| Part I:The effect and mechanism of induced pluripotent stem cell transplantation on cardiac function in a swine model of acute myocardial infarctionBackgrounds:As the leading cause of death in developed countries and one of the major disease burdens in developing countries, cardiovascular disease, especially acute myocardial infarction (AMI), seriously affect the quality of life of the patients. Previously, most of basic researches and clinical studies have shown that variety of stem cells transplantation, including the bone mesenchymal stem cells (MSC), can improve the cardiac pump function after AMI, however, none of them has been used as a standard strategy for patients. iPS cells (induced pluripotent stem cells), firstly generated by Japanese scientists in2006, share much characters with embryonic stem cells (ESC), but are relatively accessible and with less ethical issues and eliminating the problem of host immunorejection when comparing with MSCs and ESC. Several studies have showed iPS cells can restore the damaged heart in small animal model of AMI, but intramyocardial injection of iPS cells has not been assessed in large animals.Objectives:To investigate the efficacy of porcine iPS cells on cardiac function after AMI, and to explore the fate and the mechanism of iPS cells after transplantation.Methods:26pigs (24.83±2.52kg) were randomly divided into three groups:Sham group, PBS group and iPS group.1week after the AMI model creation, allogeneic undifferentiated iPS cells (2x107cells/2ml) or phosphate buffered saline (PBS,2ml) were injected into the ischemic myocardium induced by90minutes occlusion of left anterior descending (LAD) artery.1week,6weeks after cells delivery, cardiac perfusion and function were evaluated by single photon emission computed tomography (SPECT) imaging and cardiac dual-source CT (DSCT).7weeks after transplantation, histological and molecular biology analysis was used to observe infarct size, collagen deposition and distribution, blood vessel density and apoptosis in heart tissue.Results:(1). SPECT showed that the myocardial perfusion defect area was not significant different at baseline among3groups.1week after the cell transplantation, the myocardial perfusion scores between iPS and PBS group were not significant different (13.50±2.07vs.14.50±1.87, P=0.40), but they were both significantly lower than Sham group (P<0.05).6weeks after cell delivery, the myocardial perfusion scores of iPS group were significantly increased compared with PBS group (iPS group vs PBS group,19.33±4.97vs.13.67±2.94, P=0.04), but still lower than Sham group (iPS group vs of Sham control group,19.33±4.97vs.27.67±0.52, P<0.01).(2). DSCT showed that left ventricular ejection fraction (LVEF) of iPS and PBS group decreased significantly compared with Sham group (P<0.001)1week after cell transplantation, but there were no difference between iPS group and PBS group (iPS group vs PBS group,51.72±3.09vs.51.25±3.94, P=0.82).6weeks after cell transplantation, the LVEF of iPS group improved significantly compared with PBS group (iPS group vs PBS group,56.68±4.44vs.50.93±3.91, P=0.04), but still lower than the Sham control group (iPS vs. of Sham control group,56.68±4.44vs.63.37±3.26, P=0.01).(3). HE staining and Masson’s Trichrom staining showed there were more inflammatory cell infiltration, and no viable myocardium cells in the PBS group. Compared with PBS group, there were less fibrosis and inflammatory cell infiltration and more viable myocardium islands in iPS group. iPS cells transplantation decreased the fibrosis area of border zone compared with PBS group (P<0.05). Electron microscopic examination also found that iPS cell transplantation can ameliorate the alteration of ultrastructure after MI.(4). Immunofluorescence showed that iPS cells can differentiate into vascular cells, thereby promoting vascular regeneration for infarct zone and border zone. Immunohistochemistry showed the capillary density in infarct zone of iPS group was significantly higher than PBS group and Sham group (PBS group vs the iPS group,11.09±3.93vs.26.38±8.17, P<0.001; Sham group vs iPS group,2.31±2.08vs.26.38±8.17, P<0.001).(5). TUNEL staining showed that the number of apoptotic cells in both infarct zone and border zone of iPS group significantly decreased compared with PBS group. Western Blot analysis also showed that the pro-apoptotic protein Bax expression was significantly increased (P<0.05) and the antiapoptotic protein Bcl-2expression was significantly lower (P<0.05) in the border’zone of PBS group, though iPS transplantation can ameliorate these changes.(6). Pathological examination revealed that no tumors were detected in the heart, lung, liver, spleen or kidney after the morphologic and histopathology examination ever4months later after iPS transplantation.Conclusions:(1). iPS cells transplantation can improve left ventricular function and myocardial perfusion in pigs with AMI;(2). iPS cells transplantation can reduce the deposition of collagen fibers in the myocardium and reduce myocardial fibrosis after AMI;(3). iPS cells can differentiated into vascular cells in vivo and significantly promote the formation of blood vessels after myocardial infarction, which may be one of the mechanisms for improvement of cardiac function;(4). iPS cell can inhibit the cardiomyocyte apoptosis both in the infarct zone and border zone after AMI. Part II:The influence and mechanism of induced pluripotent stem cell transplantation on cardiac electrophysiology of postinfarcted swinesBackground:Although most studies have shown that stem cells transplantation can improve cardiac function of ischemic heart disease, there are still controversial about whether stem cells can improve electrophysiological matrix, reduce or promote the occurrence arrhythmia after MI. As a novel ESC-like stem cells, we must first to explore the influence of iPS on cardiac electrophysiology before it’s use on clinical application.Objective:To explore influence and potential mechanism of iPS transplantation on cardiac electrophysiology in pigs with AMI.Methods:1week after the AMI model creation, iPS cells (2x107cells/2ml) or PBS (2ml) were injected into the ischemic myocardium induced by90minutes occlusion of LAD artery.7weeks after cell transplantation, electrocardiogram parameters changes were assessed using surface12lead electrocardiogram (ECG), the electroanatomical recordings were obtained using CARTO mapping system, ventricular arrhythmias (VAs) inducibility were assessed by programmed electrical stimulation (PES), the cardiac fibrosis and collagen deposition was determined by Masson’s Trichrom staining, the Cx43distribution and the expressions of Cx43protein, autonomic nervous system-related proteins, such as tyrosine hydroxylase (TH) protein, growth associated protein43(GAP43) were studied by Western Blot.Results:(1). There were no significant difference between iPS group and PBS group, in respect of parameters such as HR, RR, PR, QRS, QTcD.(2).7weeks after cell transplantation, CARTO mapping system showed that the scar tissue and ischemia myocardium which indicated by red area and yellow area of PBS group were larger, while corresponding area of iPS group was smaller.(3). PES showed that3pigs of PBS group were induced VAs, including2of them were ventricular fibrillation, and1of iPS group was induced VAs. Totally, induced VAs was not statistically significant between iPS group and PBS Group (50.0%vs.16.7%, P=0.545).(4). After MI, Cx43protein expression of border zone of PBS group decreased significantly, while iPS cell treatment can partially correct these abnormalities.(5). After MI, the expression level of TH and GAP43protein increased significantly in border zone, simultaneously, the domination of sympathetic and parasympathetic nerves got out of balance.7weeks after transplantation, iPS cells can in part to improve the neural remodeling.Conclusion:(1). iPS cells transplantation can improve the electroanatomical recordings after AMI;(2) iPS cells did not reduce the induced VAs, but also was not proarrhythmic;(3). iPS cells can improve the expression of Cx43;(4). iPS cells can improve cardiac neural remodeling, which was important in the occurrence VAs after... |
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