Results Isolation and in vitro characterization of mouse ESC- derived CPCs The mouse ESC lines D3 [15] and Rosa26 [16] were stably transfected with DNA constructs allowing the expression of the green fluorescent protein (GFP) under the control of the mouse cardiac specific enhancer element of the Nkx2-5 transcription factor as previously described [14]. Following isolation of 50 colonies (clonal) for each cell line, stably transfected clones were identified and further used based on their capacity to express GFP selectively in the spontaneously contracting cardiomyocyte cell clusters. Mouse ESCs were induced to differentiate in suspension forming aggregates termed embryoid bodies (EBs) and initial detection of GFP coincided with initiation of Nkx2-5 expression on differentiation day 5 (Figs. 1a, b). Temporal quantitative RT-PCR analysis performed on differ- entiating ESCs indicated a time period (days 5–6) during which the CPCs were present but not yet committed into specific cell lineages (Fig. 1g). Prior to initiation of Nkx2-5 expression, coinciding with cardiac progenitor induction, the detection of Brachyury transcripts (day 4) indicated the formation of nascent mesoderm. By day 7, the CPCs underwent differentiation- commitment into cardiac cell lineages including cardiomyocytes as indicated by the initiation of Mhc6 expression along with the detection of spontaneously contracting cell clusters. The initial number and percentage of GFP(+) CPCs decreased temporally between days 5 and 10 (Fig. 1h). This observation, which has been previously reported [13], may be a result of proliferation of other cell types, epigenetic silencing of the specific Nkx2-5 enhancer element used in this set of experiments, and/or differentiation towards the vascular and smooth muscle endothe- lium cell lineages which downregulate Nkx2-5. To assay the differentiation capacity of the CPCs, following isolation of the GFP(+) progenitor population, the cells were reaggregated and the cell clusters were induced to differentiate for 7–30 days. Several foci of GFP(+) spontaneously contracting cardiomyocytes (a-Actinin expression) were detected in culture (Figs. 1c, d). The multi differentiation potential of the CPCs was determined by demonstrating cell specific expression of cardiac a- Actinin, endothelial Von-Willebrand factor, and smooth muscle actin in the differentiating clusters (Figs. 1e, f). This was also confirmed by the RT-PCR analysis (previously demonstrated [14]). No colonies of undifferentiated ESCs were detected as confirmed by the absence of Pou5f1 and Nanog expression. Determination of the electrocoupling capacity of CPCs The potential of CPC derived cardiomyocytes to form functional gap junctions was determined by utilizing a previously described in vitro NRVM co-culture assay [17,18] which allows the observation of action potential propagation by the means of optical mapping over a macroscopic (3 cm2) culture area [18]. In particular, the CPCs were selectively seeded at high density in the central region of pre-masked coverslips with the NRVMs surrounding them (Fig. 2a). The co-cultures exhibited distinct border between the CPCs and the NRVMs as assayed by GFP and a-Actinin expression (Figs. 2a, b). Immunostaining for cardio- myocyte, smooth muscle and endothelial markers after 14 days of co-culture demonstrated that the majority of the CPCs differen- tiated into cardiomyocytes (Fig. 2c) with limited smooth muscle or endothelial cell differentiation (data not shown). On the other hand, CPCs plated at low density in the central region of the coverslips formed few patches of cardiomyocytes with the majority of cells differentiating into smooth muscle cells (Fig. S1). Gap Progenitor Cell Heart Therapy junctions indicated by punctuate Connexin 43 localization at the site of cell-cell contacts were detected in the differentiated cardiomyocytes (Fig. 2d). However, unlike the NRVMs that expressed Connexin 43 in relatively long plaques at the sites of cell contacts, the CPC-derived cardiomyocytes expressed Connexin 43 sporadically and irregularly. Optical mapping in all co-cultures revealed spontaneous electrical activity at a relatively low rate (1.260.3 Hz) that originated in the CPC islands, suggesting the existence of pacemaking cells amongst the CPC-differentiated cardiomyocytes. Consistent with positive staining for Connexin 43, both sponta- neous and pacing-induced action potentials (2 Hz rate) within the CPC island propagated outwards into the surrounding NRVM area (Fig. 2e). Action potentials also propagated into the CPC island when initiated from the surrounding NRVM area (data not shown). The apparent velocity of electrical propagation inside the CPC island was significantly lower as compared to that of the surrounding NRVMs (1.660.2 vs. 16.761.5 cm/s) and dependent on the efficiency of CPC differentiation into cardiomyocytes. Specifically, lower numbers of sparsely distributed cardiomyocytes within the central island yielded significantly lower velocity of propagation (0.460.1 cm/s, Fig. S1g). CPC injection in the mouse infracted myocardium The potential of CPCs to treat myocardial infarction injury was assessed by evaluating their ability to form stable grafts in the host myocardium, differentiate into lineage-committed cells, form in vivo gap junctions, and ultimately improve the functional output of the injured hearts. To determine the extent of injury and scar tissue formation in the infarcted hearts, we performed Mason’s trichrome staining on thin heart sections that allowed us to distinguish healthy muscle tissue (red) from scar tissue (blue) (Fig. 3). The control group that underwent sham surgery displayed only minor scarring which could be attributed to injury sustained during the saline injections (Figs. 3a, b). The hearts that underwent LAD ligation and received only saline injections sustained massive infarcts that encompassed almost the entire left ventricle (including apex) and exhibited thin scarred walls and extensive remodeling (Figs. 3c, d). In contrast, the animals that underwent LAD ligations and received CPCs displayed smaller zones of scar tissue formation with decreased cardiac remodeling (Figs 3e–h). While a few hearts displayed minor tissue scarring (Figs. 3e, f) similar to that observed in the sham hearts, others displayed a certain degree of wall thinning and remodeling (Figs. 3g, h). In this case, however, the scar was localized only around the ligation site. To assess the engraftment potential and distribution of the transplanted cells irrespective of their lineage differentiation phenotype within the myocardium we compared parallel serial tissue cross-sections pre-treated either with Mason’s trichrome stain or anti b-Galactosidase, which is constitutively expressed in the Rosa26-derived CPCs (Figs. 3i, l). b-Gal(+) cells were detected within the infarcted region in an area comprising a thin muscle band underneath the scar tissue (Fig. 3i), borders between the scar tissue and healthy muscle (Figs. 3j, k), as well as within the healthy muscle in the vicinity (Fig. 3l) but not remote (not shown) from the scar. These results suggested that the transplanted cells successfully engrafted at all three injection areas including the center of the infarct. Anti b-Galactosidase-specific staining was also used in order to determine the survival rate of the transplanted cells one month post intramyocardial delivery. bGal(+) cells was detected and summed in multiple sections both within and in the surrounding area of the myocardial infarction region. The survival rate was PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11536