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Primary culture and identification of cardiac progenitor cells of neonatal rats
HOU Bin ZHANG Yong-chun CAI Xin-hua* ZHU Zhan-zhan TIAN Xiang-qing
Acta Anatomica Sinica ›› 2016 ›› Issue (2) : 216-220.
Primary culture and identification of cardiac progenitor cells of neonatal rats
Objective To primarily culture and identify the cardiac progenitor cells (CPCs) in vitro with morphological observation, immunofluorescence and tracing technology. Methods The CPCs were separated and cultivated with differential adhesion methods. The proliferation of CPCs was traced with two carboxyfluorescein succinimidyl ester(CFDA-SE) tracer, observed with a phase contrast microscope. The calcium ion concentration of CPCs was detected with Fluo-3/AM. The expressions of stem cell markers and cardiomyocyte markers were detected with immunoflurescent staining in colony proliferation cells. Results The CPCs of primary culture in vitro with differential adhesion methods showed colony proliferation, in which the undifferentiated cells appeared as circle, oval or irregular and took the arrangement of cobblestone appearance. The cell colony was bigger and the differentiated cells extended to protrude or showed spindles with the extension of incubation time. About 3 weeks, there were the beating myocardial cells in the colony proliferating cells. The CFDA-SE tracing showed that the fluorescence of colony proliferating cells decreased several times. Fluo-3/AM calcium ion inspection showed that its concentration in cardiomyocyte-like cells of colony proliferating cells was more than that in undifferentiated CPCs. Immunoflurescence staining appeared as heterogeneous populations in colony proliferating cells, including c-kit+/CD34-, Nanog+/CD34-and c-kit+/Gata4+ colony. The colony proliferation cells showed cardiac troponin T(cTnT) positive expression. Conclusion The CPCs isolated in vitro with the differential adhesion methods have the characteristic of colony proliferation and differenciation, which can differentiate into cardiomyocytes. They are the best cells to research the biological characteristic, such as, the surface marker, the proliferation mechanism and regulation mechanism.
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