| OBJECTIVE:In order to develop human Mesenchymal stem cells derived from first-trimester (6-8weeks) chorionic villi (F-CMSCs) and full-term Mesenchymal stem cells derived from term chorionic membrane (T-CMSCs) in vitro amplification system, in vitro compare the potential of human F-CMSCs, T-CMSCs differentiate to a variety of cell and the capability of formation of capillary-like structures. Two weeks after the establishment of myocardial infarction (MI) in rats, after thoracotomy in the left ventricular wall infarct zone and marginal zone injection of human F-CMSCs, T-CMSCs, observe the effect of cardiac function improvment, further investigate its mechanism as well.METHODS:Human F-CMSCs, T-CMSCs cells were isolated and cultured from fresh pregnancy tissue from6-8weeks early pregnant healthy women who underwent drug abortion and placenta from full-term c-section. Flow cytometry inspected human F-CMSCs, T-CMSCs cell phenotype, three lines differentiation potential of human F-CMSCs, T-CMSCs cells were investigated by specific staining. Two groups of MSC human cell vessel formation ability were tested by capillary formation assay in vitro. The model of rat myocardial infarction was established by ligation of the left anterior descending coronary artery, two-week after successfully build the model, underwent thoracotomy and perform the cell transplantation around the cardiac ventricular infarct zone and surrounding area. The experimental animals were divided into three groups: Control group; T-CMSCs transplantation group; F-CMSCs transplantation group. Four weeks after the cell transplantation, using fluorescence microscope to observe GFP+ F-CMSCs cell survival rate in rats myocardial infarcts location, the improvement of echocardiography in cardiac function was also observed in rats.RESULTS:1. Flow cytometry showed that, cultured P3generation human F-CMSCs, T-CMSCs were both highly expressed CD73, CD90, CD105. On the contrast, they neither expressed blood lineage cells markers CD14, CD34, HLA-DR, nor trophoblast cells marker CK18.2. three-line differentiation potential tests showed human F-CMSCs and human T-CMSCs have similar differentiation potential, after osteogenic the cells showed colony growth and the emergence of calcium nodules in von Kossa staining; after fat-induced clear showed lipid droplets in cells, oil red O staining was positive; after cartilage induced the cells stained blue by toluidine blue staining.3. Flow cytometry analysis showed that in vitro cultured human F-CMSCs express VEGF receptor1,2, FLT-1and KDR, after induced by endothelial cells, human F-CMSCs express endothelial cell markers CD31and vWF. Capillary angiogenesis experiments showed that human F-CMSCs was easier to form pipe network structure than human T-CMSCs.4. Masson staining showed:a large number of myocardial necrosis and disappeared at MI zone, replaced by collagen fibers which were dyed into blue, human F-CMSCs cell transplantation group showed infarct collagen and elastic fibers were significantly reduced in MI zone, only see a small amount of elastic fibers at epicardial.5. Immunofluorescence microscopy observed,4weeks after cell transplantation, there is still some GFP+CMSCs cell survived at myocardial infarcts location.6. Echocardiography results suggested, the left ventricular end diastolic diameter (LVEDd), left ventricular end systolic diameter were significantly reduced in GFP+F-CMSCs cell transplantation group (LVEDs) compared among all the groups, while ventricular ejection fraction (EF), shortening score (FS%) were significantly increased, heart function were improved significantly, the experimental results had statistically significant differences (p<0.05).CONCLUSION:(1) Human F-CMSCs and human T-CMSCs have a similar phenotype and differentiation potential, furthermore both of them also have a strong capillary endothelial cells differentiation capability and in vitro vascular formation capability;(2) Though thoracotomy the infarct zone and fringe human F-CMSCs direct transplantation showed a remarkable effect on cardiac function recovery with acute myocardial infarction in rats. |
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