Autologous Bone Marrow Stem Cell Transplantation For Ischemic Heart Disease

BackgroundThe loss of cardiomyocytes, scar formation and progressive ventricular remodeling caused by myocardial infarction are the major factor influencing the cardiac function and often end in refractory heart failure. Current therapeutic modalities including pharmacological treatment, percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG) mainly aim at saving the viable cardiomyocytes, improving the function of survival cardiomyocytes and reversing ventricular remodeling. The ability to regenerate damaged myocardium presents a major challenge in the treatment of myocardium infarction. Cardiac transplantation is hampered by the limited availability of donor organs, the complications of immunosuppressive therapy, and the long-term failure of grafted organs. Thus, the development of alternative therapeutic strategies to regenerate myocardium and combat intractable cardiac disease remains an important therapeutic goal.Stem cell can self-duplicate and is capable of differentiating into many other cells such as nervous cell and muscle cell. Studies using stem cell transplantation for the treatment of damaged myocardium demonstrated inspiring results that implanted stem cell might regenerate myocardium,induce neovascularization and reverse ventricular remodeling. We may be entering into a new era of heart therapy , not just preserving existing heart muscle but regenerating heart muscle with stem cells due to the success of several clinical trials using stem cell therapy.Many questions and challenges remain. What stem cell or combination will provide optimal myocardial regeneration? How will stem cell therapy be administered to ensure maximum therapeutic efficacy? Which kind of animal ischemic model will be most suitable? Also the labeling and identification of the stem cell are to be decided. We try to explore the effect of autologous bone marrow stem cell transplantation on ischemic heart disease from 3 aspects:(l) Can the implanted bone marrow mononuclear cell survive in the infarcted lesion and differentiate into cardiomyocyte? (2) What's the effect of endothelial progenitor cell inside the bone marrow on the neovascularization? (3) What's the influence of bone marrow stem cell on the global and regional left ventricular function?Aims(1) To establish the canine myocardial infarction model by the left anterior descending coronary artery ligation.(2) To explore the method of bone marrow mononuclear cell isolation and local injection.(3) To evaluate the labeling efficiency of 4',6-diamidino-2-phenylindole (DAPI) and the lasting time of the fluorescence.(4) To observe the survival and differentiation ability of the implanted bone marrow mononuclear cell in the infarcted lesion.(5) To evaluate the effect of bone marrow transplantation on angiogenic cytokines such as VEGF and IL-1β.(6) To assess the influence of bone marrow stem cell on the global and regional left ventricular function.Methods10 dogs were randomized into 2 groups, 6 dogs for the bone marrow stem cell transplantation group and 4 dogs for the control group. The left anterior descending branches (LAD) of the coronary arteries were ligated in all the 10 dogs. About 25ml of bone marrow were aspirated from the 10 dogs' iliac bone. Bone marrow mononuclear cell (BM-MNCs) suspension was prepared by density centrifugation using percoll (1.073g/ml). The BM-MNCs incubated with DAPI for at least 30 minutes and labeled by the fluorescence dye. Autologous BM-MNCs suspension was injected into the left ventricular wall with needle and cell culture medium was injected for the control dogs. Echocardiography was performed before the LAD ligation, 1 hour after the ligation and 4 weeks after the BM-MNCs transplantation. Two-dimensional, M-mode, acoustic quantification (AQ) and color kinesis (CK) were used to evaluate the global and regional left ventricular function. 4 weeks after the transplantation, dogs were sacrificed for detection. The detection contents were listed below.(1) The regeneration of cardiomyocyte and endothelium were detected by immuonofluorescence using laser confocal microscope. a-MHC and factor VIII antigen were used for cardiomyocyte and endothelium detection separately.(2) TTC stain for the assessment of infarcted areas.(3) Immuonohistochemistry for the microvessel density evaluation.(4) The mRNA expression of VEGF, IL-lp, TGF-p, bFGF, TNF-a was detected by RT-PCR.(5) Ultrastructural changes were observed by electron microscope. Main Results1. Immuonofluorescence detectionThe DAPI-labeled implanted cells could be located and viewed under laser confocal microscope. The implanted cells showed clear nuclear blue fluorescence. Combined "DAPI and FITC" in images were observed infrozen tissue sections. The amount of the blue fluorescence nuclear was more in the border zone than in the center of the infarction. Positive immunostaining for a -myosin heavy chain and factor VM related antigen in cell-transplanted areas indicated differentiation of implanted cells into cardiomyocyte and vessel endothelial cells.2. Pathological observationCells showing an immature appearance with a large nucleus-to-cytoplasm ratio were detected in the BM-MNCs transplantation group. Fibrosis was effectively inhibited in the BM-MNCs group. Vitreous degeneration and disorder of basic structure could be observed in the ischemic myocardium in the control group.3. Immunohistochemistry detectionImmunohistochemistry detection was performed with anti-factor VI antibody. The density of the microvessels was significantly higher in the BM-MNCs transplantation group than in the control group in the marginal area 4 weeks after the treatment (17.2/HPF versus 9.5/HPF, P<0.05). However, the density of microvessels in the normal and infarction areas was not significantly different between the two groups.4. TTC stainThe TTC non-staining area was the necrosis area and the dark red staining area was viable myocardium. There are 2 cases of subendocardial myocardial infarction in the BM-MNCs group. All the dogs in the control group have transmural myocardial infarction. The dark red staining area in the BM-MNCs group was significantly larger than in the control group (P<0.05), the results being similar to those found by microscope.5. Ultrastructure observationElectron microscope illustrated immature cardiomyocytes, containing scattered Z-bands and myofilaments in the electron-lucent cytoplasm, which were similar to fetal cardiomyocytes. Compared with the BM-MNCs group,mitochondria swelling, crista decreasing, glycogen aggregation were more severe in the control group. The cardiac muscle fiber breakage and fibrosis were frequently observed in the control group than in the BM-MNCs group.6. The mRNA expression of angiogenic cytokinesThe mRNA expression of VEGF^ IL-^ TGF^ bFGF was higher in the BM-MNCs group than in the control group (P<0.05), but the mRNA expression of TNF-a was not detected 4 weeks after the LAD ligation and cell transplantation in either group.7. Echocardiographic detection1 hour after the LAD ligation, the left ventricular anterior wall became thinner. Hypokinetic or akinetic and obvious ejection fraction decrease were observed in both the 2 groups.4 weeks later, the BM-MNCs group had a higher left ventricular ejection fraction. The systolic and diastolic segmental endocardial motion (SEM) increased significantly 4 weeks after the stem cell therapy than the infarction models were initially set up. 4 weeks after the LAD ligation, compared with the control group, two-dimensional, M-mode, AQ and CK echocardiography showed significant improvement in left ventricular ejection fraction and regional systolic and diastolic function in the stem cell transplantation group(P<0.05).Conclusion(1) The labeling efficiency of DAPI was more than 95% and the lasting time of the fluorescence within the body was more than 4 weeks. DAPI could be a good tracer for the bone marrow stem cell.(2) The implanted BM-MNCs could survive in the infarcted lesion and differentiate into cells expressing a -MHC.(3) The implanted BM-MNCs could also survive and differentiate into endothelium.(4) Implantation of BM-MNCs into ischemic myocardium enhances...