Progenitor Cell Heart Therapy Small bGal(+) uncommitted progenitors were also detected. Taken together, ESC-derived CPCs committed predominantly to the cardiac muscle cell fate both when transplanted in the infarcted heart and when densely seeded in co-culture with rat cardiomyo- cytes in addition to inducing vascularization. Since cardiomyocytes in the adult mouse myocardium are binucleated whereas embryonic stem cell-derived cardiomyocytes are immature and primarily uninucleated [19] we were able to delineate differentiation versus cell fusion events by determining the number of bGal(+) cardiomyocytes containing a single nucleus. In our analysis approximately 96% of the identified cells contained a single nucleus indicating that at least that number of cells were the result of direct differentiation versus cell fusion. Electrical and mechanical coupling assessment of CPCs The capacity of the differentiated CPCs to electrocouple in vivo with the host myocardium was examined by immunohistochemical analysis (Figs. 5a–d). The majority of the bGal(+) cardiomyocytes which also exhibited Connexin 43 gap-junction staining were detected at the site of the infarct (Figs. 5a, c). A small number of the donor cells were detected within the healthy myocardium (Fig. 5b) or at the border between healthy tissue and infarct (Fig. 5d). Figure 5. Electromechanical coupling capacity of CPCs. Electrical and mechanical coupling of differentiated CPCs with the host myocardium and neonatal rat ventricular myocytes. b-Galactosidase(+) cardiomyocytes form gap junctions as demonstrated by Connexin 43 immunostaining within the infarcted region (a, c), as well as in the healthy myocardium (b), and at the border area between healthy and infarcted myocardium (d). CPCs differentiate in vitro into GFP(+)/a-Actinin(+) cardiomyocytes (e) and mechanically couple with co-cultured neonatal rat ventricular myocytes as determined by N-Cadherin detection at the border region between the two cell types (f). doi:10.1371/journal.pone.0011536.g005 PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11536