Experimental Study Of Cardiomyocyte Differentiation From Porcine Bone Marrow Mesenchymal Stem Cell In Vitro And Optimized Transplantation To Treat Acute Myocardial Infarction In Vivo

Objectives: Mesenchymal stem cells (MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation. This study was to explore the proliferative potential and cardiomyogenic development of porcine bone marrow-derived MSCs stimulated with or without 5-azacytidine.Methods: In this study, the bone marrow was aspirated from Chinese mini-swine. The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in low-glucose DMEM containing 10% fetal calf serum. After primary culture, relatively purified MSCs were obtained by using the method of adherence screening. The cultured MSCs in vitro were passaged every 3 days after primary culture, and MSCs at passage 1, 4, 8 and 10 (P1, P4, P8 and P 10) were examined for their proliferation and differentiation stimulated with or without 5-azacytidine (10μM) in vitro by cell counting, immunocytochemistry, transmission electrical microscopy and RT-PCR/real time qPCR.Results: In normal growth medium, approximately 10.34±1.8% of porcine bone marrow-derived MSCs at passage 10 in vitro spontaneously take on cardiomyocyte-like phenotype and structure, and express cardimyocyte-specific genes and proteins, however, earlier passages of MSCs don't. Four weeks after induction by 5-azacytidine, MSCs at passage 1, 4 and 8 all express cardiomyocyte-specific genes and proteins and have cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However only MSCs at passage 10 express connexin 43 at the level of gene and protein, and take on relatively mature cardiomyocyte ultrastructure, including myofilaments, sarcomeres and transverse striations, especially intercalated discs in vitro. The MSC-derived myogenic cells showed cardiac myogenic but not skeletal myogenic markers at both mRNA and protein levels. In addition, the cardiomyogenic differentiation efficiency of MSCs at passage 10 are significantly higher than that of earlier passages (44.5±4.2% vs. 18.3±8.2%, 17.6±11.9%,16.5±5.0%, respectively, all P＜0.0001).Conclusion: The potency of growth and differentiation of porcine bone marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine is dependent upon the endogenous gene expression and passage condition of MSCs. The myogenic development from the subset of MSCs at higher passages provides further evidence supporting the potential application of MSCs in cardiac stem cell therapy for treatment of heart failure and myocardial infarction. Objectives: This study was to investigate: (1) the biodistribution and myocardial localization of intracoronarily delivered bone marrow-derived mononuclear cells (MNCs) after acute myocardial infarction (AMI) in vivo, and (2) the beneficial effects of intracoronary infusion of autologous MNCs on post-infarction hearts of swine.Methods: Fourteen Chinese swine were divided into two groups, including group control (n=7) and group 2 (intracoronary delivery of MNCs, n=7), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Bone marrow-derived MNCs (1×10⁹ cells per animal) were radiolabeled with 185 MBq ¹⁸F-fluoro-deoxy-glucose (¹⁸F-FDG, specific activity, 18.5 MBq/ml=SmCi/ml) and infused into the infarct-related coronary artery (n=6) 1 week after AMI. MNC biodistribution after intracoronary infusion was determined by dual-nuclide single photon emission computed tomography (SPECT), including ^99mTc-sestamibi imaging for localization and size of perfusion defect and ¹⁸F-FDG imaging for anatomic localization and distribution of radiolabeled MNCs. The potentials of differentiation of implanted cells in vivo and capillary density in both infarcted and peri-infarct region were detected by immunofluorescent analysis. Cardiac function and area of perfusion defect were detected at baseline (1 week after myocardial infarction) and endpoint (6 weeks after transplantation) by magnetic resonance imaging (MRI) and ^99mTc-sestamibi SPECT.Results: More than 90% of the total radioactivity was cell bound. Cell viability of radiolabeled MNCs was more than 97%. One hour after cell infusion, all animals underwent dual-nuclide SPECT imaging. After intracoronary transfer, 6.8±1.8% of ¹⁸F-FDG-labeled MNCs was detected in the infarcted myocardium; the remaining activity was found primarily in liver and spleen. Dual-nuclide SPECT showed that MNCs predominantly engrafted in the under-perfused region. The retention rate of infused cells in hearts has a significantly positive correlation with under-perfused area (P=0.001, Pearson correlation=0.973). At endpoint, there were severe fibrosis and inflammatory cell infiltration observed in infarcted zone with seldom surviving myocardium in group control, in contrast, there were less fibrosis and inflammatory cell infiltration in group 2 with more surviving myocardium. In group 2, the capillary density was significantly more than that in group control in both infarcted zone (P＜0.003) and peri-infarct zone (P＜0.003). MRI showed that all parameters at baseline were not significantly different between 2 groups (all P＞0.05). At endpoint, regional wall thickening, left ventricular ejection fraction were increased, while left ventricular mass index, dyskinetic segments, end-systolic volume and infarcted size were decreased in group 2 compared with control group (all P＜0.05). SPECT also showed that the area of perfusion defect significantly decreased in group 2 at endpoint compared with control group (P＜0.0001).Conclusion: The present study demonstrate: (1) ¹⁸F-FDG labeling and dual-nuclide SPECT imaging can be used to monitor biodistribution and myocardial homing of MNCs, and (2) intracoronary delivery of MNCs can decrease infarcted area, improve cardiac function and prevent ventricular remodeling after AMI. Objectives: To study whether drug-facilitation in the perioperative period of mesenchymal stem cells (MSCs) transplantation