The Study On The Therapy Of Autotransplantation With Mesenchymal Stem Cells For Myocardial Ischemia

Background: Viable cardiomyocytes after myocardial infarction (MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration. Biology therapy represents one current approach in the treatment of myocardial ischemia. However, the combination of mesenchymal stem cells(MSCs) transplantation and gene therapy remains controversial. In order to investigate whether the implantation of MSCs results in sustained engraftment and myogenic differentiation, improves the postinfarction left ventricular(LV) perfusion and cardiac function, we transplanted the autologous MSCs and the MSCs induced by 5-azacytidine(5-aza)and transfected with the porcine transforming growth factor Beta 3(pTGF-β3)cDNA in a pig MI model in this study. Part ⅠA Pig Model of Myocardial Infarction by Intracoronary Embolization With Gelatin Sponge Objective A large-animal model of myocardial infarction(MI) was produced by transcatheter embolization of the left coronary artery(LAD) using a gelatin sponge. Methods Seven pigs were underwent transcatheter embolization of LAD using gelatin sponge to produce anteroapical myocardial infarction. Coronary angiography,Echocardiography and Pathology were performed 4 weeks later. Results In the MI group, the LV end-diastolic dimension increased (control versus MI: 37.0 mm±3.4mm and 50.8 mm±6.1 mm, p<0.01), the ejection fraction(EF) decreased (control versus MI: 62.3%±2.9% and 36.6%±2.1%, p<0.001). Coronary angiography revealed the LAD remained occluded. The postmortem specimen showed a transmural MI scar in the anteroseptal and apical regions in the MI group, the scar areas was 18.4%±1.6% of total left ventricular free wall and the scar thickness was 3.5 mm±0.8mm. Conclusion This pig model of MI is reliable, reproducible, and similar to the human condition. Part ⅡCulture of Mesenchymal Stem Cells from Porcine Bone Marrow and Transformation to Myogenic Cell. Objective By establishing a method for the culture of porcine mesenchymal stem cells(MSCs) from bone marrow and the transformation to myogenic cells in vitro, a new cell source for the cellular cardiomyoplasty is provided. Methods MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer. After being co-cultured with 5-azacytidine(5-aza) for 24h, the cultured cells were evaluated by immunohistochemical stains. Results The adherent, fibroblast-like cells were confluent in single layer after plating for 10～12 days. The cell cycle analysis showed that 73% of MSCs was in G0/G1 phase. After being co-cultured with 5-aza, some of MSCs became spindle-like and were found to be stained positively with desmin, MHC, cTnI, and Cx-43. Conclusion Porcine MSCs can be isolated from postnatal bone marrow through their adherentability. Myogenic cells can be generated from MSCs in vitro. It is suggesting that MSCs may be a new cell source for the cellar cardiomyoplasty. Part ⅢClone of Porcine Transforming Growth Factorβ3(TGF-β3) cDNA and Expression in Myogenic Cells. Objective To clone the porcine transforming growth factor β3(pTGF-β3) geneand investigat the pTGF-β3 expression in transfected cells in vitro. Methods According to the nuclear acid sequence of pTGF-β3, the gene was cloned with RT-PCR from the porcine ovary and subcloned into the pMD18-T vector. An eukaryotic expression vector for pTGF-β3(pcDNA3.0/pTGF-β3) was constructed by use of recombinant DNA technique, and porcine myogenic cells were transfected with pcDNA3.0/TGF-β3 plasmid by DOTAP mediated gene transfer. The transient expression of pTGF-β3 was detected by RT-PCR method 24h later. The transfected cells selected by G418 were detected using Western Blot to investigate the stable expression of pTGF-β3 gene. Results pTGF-β3 mRNA expression could be detected distinctly in transfected cells 24h later. The expression intensity of pTGF-β3 in MSCs selected by G418 remained strong 2 weeks later. Conclusion pTGF-β3 gene could be expressed stably in transfected cells, and the cytokine gene transfer for gene therapy of MI is feasible. Part ⅣStudy on the Therapy of Autotransplantation with Mesenchymal Stem Cells for Myocardial Ischemia Objective To investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improves cardiac function in a porcin MI model, and to explore whether the combined application of MSCs and gene in the treatment of MI is feasible. Methods MI models were created by occluding the distal LAD in 30 MEISHAN pig(s21～25 ㎏) with gelatin sponge. The cardiac function was evaluated by magnetic resonance image(MRI) measurements 4 weeks later. All the pigs were divided into five groups: the control group(Group A, n=6), the DMEM group(Group B, n=6), the MSCs group(Group C, n=6), the 5-aza-induced group(Group D, n=6) and the pTGF-β3-transfected group(Group E, n=6). With the animal under general anesthesia, 30 mL iliac bone marrow was aspirated,and the MSCs were isolated and purified by centrifuge. After being co-cultured with 5-aza for 24 h to induce to a myogenic phenotype(Group D and E), the induced cells were transfected with the pTGF-β3 gene(Group E). Then neither a graft of autologous CM-DiI labeled stem cells(2×108/3 mL) was injected into the infarct by transcatheter. Another 4 weeks later, the cardiac function and regional perfusion measurements were repeated by MRI, and the histologic characteristics of the hearts were also studied to assess MSCs engraftment and myogenic differentiation in the infracted areas under the fluorescence of confocal microscope(FCM). The plasma BNP levels was also detected with ELISA. Results Before the cells implantation, the LV end-diastolic dimension(EDLVd) increased and the stroke volume(SV) decreased in the MI hearts. After the cells implantation, the MRI scans showed that the cardiac function was significantly improved(compared with pre-implantation, P<0.05), the implanted MSCs prevented the infarct region from thinning and expanding, improved contraction and increased perfusion in all groups relative to the control hearts(P<0.05). The left ventricular chamber size were smaller(P<0.05) in the hearts with transplanted cells than that in the control hearts(P<0.05). However, the improvement was even markedly greater in Group D and E(compared with Group C). And the CM-DiI labeled cells engrafted within the infarct in islands and assumed a myocyte morphology. The infarct sites had more islands of cardiac-like tissue in Group D and a greater capillary density in Group E(P<0.05) than did in Group C. Conclusion The MSCs are capable of engraftment in host myocardium, may improve the cardiac function by attenuating contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells. The induced MSCs might form more islands of cardiac-like tissue, and the pTGF-β3 might increase perfusion in the infarct by angiogenesis. The combination of them show a markedly greater improvement of cardiac function, and the extracellular matrix(ECM) may play an important role in this process.Combined gene transfer and cell transplantation strategies will be an effective approach for the treatment of MI.