Characterization and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells Ali Nsair 1,6 * . , Katja Schenke-Layland 1,2,3. , Ben Van Handel 5,6 , Denis Evseenko 4,6 , Michael Kahn 8 , Peng Zhao 1 , Joseph Mendelis 1 , Sanaz Heydarkhan 1¤a , Obina Awaji 1¤b , Miriam Vottler 2,3 , Susanne Geist 2,3 , Jennifer Chyu 1 , Nuria Gago-Lopez 1 , Gay M. Crooks 4,6 , Kathrin Plath 6 , Josh Goldhaber 7 , Hanna K. A. Mikkola 5,6 , W. Robb MacLellan 9 1 Department of Medicine and Physiology, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 2 Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany, 3 Department of Thoracic and Cardiovascular Surgery, Eberhard Karls University, Tu ¨ bingen, Germany, 4 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 5 Department of Molecular, Cell and Developmental Biology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 6 The Edythe and Eli Broad Center for Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 7 Cedars-Sinai Heart Institute, Los Angeles, California, United States of America, 8 Department of Biochemistry and Molecular Biology and Institute for Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, California, United States of America, 9 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States of America Abstract Background: Cardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency. Methodology/Principal Findings: We sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1 + /Nkx2.5 + CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1 + /Nkx2.5 + CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1 + /Flt4 + best identified and facilitated enrichment for Isl1 + /Nkx2.5 + CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1 + /Flt4 + CPCs differentiated into all three cardiovascular lineages in vitro. Flt1 + /Flt4 + CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs). Conclusion/Significance: The cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1 + /Nkx2.5 + CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts. Citation: Nsair A, Schenke-Layland K, Van Handel B, Evseenko D, Kahn M, et al. (2012) Characterization and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells. PLoS ONE 7(10): e45603. doi:10.1371/journal.pone.0045603 Editor: Maurizio Pesce, Centro Cardiologico Monzino, Italy Received February 2, 2012; Accepted August 23, 2012; Published October , 2012 Copyright: ß 2012 Nsair 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 the National Institutes of Health (5T32HL007895-10 to KS-L; P01-HL080111 and R0-HL094941 to WRM), Laubisch and Cardiovascular Development Funds (to WRM), Ruth Kirschstein National Research Service Award (GM007185 to BVH), and the Alberta Heritage Foundation for Medical Research Fellowship (AHFMR to AN). 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: ansair@mednet.ucla.edu . These authors contributed equally to this work. ¤a Current address: Department of Medicine and Physiology, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America ¤b Current address: Department of Medicine and Physiology, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e45603 9