Pre-clinical Study Of Biological Therapy Of Acute Myocardial Infarction By Autologuos Bone Marrow Mesenchymal Stem Cells Transplantation

IntroductionAcute myocardial infarction (AMI) and post infarct chronic heart failure are common and frequently occurring disease that seriously affects health of people. Despite major advances in the treatment such as medication, pecutaneously coronary intervention (PCI) and coronary artery bypass grafting (CABG) of ischemic heart disease, complete revascularization procedures still do not help a large number of patients because of poor distal vessels, total arterial occlusion, or unacceptable procedural risks due to concomitant medical conditions. Clinically there is no radical treatment to regenerate and rebuild the stricken coronary artery and the infarct myocardium. Results of experimental studies have shown that neonatal cardiomyocytes, embryonic stem cells, skeletal myoblasts cells, bone marrow hematopoietic side-population stem cells (SP) and mesenchymal stem cells (MSCs) can be used to regenerate new cardiomyocytes and induce angiogenesis after myocardial infarction, resulting in improvement of myocardial function, especially autologous bone marrow MSCs for its multi-differentiated potentiality, easy to obtain and proliferate, no immunosuppression. Our aim was to test the feasibility of autologuos MSCs transplantation by endocardium injection and the safety and effectiveness of autologous implantation of MSCs after proliferated in vitro with acute myocardial infarction, compare therapeutic efficacy of MSCs transplantation by three different ways. We hope to provide experimental evidences for the future clinical application of stem cells transplantation in ischemic heart disease.Study of cardiomyocytes-like differentiation of swine bone marrow MSCs in vitro by different concentration of 5-azacytidineObjective To establish the method for isolation, culture and proliferation ofswine bone marrow MSCs and investigate the potential and contribution of cell proliferation MSCs derived from swine bone marrow to cardiomyogenic differentiation after exposure to different concentration of 5-azacytidine (5-aza) in vitro. Methods 10ml bone marrow was taken from swine, and mononuclear cells (MNCs) were freshly isolated by 1.077g/ml Ficoll lymphocyte separating medium using density-gradient centrifugation. The 2nd generated MSCs were exposured in medium mixed with different concentration of 5-aza (3, 5, lOumol/L) for 24h cellular differentiation, than cultured in complete medium for 4 weeks together with control group. Desmin and cardiac-specific cardiac troponin I (cTnl) were examined respectively with immunohischemistry at 28 days and the ultramicrostructure of induced cells were observed by electron microscope. Results The methodology for isolation and expansion of MSCs was established. The isolated MSCs were set very well in culture of DMEM-Fn (dulbecco's modified Eagle" s medium-Fi2) supplemented with 10% selected fetal bovine serum (FBS). MSCs attached and grew as fibroblastic cells at 24 hours after initial plating and developed into visible symmetric colonies at about 36~48 hours, 7-10 days later it reached confluence while the cells were permitted to proliferate. The swine MSCs from 10ml bone marrow aspirate showed a fibroblast-like morphology with vortex distribution in their peak propagation and were harvested 3.8+0.6 X106 cells after cultured three generations over 2-3 weeks. The MSCs did not differentiate spontaneously during culture expansion and maintained normal biological features. The morphology of MSCs were augmented and lengthened growing with well and closed-up arranged after chemical inducement. Multinuclear cells and myotube structures were found more in 5> 10 u mol/L 5-aza treatment groups with inducement rate were 36% and 31% respectively (no significant difference) than in 3 U mol/L and control groups. The 3, 5, 10 u mol/L 5-aza and control groups were harvest 3.81 + 0.40 X106> 3.76±0.62X10\ 3.67 + 0.80X106 and 2.9 + 0.21 X106 cells respectively and no spontaneous beat in all groups after 28 days cultured, the generation was slow down obviously in 10 n mol/L group (P<0.05). Immunohistochemistry revealed positive expression of desmin and cTnl in 5> 10 p mol/L but no cardiac specific protein were found in neither 3 u mol/L nor in control group. Transmission electron microscope showed that differentiated cells had myofilaments, atrial granules (AG)structures and crevice connection between two cells. Conclusion The