Isolation, in vitro culture and identification of cardiac stem cells from neonatal SD rats / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To isolate and culture cardiac stem cells (CSCs) in vitro and evaluate their potential of differentiation into functional cardiac myocytes.</p><p><b>METHODS</b>Myocardial tissues obtained from neonatal SD rats were cut into pieces of 0.5-1.0 mm(3), and digested twice for 5 min at 37 degrees C; with 0.2% trypsin and 0.1% collagenase II. The remaining tissues were cultured in complete explant culture medium (CEM) at 37 degrees C; in the presence of 5% CO(2). About a week later, a layer of fibroblast-like cells was generated from the adherent explants. These cells were passaged and seeded at about 1x10(6) 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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>