The measurement of the relaxation time constant (Fig. 7c), which measures the speed of isovolumic relaxation during early diastole, revealed that the diastolic relaxation of the CPC-treated infarcted group was faster (7.660.5 msec) than that of the saline- treated infarcted group (9.161.0 msec) and comparable to the sham surgery control groups (6.260.3 msec for saline-treated and 6.561.0 msec for CPC-treated). Taken together, the treatment of infracted hearts with transplanted CPCs resulted in a significant improvement of both systolic and diastolic indices of cardiac function compared to untreated, saline injected controls (to levels comparable to those found in uninfarcted animals). The measurement of preload recruitable stroke work (PRSW) relationship, which describes the dependence of the stroke work on the end diastolic volume, demonstrated a preservation of the cardiac functional capacity in the cell-treated infarcted group (Fig. 7d). Specifically, the saline-treated group exhibited signifi- cantly lower PRSW slope (26.8614.6 mmHg) compared to cell- treated group (57.3615.3 mmHg), which in turn remained comparable to both sham groups (Fig. 7d). Discussion The main aim of this study was the determination of the therapeutic capacity of the recently described ESC-derived cardiac progenitor cells [14] to treat myocardial infarction injury. We first describe the in vitro differentiation potential of mouse ESC-derived CPCs. We further demonstrate the capacity of the CPC-derived cardiomyocytes to form functional gap junctions, propagate action potentials over a relatively large area, and electrically and mechanically connect with primary ventricular cardiomyocytes in culture. Finally, we demonstrate a stable engraftment and robust cardiomyocyte differentiation of the transplanted CPCs following myocardial infarction injury, formation of gap junctions with host cells, as well as significant preservation of cardiac function in the CPC-treated animals compared to the untreated group. A significant aspect of the study is the utilization of a novel cell progenitor: ESC-derived CPCs selected based on the activity of a cardiac specific Nkx2-5 [22] enhancer. We recently reported that the CPCs closely recapitulate the earliest stages of in vivo cardiac development and are capable of multipotential differentiation into cell types that contribute to the heart [14]. Success of cell-based cardiac therapies is dependent not only on the ability of transplanted cells to differentiate into cardiomyocytes and functionally integrate with host cells, but also on the adequate vascularization of the newly formed tissue. The cardiovascular differentiation potential of the Nkx2-5(+) CPCs and the relative ease to obtain large cell numbers represent major advantages for their potential use as a single therapeutic cell source. Importantly, since these CPCs are capable of generating both cardiomyocytes and vascular cells, they may obviate a need for use of additional vascular cell types [23] or different gene manipulation strategies [24] to facilitate vascularization of the newly formed cardiac muscle. One of the major concerns in using ESCs or their differentiated progeny for cell-based interventions is the risk of neoplastic tumor or teratoma [10] formation due to undifferentiated ESCs. Recent reports describe extensive teratoma formation following injection of ESCs [25,26]. In one such study, the authors reported tumor formation in 21% of the mice [26] receiving cells. We have previously demonstrated that no expression of Nanog or Pou5f1 was detected following Nkx2-5(+) CPC derivation and extended culture in vitro. In contrast, colonies of undifferentiated ESCs and high expression levels of Nanog and Pou5f1 were found in cultures of the Progenitor Cell Heart Therapy sorted GFP(2) cells [14]. Importantly, in this study we observed no evidence of neoplastic cell growth in the mouse hearts as long as 1 month after cell injections. While these results, along with similar findings in our previous study [14], strongly suggest that the narrow differentiation potential of CPCs could be safe for cell- based therapies, longer grafting times must be studied to further validate these conclusions. A major drawback of any allogeneic transplantation therapy is the immune response which leads to the rejection of transplanted tissue. In our study we successfully utilized animals with common genetic background to the donor cells [27] trying to avoid immunorejection or the need for immunosuppressive therapy. Even though it has been argued that ESCs and their differentiated progeny are capable of evading immunorejection [7], two reports have demonstrated large-scale host immune response against transplanted ESCs [10,28]. In a clinical setting this issue must be addressed. Recent studies have proposed several alternatives including somatic cell nuclear transfer [29], and induced pluripotent stem cells [30]. The success of any cell-based therapy for myocardial infarction injury is ultimately judged by the improvement of the heart function, i.e., decreased heart dilatation and increased cardiac contractility and output. Based on the optical mapping of electrical propagation in co-cultures of CPCs and primary ventricular cardiomyocytes, the observed improvement in cardiac mechanical indices in treated hearts could be attributed in part to the functional integration of CPC-derived cardiomyocytes with the host cells. Of note, however, is the relatively low velocity of impulse propagation measured within the CPC islands in vitro. This low conduction velocity was consistent with observed sparse Connexin 43 expression and possibly contributed by immature (or mixed from nodal, atrial, and ventricular cells) electrophysiological phenotype of CPC-derived cardiomyocytes [31]. Conceivably, more efficient cardiogenic differentiation within the environment of infracted heart may have yielded higher Connexin 43 expression and maturation of ion currents in engrafted cardiomy- ocytes resulting in improved functional in vivo integration. In support, although no systematic studies were done to assess the arrhythmogenic risk of implanted cells, we observed no difference in the incidence of sudden cardiac death between cell-treated and untreated mice. Moreover, we demonstrate in vivo electrical coupling through gap junction formation of the differentiated CPCs with the host myocardium. It is interesting that CPCs plated at high density within neonatal rat ventricular myocytes primarily differentiated into the cardio- myocyte lineage, while abundant smooth muscle cells were found within the sparsely plated islands [32]. On the other hand, CPCs transplanted in the infarcted hearts primarily differentiated into cardiomyocytes, indicating that the stiffness of the cell attachment substrate [33], density of injected cells, and/or other differences between in vivo and in vitro microenvironments may all significantly affect their lineage commitment. The systematic studies to understand the factors that control lineage commitment are currently underway. We detected a number of bGal(+) cardiomyocytes (a-Actinin(+) or MYH6(+)) in the host myocardium suggesting the direct differen- tiation of the delivered CPCs towards the cardiac muscle lineage. Admittedly there is a possibility that some of the detected differentiation events are the result of cell fusion between the host cardiomyocytes and the CPCs. Recently Kolossov et al. examined the capacity of both bone marrow derived cells and embryonic stem cell derived cardiomyocytes to engraft into the infracted mouse myocardium [34]. Following bone marrow cell transplan- tation they detected rare fusion events of the delivered cells with PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11536