host cardiomyocytes. On the other hand the mouse embryonic stem cell-derived cardiomyocytes colocalized in clusters and could clearly be distinguished from the native cardiomyocytes as a result of their reduced size, distinct cell shape, and incomplete myofibrils, which they concluded was strong evidence that the detected cells were not the result of fusion. Similarly, for the purpose of our study during an early point analysis of the injected myocardium (day 3 post infraction and cell transplantation, data not shown) we detected areas with clustered GFP(+) cells. Although these cells expressed cardiac markers as determined by immunocytochemis- try they were small in size and resembled immature cardiomyo- cytes. Most importantly, one month, post injection 96% of bGal(+) cells contained a single nucleus suggesting their direct differenti- ation into cardiomyocytes versus their fusion with the host cells. Importantly, through the systematic assessment of cardiac contractile function, we demonstrated a superior systolic and diastolic performance of the CPC-treated relative to untreated hearts. In particular, while the function of untreated hearts was significantly compromised as early as one week after the infarction (based on echocardiographic parameters), the functional indices in CPC-treated hearts remained relatively constant. The preserved cardiac function was independently confirmed at 4 weeks post- infarction using pressure-volume analysis. Mechanistically, the cardiac function was maintained not only due to attenuated ventricular remodeling including a decrease of scar size and relatively preserved ventricular volume but more importantly due to independently preserved contractile function as evidenced from a higher PRSW slope (a volume independent factor in evaluation of cardiac function in both systole and diastole). These findings are consistent with previous reports in demonstrating that transplantation of ESC-derived cells leads to the in vivo cardiac improvement following myocardial infarction [35,36]. It is important to note, however, that the mechanisms mediating this effect are currently not known [37]. We acknowledge certain limitations in our work which we plan to further investigate in the future. In particular we are interested in addressing the therapeutic mechanism of the CPCs. More specifically in addition to their direct differentiation capacity into cardiomyocytes, and vascular cells, the CPCs may induce a cardioprotective paracrine effect which in turn induces the host myocardium to regenerate. A genomic expression analysis performed on the CPCs and reported in our previous work indicated that Vegf, Igf1, and Notch signaling pathways are active in the cells both of which have been shown to have pro-vascular and cardioprotective effects [38,39]. Moreover, in order to be able to assay in vivo functional integration of the donor cells with the host myocardium we plan to utilize genetic calcium indicators and two- photon laser scanning microscopy [40,41]. An additional important determinant of a successful long-term cell-based cardiac therapy involves the level of cell survival following direct intramyocardial delivery. Previous studies have demonstrated a clear correlation between graft size and the level of functional improvement [42,43]. Thus cell survival determines graft size and ultimately the level of the therapeutic effect. In general cell survival depends primarily on the initial number of cells delivered, the local cell donor density per injection point, and the status of the tissue in which the cells are being injected (ischemic/inflamed versus normal) [44]. For this study the cell survival capacity of the transplanted CPCs was determined to be 12.762.1% which is similar to previous reports utilizing a similar injury and which determined the cell survival to be between 5– 9% up to a month following initial cell injections [45,46] [47]. Finally, in order to maximize the therapeutic effect of the delivered CPCs through increased cell survival it may be Progenitor Cell Heart Therapy necessary to modify the delivery method in order to ensure decreased apoptosis and localized inflammatory response as well as prime the cells to cope with the increased stress associated with the entire process [36]. The recent identification and characterization of adult [48] and ESC-derived CPCs with multipotential but cardiac tissue-restrict- ed differentiation capacity has reinvigorated the field of cell-based cardiac therapies. Taken together, our results clearly demonstrate the therapeutic value of the ESC-derived CPCs in the infarcted mouse heart. While potential strategies to control differentiation fate of CPCs towards blood vessels or cardiac muscle upon implantation remain to be explored, this study establishes the foundation and the rationale for the future testing of human embryonic or induced pluripotent stem cell-derived CPCs for repair of cardiac infarction. Materials and Methods Cell culture and differentiation Mouse ESCs (D3 [15] and Rosa26 [16] lines) were cultured and differentiated as previously described [14]. Further detail is provided in File S1. DNA plasmids and cell transfection The previously described [22] cardiac specific enhancer element (fragment 8) of the mouse Nkx2-5 transcription factor along with the Hsp68 minimal promoter [49] were excised from the provided plasmid (XhoI/NcoI) and inserted upstream of the enhanced GFP gene in the Bluescript vector. This in conjunction with a plasmid DNA containing the polymerase II promoter at the 59 end the hygromycin phosphotransferase gene were used to stably transfect mouse ESCs in the presence of hygromycin. A stable clone from each cell line was selected for any further experiments. In vitro CPC differentiation capacity To assay their differentiation capacity, sorted GFP(+) CPCs were re-aggregated for 24 hours in suspension and re-plated on fibronectin coated tissue culture dishes and further maintained for seven days in ESC differentiation medium. Quantitative RT-PCR Embryoid bodies cultured in hanging droplets or in suspension were harvested every 24 hours in order to isolate total RNA. For the quantitative RT-PCR analysis total RNA (2 mg) was reverse transcribed using random decamers as primers (Ambion, cat.# 57226), and the MMLV-RT enzyme (Invitrogen, 28025-013). Relative RNA expression levels were measured using quantitative RTPCR. Detection of the described markers was achieved with FAM/MGB probes (Applied Biosystems): Nkx2.5 (Mm00657783_m1), Myh6 (Mm00440354_m1), Brachyury (Mm00436877_m1). The instrument used was the Applied Biosystems ABI Prism 7900HT sequence detection system and the software for data collection and analysis was the SDS2.1. An 18S RNA probe (Hs99999901_s1) was used in order to normalize the data. Co-culture of CPCs and NRVMs Fibronectin-coated coverslips and polydimethylsiloxane (PDMS) rings were used to selectively plate isolated CPCs only in the central region (5 mm diameter) of the coverslips (low density: 56104 cells, high density: 2.56105 cells). Following CPC attachment floating cells were washed off and the PDMS ring was removed. Isolated NRVMs were subsequently plated around the CPCs. Washing of unattached NRVMs within 6–8 hours following plating ensured minimum NRVM attachment on top of the CPCs. PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11536