Progenitor Cell Heart Therapy Figure 3. Scar tissue formation and CPC detection. Histological analysis (Mason’s trichrome staining) and immunohistochemical analysis of the infarcted myocardium demonstrating the extent of scarring (blue) in the myocardium of the three experimental groups at two parallel cross-sections of the myocardium. Sham surgery and saline injections (a–b). Myocardial infarction and saline injections (c–d). Myocardial infarction and CPC injections (e–h). Scale bar: 1 mm. High magnification panel pairs show parallel thin cross-sections stained either with Mason’s trichrome stain or with an antibody against the b-Galactosidase protein which is constitutively expressed in the injected cells (Rosa 26 mouse ESC line). Scar region (i), Interface between the scar and healthy myocardium (j–l). Healthy myocardium (k). Black arrowheads denote b-Galactosidase(+) cells within the myocardium. doi:10.1371/journal.pone.0011536.g003 calculated by extrapolating the number of bGal(+) cells over the entire estimated area of the affected myocardium (<4 mm) and determined to be 12.762.1% of the initial total number of cells injected. We proceeded to examine the differentiation commitment of the transplanted cells. Large tissue areas surrounding the injury site co-stained for b-Galactosidase and a-Actinin (Figs. 4a, b) indicating robust differentiation of the transplanted cells into cardiomyocytes. At higher magnification, a large number of individual or clustered CPC-derived cardiomyocytes were detected incorporated within the host cardiac cell network (Figs. 4c–f). While the presence of rod cell shape and cross-striations (Fig. 4e) indicated advanced cell differentiation, smaller size of CPC- derived cardiomyocytes compared to host cardiomyocytes sug- gested maturation level inferior to that of the adult cells. The majority of the b-Gal staining also co-localized with another cardiac muscle marker Myosin Heavy Chain (MHC, Figs. 4d, f). In the infarcted region of animals which received CPC injections several blood vessels comprised of both donor and host cells were detected (Figs. 4g–j). The bGal(+) CPCs readily differentiated into vascular smooth muscle and incorporated in large and small vessels with bGal(-) host cells (Figs. 4g, h). The CPCs also differentiated into endothelial cells in some of the detected blood vessels as determined by the detection of Vwf and Kdr expression (Figs. 4i, j). Figure 4. Differentiation potential of CPCs in the infarcted myocardium. Low magnification images demonstrating large-scale engraftment and differentiation of the injected CPCs into the cardiomyocyte cell lineage (a–b). Panels (a) and (b) show parallel thin sections of the infarcted myocardium. Yellow arrowheads denote the newly formed b-Gal(+) and a-Actinin(+) myocardium. High magnification images demonstrating that the transplanted CPCs primarily differentiated into cardiomyocytes (c–f). Large and small newly formed vessels located within the infarcted region of the myocardium contain a combination of host and donor cells. Yellow arrowheads denote the b-Gal(+) donor cells expressing smooth muscle actin (g, h), Von Willebrand factor (i), and KDR (j). doi:10.1371/journal.pone.0011536.g004 PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11536