can improve survival, differentiation and subsequent activities of implanted cells in swine hearts with acute myocardial infarction (AMI).Methods: Fifty-six Chinese mini-pigs were divided into 8 groups (n=7 in every group), including group 1 (control), group 2 (MSCs transplantation alone), group 3 (Tongxinluo administration, TXL), group 4 (TXL + MSCs), group 5 (simvastatin),group 6 (simvastatin + MSCs), group 7 (atorvastatin), group 8 (atorvastatin + MSCs), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Then autologous bone marrow MSCs (3×10⁷ cells per animal) were injected into infarcted and peri-infarct myocardium immediately after AMI and reperfusion. The survival and differentiation of implanted cells in vivo were detected with immunofluorescent staining. The data of cardiac function were obtained at baseline (1 week after transplantation) and endpoint (6 weeks after transplantation) with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The cellular apoptosis in the peri-infarction region was detected with TUNEL assay and oxidative stress level was investigated in the post-infarct myocardium. Inflammatory cytokines within the post-infarcted myocardium, including interleukin-1β(IL-1β), IL-6 and tumor necrosis factor (TNF-α), were detected with Western blotting at the protein level.Results: Baseline SPECT showed that the area of perfusion defect was not significantly different among all groups (51.8±16.5%, 46.5±9.2%, 47.9±12.2%, 52.7±15.5%, 49.7±16.8%, 53.7±14.9%3 48.9±15.9% vs 50.7±14.5%, all P＞0.05). At endpoint, perfusion defect in group MSCs and TXL was not different compared with group control (47.3±13.2%, 44.8±13.9% vs 47.8±11.1%, both P＞0.05). However, it was significantly decreased in group simvastatin, atorvastatin and three drug-facilitating MSCs groups compared with group control (39.3±12.4%, 38.7±13.1%, 27.6±7.4%, 28.3±7.7%, 29.3±9.0% vs 47.8±11.1%, all P＜0.05). Baseline MRI indicated that there were not significant differences in cardiac function among all groups (all P＞0.05). At endpoint, every cardiac function parameter in group MSCs and TXL was not significantly different compared with group control (all P＞0.05), however, the number of dyskinetic segments (P=0.039) and infarcted size (P=0.0061) in group simvastatin, and the number of dyskinetic segments (P=0.04) in group atorvastatin, were remarkably decreased compared with group control. Except end-diastolic volume (EDV), other parameters of cardiac function in the three drug-facilitating MSCs transplantation groups were significantly improved compared with group control (all P＜0.05).H&E and Masson's Trichrome staining showed that there were serious and extensive fibrosis and inflammatory cell infiltration with seldom surviving myocardium within infarcted regions in group control, MSCs and TXL. However, fibrosis and inflammation were slighter in group simvastatin and atorvastatin than that in group control. Furthermore, inflammation and fibrosis were significantly decreased in the three drug-facilitating MSCs transplantation groups. The implanted MSC survivals in the three drug-facilitating MSCs transplantation groups were significantly more that that in group MSCs (223.1±26.9, 310.6±83.8, 373.9±90.3 vs 73.2±21.3/HPF, all P＜0.0001), and were the same as the cardiomyogenic differentiation efficiencies (55.6±12.1%, 46.0±5.1%, 44.4±12.5% vs 8.7±2.4%, all P＜0.0001) and connexin 43 expression (22.4±6.6, 17.0±1.6, 14.2±2.6 vs 4.7±1.8, all P＜0.0001). In addition, the capillary densities within infarcted and peri-infarction regions in group MSCs, TXL, simvastatin and atorvastatin were not significantly different from that in group control (all P＞0.05). However, they were significantly more in the three drug-facilitating MSCs transplantation groups than that in group control (all P＜0.0001).At endpoint, the apoptotic index in group control was significantly increased compared with that in group sham (10.1±1.8 vs 0.9±0.3, P＜0.0001). There was not significant difference in the apoptotic index between group control and MSCs (9.2±1.4, P＞0.05), however, it was remarkably decreased in group TXL (5.6±1.7), simvastatin (5.7±1.5) and atorvastatin (5.2±1.9) compared with group control (all P＜0.0001). Based on the drug application, it was less in the three drug-facilitating MSCs transplantation groups than that in group control (2.4±0.5, 2.3±0.3, 1.9±0.3, P＜0.0001). In addition, Western blotting indicated that Bax increased and Bcl-2 decreased significantly in group control compared with group sham (both P＜0.0001). However, Bax was decreased and Bcl-2 increased in group MSCs, three drug application and drug-facilitating MSCs transplantation groups (all P＜0.0001).In addition, SOD activity in group control was significantly decreased compared with group sham (82.5±8.2 vs 122.5±12.2 U/mg protein, P＜0.0001) and MDA contents decreased (9.0±0.8 vs 5.5±0.9nmol/mg protein, P＜0.0001). They were not remarkably different between group control and MSCs (P＞0.05). However, the three drug application and three drug-facilitating MSCs transplantation significantly increased SOD activity (all P＜0.0001) and decreased MDA contents (all P＜0.0001).Furthermore, the expressions of IL-1β, IL-6 and TNF-αin group control were significantly higher than that in group sham (all P＜0.0001), however, they were remarkably decreased by the three drug application and three drug-facilitating MSCs transplantation (all P＜0.0001).Conclusion: Our study demonstrated (1) immediately intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival and differentiation potential of implanted cells in vivo, without significant benefits in cardiac function; (2) TXL-, simvastatin- and atorvastatin-facilitation resulted in significantly powerful survival and differentiation potential of implanted cells in vivo via inhibition of apoptosis, oxidative stress and inflammatory responses, accompanying by significant benefits in cardiac function.