An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors Carissa Ritner1., Sharon S. Y. Wong1., Frank W. King1., Shirley S. Mihardja1, Walter Liszewski1, David J. Erle1,2, Randall J. Lee1,2,3, Harold S. Bernstein1,2,3,4* 1 Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 3 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, United States of America, 4 Department of Pediatrics, University of California San Francisco, San Francisco, California, United States of America Abstract Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an a-myosin heavy chain (aMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-b signaling pathways in CM development. The identification of hMPs using this aMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. Citation: Ritner C, Wong SSY, King FW, Mihardja SS, Liszewski W, et al. (2011) An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell- Derived Myocardial Precursors. PLoS ONE 6(1): e16004. doi:10.1371/journal.pone.0016004 Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America Received August 4, 2010; Accepted December 3, 2010; Published January 4, 2011 Copyright: 2011 Ritner 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 supported by a Public Health Service Grant (HL085377) from NHLBI, a Comprehensive Research Grant (RC1-00104) from the California Institute for Regenerative Medicine, and a gift from the Pollin Foundation to H.S.B., and funds from an NIH/NCRR UCSF-CTSI Grant (UL1 RR024131) to D.J.E. S.S.Y.W. and F.W.K. were supported by a National Research Service Award (HL007544) from NHLBI. 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: harold.bernstein@ucsf.edu . These authors contributed equally to this work. Introduction Over five million people in the United States alone suffer with heart failure [1] because unlike some organs, the heart is unable to repair itself after injury [2]. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat a variety of human diseases characterized by tissue loss or insufficiency. Animal studies have shown that pluripotent hESCs have a high risk of tumor formation [3], while fully differentiated hESC-derived cardiomyocytes (CMs) confer only modest functional benefit [4]. This suggests that from a developmental standpoint, mature CMs may be beyond the ability to fully incorporate into existing muscle. Therefore, the identification of hESC-derived myocardial precursors that are committed to the cardiac lineage, but retain the plasticity to facilitate complete engraftment has been an important goal [5]. Work over the past decade has shown that hESCs differentiate into a heterogeneous population of CMs in culture, with gene expression patterns and electrophysiological properties reminis- cent of embryonic atrium, ventricle and specialized conduction tissue [6,7,8,9,10]. The mechanisms that drive CM subtype specification, however, are not well understood. To approach both the need for a model system with which to elucidate the process of human CM subtype specification, and provide a source of human myocardial precursors for cell therapy studies, we engineered a hESC line that identifies multipotent myocardial precursor (hMP) cells. These cells give rise to multiple CM subtypes, and is therefore uniquely suited to address both of these needs. Results Construction of an a-myosin heavy chain human embryonic stem cell reporter line The a-myosin heavy chain (aMHC) gene has been shown to be expressed both early and late during murine cardiac development [11,12]. Taking advantage of this developmentally broad expres- PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16004