5-aza was used.Immunohistochemistry analysis was used to test MHC and cTnI expression of the induced BMSCs expanded by M-CSF.Patch-clamp recording was used to analyze electrophysiological characteristics of the BMSCs expanded by M-CSF.Result:M-CSF markedly accelerated proliferation of the BMSCs at the concentration of 5ng/ml of M-CSF.FCM analysis showed the BMSCs expanded by M-CSF were CD44,CD29 positive,and CD45,CD11 negative.S phase cells of the BMSCs cultured by M-CSF is markedly higher than those of the control.The BMSCs cultured by M-CSF induced by 5-aza could express MHC and cTnI.The inward I_Ca2+ and I_Na+ currents of BMSCs expanded by M-CSF were recorded after BMSCs were treated with 5-aza three weeks.Conclusion:BMSC proliferation could be markedly accelerated by M-CSF,and M-CSF stimulated BMSCs could retain the cardiomyocyte differentiation potential in Vitro.PartⅢBMSCs expanded by M-CSF as Transplantation Cells for Myocardial Ischemia TherapyObjective:To test the cardiac environment for BMSCs expanded by M-CSF and evaluate the effect of the cardiac environment on cardiomyocyte differentiation potential of the BMSCs.To investigate BMSCs expanded by M-CSF as transplantation cells to improve cardiac functions in rat MI model,and to explore the underlying mechanism.Methods:The BrdU-BMSCs expanded by M-CSF were co-cultured with rhythmically beating CM or cultured in the presence of conditioned media for one week. Immunohistochemistry analysis was used to test cTnT of the BrdU-BMSCs.Rat myocardial infarction model was established by ligating the left coronary artery.Cardiac function of MI was assessed by echocardiographic-Doppler.BrdU labeled BMSCs were injected directly into cardiac muscle around the infarct zone.After 20 days, immunohistochemistry analysis was used to test the cTnT expression of the transplantation BMSCs and capillary density in the infracted area.Results:The M-CSF expanded BrdU-BMSCs which were co-cultured with rhythmically beating CM showed cTnI but BrdU-BMSCs cultured with culture media without cardiomyocytes did not.Before the cellular implantation,the stroke volume(SV) and ejection fraction(EF) decreased in the MI heart(SV=0.382±0.033 ml,EF=(0.562±0.052)%) compared with the control group(SV=0.459±0.052ml,EF=(0.646±0.025) %).Twenty days after the M-CSF expanded BrdU-BMSCs implantation,the cardiac function was significantly improved in both induced(SV=0.641±0.065ml,EF=(0.756±0.058)%) and uninduced groups(SV=0.651±0.062ml,EF=(0.762±0.069)%) compared with the control(SV=0.459±0.052ml,EF=(0.646±0.025)%).However, cardiac functions didn't differ between the induced and uninduced groups.The infarct site had more cardiac-like tissue islands.The transplanted BMSCs were found in the infarct zone and the capillary desity was markedly higher than that of the control.Conclusions:Direct cell-cell contact between BMSCs and CMs was the prerequisite for the cardiomyocyte differentiation of BMSCs expanded by M-CSF.The M-CSF expanded BMSCs as transplantation cells can improve the cardiac function of MI. No matter whether the BMSCs were induced or not,the improvement of the cardiac function might result from myocardial regeneration and renascent capillary vein by engrafted cells.In summary,BMSCs have cardiomyocyte differentiation potential and can develop into excitability cells.BMSCs showed the inward currents of I_Ca2+ and I_Na+ induced by 5-azacytidine.M-CSF is an efficient growth factor in vitro for BMSCs and can retain the cardiomyocyte differentiation potential in Vitro.Direct cell-cell contact between BMSCs and CMs was the prerequisite for the BMSCs cardiomyocyte differentiation.The BMSCs expanded by M-CSF can improve the cardiac function after the cells were implanted around the infarct zone and its mechanism might result from myocardial regeneration and renascent capillary vein by engrafted cells.