cells except CSL2 showed similar short doubling-time to embryonic stem cells, only CSL2 showed significantly longer doubling-time compared to other cells (Figure 1B). Non-colony- derived Sca-1 cells, MSH, also showed similar doubling-time. These results were consistent with the previous reports [18]. Molecular characteristics and clonogenicity of cardiac cell populations Gene expression profile showed that these cell populations were positive for pluripotent stem cell markers Nanog, Telomerase reverse transcriptase (TERT, which is usually not detectable in cardiac fibroblasts) [19], and Bcrp1/ABCG2 (which play central role to form stem cell-enriched ‘‘side population’’) [6] (Figure 1C). Bmi1 gene, a determinant of self-renewal of hematopoietic and neural stem cell was also detected [20]. These pluripotency markers were detectable in all the cell populations, however, the expression of Nanog and Bcrp1 was obviously weaker in CSH1 cells. These data suggested that these cells had different genetic characteristics compared to the cardiac fibroblasts or endothelial cells. Sphere formation assay ascertained the self-renewal capacity of the isolated cells [8] and showed sphere-like cell aggregation. However, sphere formation with homogeneous positive staining for Ki67 was detected in CSH2 cells only (Figure 1D). Differentiation potential of the cells into cardiac cell lineage We assessed the differentiation potential of these cells into smooth muscle, endothelial, and cardiac phenotypes. After stimulation with platelet derived growth factor-BB (PDGF-BB), these cells showed significant increase in calponin positive cell count (Figure S1 A–B) which was confirmed by RT-PCR (Figure S1 C). Two weeks after induction for endothelial cell differenti- ation, vWF+ cells were detected only in CSH2, CSL2, and MSH populations (Figure S2A) whereas we did not observe expression of endothelial specific markers in any of the cell populations before induction of differentiation (Figure S2B–C). However, a statisti- cally significant increase in vWF+ cells was only observed in CSH2 cells (Figure S2). The expression of endothelial cell specific genes, CD31, VE-cadherin, and vWF was confirmed by RT-PCR in CSH2, CSL2, and MSH populations. Cardiomyogenic differentiation Quantitative analysis of cardiac MHC+ cells revealed highest differentiation potential in CSH2 population (Figure 2A, 2B). At the baseline, the early cardiac marker gene, Nkx2.5 was detected only in MSH population (Figure 2C) whereas GATA4 and MEF2C were detected in all cell populations (Figure 2C). On the contrary, mature cardiomyocyte marker genes, cardiac a-myosin heavy chain (a-MHC), cardiac b-myosin heavy chain (b-MHC), myosin light chain 2a (MLC2a), and myosin light chain-2v (MLC2v) were undetectable in all cell populations. Cardiogenic differentiation was attempted with dexamethasone, DMSO, 5- azacytidine, oxytocine, TGF-b, Wnt1, Wnt5a, BMP2, and FGF4, individually or in combination with two or more agents. Although these agents failed to generate mature beating cardiomyocytes invariably in all the purified cells, stimulation with BMP2 combined with FGF-4 induced cardiac mature marker genes in several cell populations (Figure 2C). Only CSH2 cell population showed multiple cardiac genes (Nkx2.5, b-MHC, and MLC2a) after induction (Figure 2C). MSH population showed only limited cardiomyogenic gene expression (Figure 2C) despite the expression of early cardiogenic marker genes Nkx2.5, GATA4, and MEF2c prior to induction. Multi-Potent Sca-1+ CPCs Genetic background and differentiation potential of Sca-1+ cells Sca-1 is commonly used for enrichment of haematopoietic stem cells [21]. Certainly, Sca-1 high expressing population, CSH2, showed obvious and unique stem/progenitor cell characteristics in all analyses. However, Sca-1 high expressing population, CSH1, showed poor potential inspite of similar isolation method, growth function, and high expression of Sca-1 antigen compared to CSH2. Differentiation assay showed that these cells failed to differentiate into adipogenic lineage, whereas CSH2 and MSH showed obvious differentiation potential into osteogenic and chondrogenic lineages (Figure 3A). CSH1 did not differentiate into any of these three different cell lineages. We also performed gene expression analysis to determine additional difference between Sca-1 high cell populations. The gene expression of LIM homeodomain transcription factor islet-1 (Isl1), the marker of cardiac precursor in the developing as well as postnatal heart [5], was detected in CSH2 and MSH but not in CSH1 cells (Figure 3B). Since Isl1 is the marker of cells from secondary heart field in developing heart [9], we checked the expression of FGF10 and dHand gene which are expressed in the cells from secondary heart field. All of these three cell populations were positive for FGF10 and dHand (Figure 3B). On the contrary, Tbx5 and eHand genes, which are expressed in the cells from primary heart field [22], were detected only in MSH (Figure 3B). The expression of neural RNA-binding protein Musashi1, marker of neural stem/progenitors [22], was also detected in CSH2 and MSH populations but not in CSH1 cells. In accordance with the recent publications that the heart harbors neural crest-derived multipotent stem/progenitor cells, we analyzed the expression of neural crest-related marker genes AP2a, Pax3, and ErbB3 [23]. Only MSH cells were positive for the three neural crest-related marker genes. Although all cell populations expressed epithelial- mesenchymal transition marker genes Snail and Twist [24], expression of Snail was relatively poor in CSH1 cells. Transplantation of Sca-1+ cells into the infarcted heart Cardiac function. Based on our in vitro data, CSH2 were used for transplantation studies in experimental mouse model of MI (Figure 4A). There was no significant difference between cell injected and control groups in the cardiac function indices (LVFS and LVEF), dimensions of LV during diastole (LVDd), and anterior wall thickness (AWT) at base-line levels (Figure 4B). One week after LAD ligation, both groups showed deterioration of cardiac function, slight dilatation of LV dimensions, and decrease of LV wall thickness. However, 4-weeks after LAD ligation, cell injected group showed significantly improved cardiac function and increased wall thickness in the infarct area as compared to control group. Cell transplantation had no significant effect on LV dilatation after MI. Four weeks after their respective treatment, significantly attenuated infarct size was observed in the animals treated with cardiac stem cells (Figure 4C). The infarct size was 28% of LV wall in the cell injected group which was significantly smaller as compared to the control animal hearts wherein the infarct size was 40% of LV wall (n = 3/group). On the other hand, the percent fibrosis area calculated with computer software from the Masson- trichrome stained heart tissues showed weak tendency of reduction in the cell transplanted animal hearts, however, reduction was insignificant as compared to control animals (Figure 4C). Neovascularization. The number of capillaries and their number distribution per myocyte were counted at high power magnification field. Capillary density was increased in border and infarcted zones of cell transplanted group as compared to control PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25265