Experimental Study On Repair Of Myocardial Injury By Stem Cells

Section I Monocyte chemotactic protein-1 promotes the myocardial homing of mesenchymal stem cells in dilated cardiomyopathyBackground:Dilated cardiomyopathy (DCM) is the most common form of non-ischemic cardiomyopathy that leads to heart failure. Mesenchymal stem cells (MSCs) are under active investigation currently as a potential therapy for DCM. However, little information is available about the therapeutic potential of intravenous administration of MSCs for DCM. Moreover, how MSCs home to the myocardium in DCM is still unclear.Methods:MSCs were isolated from bone marrow of C57/BL6 mice and expanded in vitro. DCM was induced by intraperitoneally administering Doxorubicin and MSCs or vehicles were infused through the internal jugular vein. Cardiac functions including the percentage of fractional shortening, left ventricular diastolic dimension, left ventricular end-diastolic pressure, and left ventricular maximum dp/dt were evaluated by echocardiographic and hemodynamic studies. Fibrosis was determined by Masson’s trichrome staining. The mRNA expression levels of monocyte chemotactic protein-1 (MCP-1), stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-la (MIP-lα), and monocyte chemotactic protein-3 (MCP-3) were determined using real time polymerase chain reactions and the protein expression level of MCP-1 was detected with Western blot. The MSCs expression of C-C chemokine receptor type 2 (CCR2), a MCP-1 receptor, is confirmed by Western blot and flow cytometry analysis. The chemotactic effects of MCP-1/CCR2 were checked by assessing the migration in vitro and in vivo.Results:MSCs transplantation improved the cardiac function and decreased the myocardial fibrosis of mice with DCM. MCP-1 was up-regulated in dilated myocardial tissue both at the mRNA and protein level while SDF-1, MIP-1α and MCP-3 remain unchanged. CCR2 was present in MSCs. MCP-1 promoted MSCs migration in vitro while CCR2 inhibition decreased the migration of MSCs to the dilated heart.Conclusions:This study shows that peripheral intravenous infusion of MSCs can support the functional recovery of DCM, and this effort is caused by the myocardial homing of MSCs involving of MCP-1/CCR2 pathway. Modulation of MCP-1/CCR2 signaling system could be as a novel therapeutic target for DCM.Section Ⅱ Generation of human induced pluripotent stem cells from adult cardiac fibroblast and the application in the mouse model of myocardium infarctionBackground:Induced pluripotent stem cells (iPSCs) retain epigenetic characteristics from their tissues of origin. This epigenetic memory can influence iPSC differentiation and the subsequent function of iPSC-derived cells. Thus, we assume that iPSC-derived cell therapy may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells.Methods:Cardiac fibroblasts from patient with heart failure were used to generate human iPSC lines (hciPSC) by Sendai virus transduction using Oct3/4, Sox2, Klf4 and cMyc as programming gene. After the establishment and biology identification, hciPSC were induced to differentiate into cardiomyocyte (hciPSC-CMs) via cytokine including Activin A, BMP4 and bFGF in vitro. Myocardial infarction (MI) was created by occluding the left anterior descending artery (LAD) in immunodeficient mice. Four weeks after transplantation of hciPSC-CM sheets, immunistaining of human specific nuclei antibody (HNA) and human specific cardiac troponinT (hcTNT) were performed to evaluate the engraftment rate and cell maturation, the heart function was detected by echo with the measurements of left-ventricular ejection fraction (EF%) and fractional shortening (FS%). TUNEL assay was used for the assessment of cell apoptosis and vascular density in the heart tissue was measured by CD31 staining.Results:We showed that human cardiac iPSCs can be generated from adult cardiac fibroblasts, and the biology identification illuminated that iPSCs displayed a normal human chromosomal structure, expressed the pluripotency markers Tral-60, SSEA4, and Oct4 and the mRNA levels of pluripotency gene such as Oct3/4, Sox2, and Nanog were similar to the levels observed in a control line of iPSCs. Pluripotency of the hciPSCs was confirmed via the formation of teratoma which contained cells of all three developmental germ layers. HciPSC can be differentiated into cardiomyocytes (CMs) with greater than 92% CM purity. Four weeks after hciPSC-CMs transplantation into the mice with myocardium infarction, more than 30% of the original cells survived in the mouse heart based on the number of hcTNT+ and HNA+ cells and the cell sheets had become structurally organized, with aligned myocytes. The EF% and FS% were improved in the cell transplantation group compared with MI group, p<0.05. TUNEL assay showed that the apoptotic cells were 45% less in myocardium underneath of the cell sheet in the hciPSC-CM group than myocardium of the MI group, p<0.05. The myocardial vascular density was significantly higher in the peri-infarct region of hciPSC-CM hearts than in the corresponding regions of hearts from MI animals, p<0.05.Conclusions:Human cardiac iPSCs can be generated from adult cardiac fibroblasts successfully and differentiate into cardiomyocytes with high efficiency. HciPSC-CMs can survive and mature in the mice of myocardium infarction on the 4 weeks after transplantation and improve the heart function which is likely secondary to a combination of the reduction of apoptosis and increased vascular density.