MSCs from swine bone marrow could be isolated, cultured and generated in vitro by 1.077g/ml Ficoll lymphocyte separating medium using density-gradient centrifugation and adherence screening method. Purified MSCs from swine bone marrow displayed a stable phenotype, biological and cellular features and can be selectively differentiated into cardiac-like muscle cells with 5> 10 U mol/L 5-aza inducement in vitro and the 5 u mol/L 5-aza concentration had little effect on cell proliferation.Autologuos bone marrow MSCs transplantation in AMI therapy using self-made endocardium injection catheterObjective In the present study, we demonstrate the therapeutic potential, safety and technical feasibility of percutaneous, intramyocardial transplantation of autologous mesenchymal stem cells (MSCs) in the setting of AMI. Methods 40ml bone marrow aspirate were taken suction from each 10 swines and the MNCs were freshly isolated from the bone marrow blood by 1.077g/ml density-gradient centrifugation. Two to three weeks after cultured in the complete cell-culturing medium and labeled by DAPI (4', 6-diamidino-2'-phenylindole) the purified MSCs or no serum medium were autologously transplanted into target ischemia endocardium in 1 hour by reconstructed injection catheter. The adjusted injection catheter was reconstructed by 6FJR4.0 angio-catheter, 8FJR4.0 guiding catheter, skin test noodle No.27 and 7F sheathing canal. Acute myocardial ischemia was induced by reperfusion after Hgating the left anterior descending coronary artery (LAD) and reopened in 1 hour of swine. The infarcted region was marked by suturing the non-penetrated part of 0.014 " BMW/ACS guide wire on the surface of epicardium. The DAPI labeled MSCs were injected into the marked infarct region after 2-3 weeks using the self-made injection catheter. We examined fluorescence microscope with immunohischemistry around infracted region by myocardium frozen section on desmin and cTnl 3 months after transplantation. LV function was estimated by echocardiogram and blood biochemistry was examined at before and 6 weeks, 3 months after transplantation. Results The swine MSCs showed a fibroblast-like morphology with vortex distribution in their peak propagation and were harvested 3.81 ±0.09X107 cells after cultured three generations over 2-3 weeks. DAPI label rate was 100%. DAPI-labeled MSCs in lml medium andmedium only were multi-sites injected into the ischemia LAD territory with injection catheter. lOOul per sites and totally 11 sites with 10.04 + 0.02 sites (n=5) and 9.03+0.14 sites (n=5) were injected in transplantation group and control group respectively. The residual cells suspension was 0.5ml in catheter. Left ventricular function was improved significantly of which LVEF were 46.6 + 3.5% and 56.7+ 0.8% vs. 34.9+0.9% (P<0.05) at 6 weeks and 3 months vs. post-AMI respectively. Blue fluorescence nuclear cells marked all autologously transplanted cells around infracted myocardium and express markers of myocardium phenotype, which showed positive, stain of desmin and cTnl. Conclusion The generated MSCs were safe for regeneration therapy in vivo. The experimental endocardium injection catheter can be used in experimental study for its manipulate conveniently, material easy obtained, cost price low, safe and utility. The transplanted MSCs located definitely, expressed markers of myocardium phenotype result in the heart function improved significantly. The reconstruct endocardium injection catheter had an important application value in the further study of regeneration therapy of cardiovascular disease in vitro.Compared therapeutic efficacy of autologuos bone marrow MSCs transplantation in AMI by three different deliver waysObjective Compared the therapeutic efficacy of autologuos bone marrow MSCs in AMI by three different ways. Methods The AMI model was induced by reperfusion after ligated or bloon blocked LAD for 1 hour of 12 swine. Purified and DAPI-labeled MSCs or no serum medium were autologously transplanted into target ischemia myocardium by ways of intra-coronary artery, endocardium and epicardium injection. LVEF and LV hemodynamics were measured by echocardiogram and electrophysiolograph before and after cells transplantation. Immunohischemistry around infracted region on desmin and cTnl were examed 3 months later. Results The number of generated MSCs, DAPI label rate and preservation condition had no differences between three groups. The transplanted cell suspensions were lml and lOOul/sites injected in both endocardium and