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Cardiac progenitor cells (CPCs) Sca1+ were isolated by selection of single-cell colonies derived from hearts of male C57BL6 mice that express high levels of the protein marker Sca-1. The colony-derived cells have a short doubling-time (similar to embryonic stem cells), self-renewal capacity (demonstrated using a sphere-formation assay) and sphere-like cell aggregation with homogeneous, positive staining for Ki67. In addition, the cells demonstrate ability to differentiate into osteogenic, chondrogenic and cardiac cell lineages.
Hearts of male C57BL6 mice (12-weeks-old) are removed from their aorta, pulmonary artery, and pericardium and digested with collagenase type-II and DNAse-I. The cells are then fractionated with Percoll (70%) and cultured in serum-free maintenance medium containing DMEM/F12 supplemented with B27, epidermal growth factor (EGF; 20 ng/ml), and basic fibroblast growth factor (bFGF; 40 ng/ml). After 7 days, the cells are re-seeded in the maintenance medium at a low density (100 cells/cm2) to initiate colony formation. After 16 days, the obtained colonies are transferred into larger wells and cultured individually in expansion medium containing DMEM/F12, supplemented with FBS (2%), B27-Supplement, EGF (20 ng/ml), bFGF (40 ng/ml) and leukemia inhibitory factor (LIF; 10 ng/ml). After reaching 90–100% confluence the colony-derived cells are re-seeded on new dishes and maintained in expansion medium. Colonies with high Sca-1-expressing cells (tested using FACS analysis) are selected and further cultivated in expansion medium.