Multi-Potent Sca-1+ CPCs Figure 2. Differentiation potential of isolated Sca-1 high or low expressing cell populations. A, All cell populations were stained for cardiomyocyte specific marker cardiac MHC (green) and DAPI (blue) after induction. White arrow indicated the cells stained positively for cardiac MHC. Magnification =6400. B, Cardiac MHC positive cells were calculated before induction (control) and after induction (Induction). The positive rate was presented as ratio of MHC positive cell number to total cell number (*p,0.01 vs control; **p,0.05 vs control; { p,0.05 vs CSL1, CLS2, and MSH). C, Expression of cardiomyocyte specific genes analyzed by RT-PCR. RNA extracted from whole adult mouse heart (mH) was used as positive control. Arrow indicated the change of cardiomyocyte specific gene expression after induction. doi:10.1371/journal.pone.0025265.g002 group (Figure 5A) whereas no significant difference in capillary density in the remote zones. To evaluate whether the neovascularization was induced by some paracrine factors from transplanted cells, CSH2 cells were stimulated by hypoxia to mimic the in vivo ischemic environment. After 2-hours hypoxia, there was 2-fold increase in VEGF gene expression in CSH2 cells as compared to CSH2 cells cultured under normoxia (Figure 5B). We also observed that 1-week after cell transplantation, nanocrystal (red) labeled transplanted cells were clearly incorporated into vWF+ microvessels located between cardiomyofibers (Figure 5C). Two and 4-weeks after cell transplantation, cells positive for both nanocrystals (red) and vWF+ (green) were detected in infarcted area. Myogenesis. We tracked cardiomyogenic fate of the transplanted cells using red fluorescence nanocrystals in vivo for up to 4-weeks after transplantation. The transplanted cells were observed only in infarcted area but were not detected in non- infarcted area (Figure 6A). One week after cell transplantation, extensive presence of the transplanted cells was observed at the site of the cell graft which appeared small and round with weak positivity for cTn-I (Figure 6B). Two and 4-weeks later, many nanocrystal- positive cells were aligned parallel to the native microfibers and expressed cTn-I (Figure 6C). These findings indicated slow and progressive morphological transformation of transplanted cells into cardiomyocytes. Despite clear evidence of differentiation potential of CSH2 cells into mature cardiomyocytes in vivo, this cell population failed to adopt mature cardiac phenotype in vitro even if they expressed cardiomyocyte lineage specific genes and proteins. However, CSH2 cells co-cultured with cardiomyocytes indeed transformed into cardiomyocytes (Figure 6D). Discussion We report successful isolation of Sca-1+ cells from adult mice heart based on their functional competence. The isolated Sca-1+ PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25265