Implantation of Mouse Embryonic Stem Cell-Derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts Nicolas Christoforou 1 * . , Behzad N. Oskouei2., Paul Esteso3, Christine M. Hill3, Jeffrey M. Zimmet4, Weining Bian1, Nenad Bursac1, Kam W. Leong1, Joshua M. Hare2, John D. Gearhart3 1 Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, North Carolina, United States of America, 2 Miller School of Medicine, University of Miami, Miami, Florida, United States of America, 3 Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 4 Interventional Cardiology, University of California San Francisco, San Francisco, California, United States of America Abstract Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart. Citation: Christoforou N, Oskouei BN, Esteso P, Hill CM, Zimmet JM, et al. (2010) Implantation of Mouse Embryonic Stem Cell-Derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts. PLoS ONE 5(7): e11536. doi:10.1371/journal.pone.0011536 Editor: Arnold Schwartz, University of Cincinnati, United States of America Received February 27, 2010; Accepted June 16, 2010; Published July 12, 2010 Copyright: 2010 Christoforou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded with resources from the Institute for Cell Engineering at Johns Hopkins University. National Heart, Lung and Blood Institute grant HL083342 to N.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nc28@duke.edu . These authors contributed equally to this work. Introduction Cardiomyocyte loss as a result of myocardial infarction (MI) injury is considered irreversible with the heart lacking sufficient capacity for self-regeneration. Cell-based cardiac therapies are proposed as an attractive therapeutic alternative to reverse cardiomyocyte loss, repair the injured myocardium and ultimately prevent heart failure. To date, a variety of cell sources, both of adult and embryonic origin, have been investigated for use in heart repair with mixed outcomes [1,2]. The use of adult cells is attractive because of their immunocompatible nature, ease of isolation, restricted differentiation potential, and capacity to proliferate rapidly. However, inadequate potential for cardiac differentiation or integration with host cells, limits the benefit of these cells mainly to their paracrine action. On the other hand, embryonic stem cells (ESCs) are able to differentiate into relatively large numbers of early stage cardiomyocytes that functionally integrate with host heart cells [3,4,5]. While ESC-derived cardiomyocytes have been successfully applied for the treatment of myocardial infarction in animal models [6,7,8,9], their clinical application is currently hampered by their neoplastic and immunogenic potential [10]. We and others have recently described the identification, isolation, and characterization of the novel mouse ESC-derived cardiac progenitor cells (CPCs) on the basis of Brachyury/Flk1 [11], Isl1/Flk1/Nkx2-5 [12], cKit/Nkx2-5 [13], or Nkx2-5 [14] expression. These cells represent a promising source for heart repair as they have the restricted capacity to differentiate into cardiac muscle, smooth muscle, and vascular endothelium [11,12,13,14]. In this study we hypothesized that mouse ESC- derived CPCs will exert functional improvement after myocar- dial infarction primarily through their multipotential differen- tiation capacity as well as through the formation of stable and integrated grafts within the host myocardium. We found that when co-cultured with neonatal rat ventricular cardiomyocytes (NRMVs), the CPCs differentiated into cardiomyocytes, formed gap junctions with the rat cells, and supported electrical propagation over a centimeter-scale distance. Temporal assess- ment performed as long as one month after injection into the infarcted region of the murine myocardium, demonstrated that the CPCs engrafted and differentiated into cardiomyocytes, as well as contributed to neovascularization in the infarcted region. The differentiated cardiomyocytes also formed gap junctions with the host myocardium. The animals that received the CPCs demonstrated significantly improved cardiac function as assess- ed by echocardiography and pressure/volume (PV) loop analysis. No teratoma formation was observed following cell transplantation. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11536