Cardiac Differentiation Of Human Cardiac Stem Cells In Vitro

BackgroundIschemic cardiomyopathy caused by myocardial infarction is one of the mostcommon reasons of heart failure. Drug therapy can attenuate symptoms and reduce therisk of coronary heart disease. Early PCI can open blood vessels and reduce mortality inmyocardial infarction patients. Coronary artery bypass grafting can improve blood supplyin the distance area of the occlusive vascular. Heart transplantation can fundamentallyachieve the purpose of treatment, but it is hindered by immune rejection, scarce donorsource and heavy financial burden. At present, neither drugs, PCI or surgery treatment canfundamentally solve the problem of loss of cardiomyocyte, as well as meet therequirements of heart cells for rebuilding. Cardiac stem cells (CSCs) have beencharacterized of self-renewal, clonogenic, and multipotent capacity, and able todifferentiate into three major cardiac cell populations, including cardiomyocytes, smoothmuscle cells and endothelial cells. Autologous CSCs transplantation has no ethical issue orimmunological rejection, and is the ideal seed cells for cell therapy of ischemiccardiomyopathy. Recently, several experiments focus on animals, in which a lot of methods can induce CSCs with various efficiency. Human cardiac stem cells have greatclinical potential, and they are expected to becthe ideal seed cells for cell theray ofcardiovascular diseases.Objectives1. Isolation,culture of human cardiac stem cells and study its morphology.2. Exploration of cardiac stem cells purification and identification method3. Exploration of5-AZA, Ang II and combined induction of cardiac stem cellsdifferentiate into cardiomycytes and the method to increase its efficiency.Methods1. Myocardial tissues obtained from the right atrial appendage of patients duringsurgery were digested with collagenaseⅡwith informed concentration. The isolated cellswere inoculated in culture flask and Cells of90%confluence were passaged. P2～P4cellswere collected for identification of surface marker CD117(c-kit), CD31, CD45by flowcytometry.2. P2to P8cells were collected and incubated with monoclonal anti-humanPE-CD117specific antibody, then sterile sorting with flow cytometry. Then purified CSCswere collected for cultivation and research for cell morphology and immunocytochemistry(ICC).3. After confluence around90%, purified c-kit+CSCs were collected fordifferentiation and divided into four groups:①Control group(cells culture medium),②5-AZA group (10μmol/L),③Ang-Ⅱgroup (0.1μmol/L),④5-AZA combined Ang IIgroup（10μmol/L+0.1μmol/L.5-AZA,Ang II single induced group changed for the cellsculture medium after inducing for24h, and combined group changed for Ang II inductionmedium after induced5-AZA for24h, then changed for the cells culture medium afterinducing24h, and continue culture. Observations of the cell morphology variation wereperformed on inverted microscope in96h and4W respectively. Determined theexpression of protein of connexin43(Cx43) and cardiac Troponin I (cTn I) by WesternBlot and immunofluorescence, and determined the cardiomyocyte differentiation rates with flow cytometry.Results1. Characteristic of primary cells: The primary cells are adherent growth after10days,and adherent cells is triangular, fusiform, polygonal. Cells rapidly proliferate afteradherent and reach80%confluence in20days.2. Identification of cellular surface marker with flow cytometry: The surface labeledantigen of CD117(c-kit+),CD31and CD45. positive expression rate of CD117was (26.4±7.52)%, positive expression rate of CD31was (8.5±3.81)%, positive expression rateof CD45was (1.85±0.51)%.3. Human CSCs purification and identification:98.5%purity of CD117(c-kit)positive CSCs can be get by sterile sorting with flow cytometry. The sored cells areadherent growth about after24h, and cells volume are smaller and approximate shape,elongated fusiform, short fusiform.Cells rapid expansion approximately after7days, andabout14days can fuse to80%. Cells morphology is similar by Immunofluorescencemicroscopy and by inversion phase contrast microscope, and a large amount of PE-CD117surface markers of CSCs can be observed by Immunofluorescence microscopy.4. Morphological characteristics of induced human CSCs: After induction for96hours, cell morphology of the Control group showed no significantly change, arranged inno apparent regularity. The cells of5-AZA group died and fall off significantly, andarranged mainly clusters. The cells’ death in5-AZA combined Ang II group is less, andarranged mainly fusiform or cluster. After induction4W, the volume of cells in Controlgroup heterogeneity of elongated fusiform, short fusiform and polygonal. A small numberof cells’ volume is longer than before, and arranged in no apparent regularity. Cellsvolume of each induced groups are longer than before, mainly fusiform, and arranged inbeam. The morphology and arrangement of cells in the5-AZA combined Ang II groupchanged most significantly.5. Identification of induced human CSCs cTn I, Cx43protein expression: Determinedthe expression of cTn I and Cx43protein of each group by immunofluorescence staining,and the results showed that each group expressed protein of cTn I and Cx43, and comparatively low expression was observed in control group, while high expressionwere observed in5-AZA combined Ang II group. Statistics of Western Blot showed thateach group expressed the protein of cTn I and Cx43; the5-AZA combined Ang II groupand the5-AZA group expressed significantly more than the Control group (P <0.01); the5-AZA combine Ang II group expressed significantly more than the Ang II group (P <0.01); the5-AZA combined Ang II group expressed more than the5-AZA group (P <0.05); the5-AZA group expressed more than the Ang II group (P <0.05)；the Ang IIgroup expressed more than the Control group (P <0.05).6. Identification of induced human CSCs differentiation rates: The results of flowcytometry detected the cardiac differentiation rate of each group as follows: the Controlgroup is (28.86±4.62)%; the5-AZA group is (53.71±7.5)%; the Ang II group is(41.48±7.12)%; the5-AZA combined Ang II group is (65.74±6.21)%. Statistics of thedifferentiation rates between other groups had significant differences (P <0.05). The5-AZA combined Ang II group, and the5-AZA group expressed significantly more thanthe Control group (P <0.01).Conclusion1. Cardiac stem cells could be isolated from the right atrial appendage tissue ofpatients through the enzyme digestion.2. High purity cardiac stem cells can be isolated by sterile FAC sorting Cardiac stemcells can rapid grow and proliferate in CSCs culture medium.3. Cardiac stem cells can slowly differentiate into cardiomyocyte withoutintervention.4.5-AZA, Ang II can induce the cardiomyocyte differentiation of cardiac stem cells,and the5-AZA combined with Ang II seems to be more effective compare with singleinducer during the cardiac differentaion process.