Multi-Potent Sca-1+ CPCs Figure 5. Neovascularization of the infarcted myocardium by cell transplantation. A, Left panels show endothelial cell immunostaining with anti-vWF (green) and DAPI (blue) of control and cell injection group in border zone and remote zone of infarcted heart. Middle panel shows quantitative estimation of capillary density expressed as the number of capillaries per high power magnification field. Right panel shows quantitative estimation of capillary density expressed as capillaries per myocyte. (*p,0.05 vs control). B, Relative mRNA expression level of VEGF under normoxic and anoxic conditions analyzed by real-time RT-PCR. (*p,0.05 vs control). C, Representative immunostaining of endothelial cells with anti-vWF (green) one, two, and four weeks after transplantation of nanocrystal-labeled cells (red) to detect direct incorporation of transplanted cells into newly formed vessels. Bar = 50 mm. doi:10.1371/journal.pone.0025265.g005 embryoid bodies differentiated into three cardiac cell lineages [30]. Similarly, cardiac progenitor cells from developing mouse embryo differentiated into smooth muscle cells and cardiomyocytes [26]. Although the definite hierarchy of these reported cardiac precursor/progenitor cells remains unclear, it is suggested that Bry+/Flk-1+ cells may precede the separation of Isl-1+ cells and, the Nkx2.5 expressing progenitor cells may be downstream of the Bry+/Flk-1+ cells and Isl-1+ cells because of their bi-potential differentiation capacity [25]. In accordance with our data, CSH2 cell population is positioned between Isl-1+ and Nkx2.5 due to expression of Isl-1 and Flk-1, and lack of Nkx2.5 during the undifferentiated state while these cells expressed Nkx2.5 after differentiation respectively. Our in vivo studies in the experimental animal model provided a clear evidence of differentiation potential of CSH2 cells into mature cardiomyocytes. We believe that rate of cardiomyogenic PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25265