cardiomyocytes is a pre-requisite and critical for their differenti- ation [5]. These cells co-cultured with cardiomyocytes were indeed transformed into myocytes (Figure 6D). Although we only considered a-sarcomeric actinin positive cell with single nucleus as the differentiated cell, we could not rule out the possibility that these a-sarcomeric actinin positive cells were formed by fusion with co-cultured cardiomyocytes. In order to exclude the possibility of cell fusion between multipotent cardiac stem/ progenitor cells and living cardiomyocytes, we co-cultured cardiac stem/progenitor cells with paraformaldehyde-fixed neonatal rat cardiomyocytes with cardiomyocytes conditioned medium [5]. These results showed that both secreted factors in the cardiomy- ocyte conditioned medium and the membrane bound factors in the cardiomyocytes were essential for induction of cardiomyogenic differentiation of multipotent stem cells. One week after co- culture, small number (similar to co-culture with living cardiomy- ocyte) of CSH2 cell population stained positively for a-sarcomeric actinin (Figure 6D). These data suggested that CSH2 cell population really harbored true immature cell population which differentiated into cardiomyocytes under optimal conditions. Despite promising data, the study has some limitations. Although the cells showed cardiomyogenic regeneration capacity and therapeutic effects post-engraftment, the frequency of cardiomyogenic differentiation of CSH2 cells in vitro conditions was lower than our anticipation. Although the underlying reason is unclear, a recently published study using four cardiac progenitor cell types including Isl-1+/Nkx2.52, Isl-12/Nkx2.5+, Isl-1+/ Nkx2.5+, and Isl-12/Nkx2.52 cells to compare their differentia- tion capacity into cardiomyocytes and smooth muscle cells under two different culture conditions [32]. Interestingly, only Isl-1+/ Nkx2.52 cardiac progenitor cells showed significant increase in cardiomyogenic differentiation under specific culture conditions. The authors suggested that the cardiomyogenic potential of Isl-1+/ Nkx2.52 was modulated by microenviromental geometric cues. These observations supported our results that CSH2 with Isl-1+/ Nkx2.52 cells represented a true population of cardiac progenitor cells. Secondly, inspite of immunohistological evidence that about cardiomyogenic differentiation, future studies would be required showing functional integration of the differentiated cells. Lastly, a direct comparison of the clonally selected Sca-1+ cells with MACS sorted Sca-1+ cells would further show the significance of our new method of Sca-1+ cell purification. In conclusion, we have demonstrated that adult mouse heart harbors Sca-1+/Isl-1+/Nkx2.52 cells with their unique functional features, clonality and self-renewal capacity. This cell population originated from cells of secondary heart field and showed multilineage differentiation potential. Transplantation of these References 1. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, et al. (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114: 763–776. 2. Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, et al. (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13: 970–974. 3. Wang X, Hu Q, Nakamura Y, Lee J, Zhang G, et al. (2006) The role of the sca- 1+/CD312 cardiac progenitor cell population in postinfarction left ventricular remodeling. Stem Cells 24: 1779–1788. 4. Matsuura K, Nagai T, Nishigaki N, Oyama T, Nishi J, et al. (2004) Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem 279: 11384–91. 5. Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, et al. 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Magnification = 6400. B, Calponin positive cell number was calculated in all cell populations both before induction (Control) and after induction (Induction). The positive rate was presented as the ratio of calponin positive cell number to total cell number (*p,0.01 vs Control; **p,0.05 vs Control; {p,0.01 vs all other cell populations; {p,0.01 vs CSH1, CSH2, and CSL1; 1p,0.01 vs CSH2 and CSL1). C, Expression of smooth muscle cell specific genes analyzed by RT-PCR. RNA extracted from whole heart of adult mouse (mH) was used as positive control. (TIF) Figure S2 Differentiation potential of isolated Sca-1 high or low expressing cell populations into endothelial cell lineage. A, All cell populations were stained for endothelial cell specific marker von Willebrand Factor (green) and DAPI (blue) after induction. Magnification = 6400. B, vWF positive cell number was calculated before induction (Control) and after induction (Induction). 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