epicardium groups, 10.04±0.02 sites in endocardium group and 9.03 + 0.14 sites in epicardium group averagely. Compared with control group the left ventricular end-systolic pressure (LVESP) decreased (PO.05), left ventricular end-diastolicpressure (LVEDP) markedly increased (P<0.01), LV + dp/dtmax significantly decreased (P<0.0\), LVEF augmented decreased measured by echocardiogram and improved after MSCs transplantation both in three groups, with furthermore improved in endocardium injection group and pericardium injection group than intra-coronary artery group. Specifically increasing LVEDP degraded (both PO.05) in two myocardium injection groups, LV+dp/dWx increased to 2905 + 78 mmHg/s vs. 3289+102 mmHg/s and 3444 + 89 mmHg/s (both ^O.05), LVEF augmented to 40.5 + 1.1% vs. 56.7+0.8% and 51.0+2.7% (both P<0.05) in intra-coronary artery vs. endocardium and epicardium injection groups. It was found scar area diminution, neovascular regeneration in all three groups, which showed significantly changes in myocardium injection groups than intra-coronary artery group and no differences between myocardium injection groups. Although left ventricular function was improved compared with control group, no exactly exogenous cells but only non specific fluorescence stain were found around the infracted myocardium by the way of intra-coronary artery transplantation. DAPI-labeled MSCs with oval nucleus were widely distributed and stained positively of cardiac specific proteins of desmin and cTnl both in endocardium and epicardium injection group. Conclusion LV function of AMI can be improved by different ways of autologuos bone marrow MSCs transplantation. But it was more effective by the way of myocardium injection than intra-coronary artery injection.Security and effectiveness of autologuos bone marrow MSCs transplantation by endocardium injection in the treatment of AMIObjective In the present study, we demonstrate the therapeutic potential, safety and technical feasibility of percutaneous, intramyocardial transplantation of autologous MSCs in the setting of AMI. Methods AMI was induced by reperfusion after ligating the LAD for 1 hour of swine. MNCs were freshly isolated from the bone marrow blood by 1.077g/ml density-gradient centrifugation. Two to three weeks after cultured in the complete cell-culturing medium the purified MSCs or no serum medium were autologously transplanted into target ischemia endocardium by reconstructed injection catheter. LV function was estimated by echocardiogram and emission computerized tomography (ECT) and blood biochemistry was examined at 6 weeks and 3 months after transplantation. We examined fluorescence microscopewith immuno-hischemistry around infracted region on desmin and cTnl 3 months after transplantation. Results The swine MSCs showed a fibroblast-like morphology with vortex distribution in their peak propagation and were harvested 3.81 ±0.09X 107 cells after cultured three generations over 2-3 weeks. DAPI-labeled MSCs in lml medium and medium only were multi-sites injected into the ischemia LAD territory with injection catheter. Left ventricular function was improved significantly of which LVEF were 46.6+3.5% and 56.7+0.8% vs. 34.9+0.9% (PO.05) at 6 weeks and 3 months vs. post-AMI respectively. cTnl and specific myocardium enzyme increased after AMI (P <0.001), and lasted for 3 month. PaCh decreased, PaCO2 remained slightly, respiratory acidosis when breathing machine removed for 1-2 hour and recovered at 6 week and 3 months, no gas exchange abnormal appeared. The biochemical examination of liver and kidney function, blood glucose was normal in anterioposterior, AMI, at 6 week and 3 months after transplantation. No arrhythmias were found in both two groups in ECG follow up. Blue fluorescence nuclear cells marked all autologously transplanted cells around infracted myocardium and express markers of myocardium phenotype, which showed positive, stain of desmin and cTnl. The HE stains of microstructure of important organs such as liver, spleen and kidney were normal although the fluorescence stain in lung was stronger than the other organs. Conclusion The generated MSCs were safe for regeneration therapy in vivo manifested that the structure and function of main important organs were normal. The experimental endocardium injection catheter can be used in experimental study for its manipulate conveniently. The transplanted MSCs located definitely, expressed markers of myocardium phenotype result in the heart function improved significantly.