Abstract: Objective To isolate and culture cardiac stem cells(CSCs) in vitro and evaluate their potential of differentiation into functional cardiac myocytes.Methods Myocardial tissues obtained from neonatal SD rats were cut into pieces of 0.5-1.0 mm3,and digested twice for 5 min at 37 ℃ with 0.2% trypsin and 0.1% collagenase II.The remaining tissues were cultured in complete explant culture medium(CEM) at 37 ℃ in the presence of 5% CO2.About a week later,a layer of fibroblast-like cells was generated from the adherent explants.These cells were passaged and seeded at about 1×106 cells/ml in poly-D-lysine-coated multi-well plates in cardiosphere-growing medium.When beating of the cultured cells was observed(at week 2),flow cytometry and immunohistochemistry were performed for identification of the primary and passaged cells.Results The primary cells were successfully cultured from the digested myocardial tissue,and flow cytometry demonstrated the phenotype of c-kit+CD31+CD34-CD45-CTnT-.After cell passage for about two weeks,single beating cells and cell clusters with synchronized contraction were seen microscopically,and their phenotype was converted to c-kit+CD31-CD34-CD45-CTnT+.Immunohistochemistry staining identified CTnT expression in the passaged cells but not in the primary cells.Conclusions A cell population with the phenotype c-kit+CD31+CD34-CD45-CTnT-has been obtained from neonatal SD rat heart,which possesses the potential to differentiate in vitro into beating cardiac myocytes and express CTnT protein.