Fibroblast Growth Factor-10 Promotes Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells Sunny Sun-Kin Chan1., Hui-Jing Li1., Ying-Chang Hsueh 2 , Desy S. Lee1, Jyh-Hong Chen 3 , Shiaw-Min Hwang 4 , Chen-Yun Chen5,6, Emily Shih7, Patrick C. H. Hsieh1,2,5,6* 1 Institute of Clinical Medicine and Research Center for Clinical Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan, 2 Institute of Basic Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan, 3 Department of Medicine, National Cheng Kung University and Hospital, Tainan, Taiwan, 4 Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, 5 Molecular Medicine Program, National Yang-Ming University, Taipei, Taiwan, 6 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 7 Department of Biochemistry, University of Washington, Seattle, Washington, United States of America Abstract Background: The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Methodology/Principal Findings: We used mouse ES cells stably transfected with a cardiac-specific a-myosin heavy chain (aMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co- administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation. Conclusion/Significance: FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications. Citation: Chan SS-K, Li H-J, Hsueh Y-C, Lee DS, Chen J-H, et al. (2010) Fibroblast Growth Factor-10 Promotes Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells. PLoS ONE 5(12): e14414. doi:10.1371/journal.pone.0014414 Editor: Jeffrey A. Whitsett, Cincinnati Children’s Hospital Medical Center, United States of America Received July 3, 2010; Accepted November 29, 2010; Published December 28, 2010 Copyright: 2010 Chan 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 grants from the National Science Council [NSC 97IR082, NSC 96-2314-B-006-032] (http://web1.nsc.gov.tw), the National Health Research Institutes [NHRI EX97-9722SI] (http://www.nhri.org.tw), and the National Cheng Kung University Landmark Project [97I006] (http://web.ncku.edu. tw). 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: phsieh@mail.ncku.edu.tw . These authors contributed equally to this work. Introduction Heart failure is a leading cause of morbidity and mortality throughout the world [1,2]. The dominant cause of heart failure is the loss of myocardium due to coronary artery disease. In the ischemic heart, cardiomyocytes (the contracting heart muscle cells) undergo necrosis, autophagy, and apoptosis, ultimately leading to scar formation. Therefore, strategies to replace damaged cardio- myocytes may prevent heart failure and save lives. Stem cell therapy is an attractive approach for cardiomyocyte replacement. Recently, mesenchymal stem cells [3], cardiac progenitor cells [4], embryonic stem (ES) cells [5], and induced pluripotent stem (iPS) cells [6] have been reported to be capable of cardiomyocyte differentiation. Amongst them, ES cells and iPS cells may have the best potential. However, the conventional protocol using the hanging drop method to induce cardiac differentiation from ES cells or iPS cells is extremely inefficient [7– 9], which is a severe obstacle to the use of ES/iPS cells for cardiomyocyte replacement therapy. Accordingly, studies to dissect the molecular pathways mediating ES/iPS cell differenti- ation into the cardiomyocyte lineage are essential. The fibroblast growth factor (FGF) family is composed of multifunctional proteins regulating organogenesis, tissue develop- ment, and stem cell differentiation. Recent studies have also shown a crucial function of the FGF family during cardiogenesis [10,11]. For example, the expression of FGF-2 stimulates proepicardial cell PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14414