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Background. Human cardiac-derived progenitor cells (hCPCs) have shown promise in treating heart failure (HF) in adults. The purpose of this study was to describe derivation of hCPCs from pediatric patients with end-stage HF. Methods. At surgery, discarded right atrial tissues (hAA) were obtained from HF patients (n = 25; hAA-CHF). Minced tissues were suspended in complete (serum-containing) DMEM. Cells were selected for their tissue migration and expression of stem cell factor receptor (hc-kit). Characterization of hc-kitpositive cells included immunohistochemical screening with a panel of monoclonal antibodies. Results. Cells, including phase-bright cells identified as hc-kitpositive, spontaneously emigrated fromhAA-CHF in suspended explant cultures (SEC) after Day 7. When cocultured with tissue, emigrated hc-kitpositive cells proliferated, first as loosely attached clones and later as multicellular clusters. At Day 21∼5% of cells were hc-kitpositive. Between Days 14 and 28 hc-kitpositive cells exhibited mesodermal commitment (GATA-4positive and NKX2.5positive); then after Day 28 cardiac lineages (flk-1positive, smooth muscle actinpositive, troponin-Ipositive, and myosin light chainpositive). Conclusions. C-kitpositive hCPCs can be derived from atrial tissue of pediatric patients with end-stage HF. SEC is a novel culture method for derivation ofmigratory hc-kitpositive cells that favors clinical translation by reducing the need for exogenously added factors to expand hCPCs in vitro. [ABSTRACT FROM AUTHOR]