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Recent remarkable studies have reported that clonogenic putative cardiac stem cells (CSCs) with cardiomyogenic potential migrate from heart tissue biopsies during ex vivo culture, and that these CSCs self‐organize into spontaneously beating cardiospheres (CSs). Such data have provided clear promise that injured heart tissue may be repaired by stem cell therapy using autologous CS‐derived cells. By further examining CSs from the original CS protocol using immunofluorescence, quantitative reverse transcription‐polymerase chain reaction, and microscopic analysis, we here report a more mundane result: that spontaneously beating CSs from neonatal rats likely consist of contaminating myocardial tissue fragments. Thus, filtering away these tissue fragments resulted in CSs without cardiomyogenic potential. Similar data were obtained with CSs derived from neonatal mice as wells as adult rats/mice. Additionally, using in vitro culture, fluorescence‐activated cell sorting, and immunofluorescence, we demonstrate that these CSs are generated by cellular aggregation of GATA‐4 + /collagen I + /α‐smooth muscle actin (SMA) + /CD45 −  cells rather than by clonal cell growth. In contrast, we found that the previously proposed CS‐forming cells, dubbed phase bright cells, were GATA‐4 − /collagen I − /α‐SMA − /CD45 +  and unable to form CSs by themselves. Phenotypically, the CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential. Our data imply that the murine CS model is unsuitable as a source of CSCs with cardiomyogenic potential, a result that is in contrast to previously published data. We therefore suggest, that human CSs should be further characterized with respect to phenotype and differentiation potential before initiating human trials. STEM CELLS 2009;27:1571–1581

Murine “Cardiospheres” Are Not a Source of Stem Cells with Cardiomyogenic Potential