Treatment with hESC-Derived Myocardial Precursors Improves Cardiac Function after a Myocardial Infarction

RESEARCH ARTICLE Treatment with hESC-Derived Myocardial Precursors Improves Cardiac Function after a Myocardial Infarction Jianqin Ye1☯, Meenakshi Gaur1☯, Yan Zhang1, Richard E. Sievers1, Brandon J. Woods1, Julian Aurigui2, Harold S. Bernstein2,3¤, Yerem Yeghiazarians1,2,3* 1 Department of Medicine, University of California San Francisco, San Francisco, California, 94143, United States of America, 2 Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, 94143, 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, 94143, United States of America ☯ These authors contributed equally to this work. ¤ Current Address: Merck Sharp & Dohme Corp., Kenilworth, New Jersey, 07033, United States of America * Abstract OPEN ACCESS Citation: Ye J, Gaur M, Zhang Y, Sievers RE, Woods BJ, Aurigui J, et al. (2015) Treatment with hESCDerived Myocardial Precursors Improves Cardiac Function after a Myocardial Infarction. PLoS ONE 10 (7): e0131123. doi:10.1371/journal.pone.0131123 Editor: Yaoliang Tang, Georgia Regents University, UNITED STATES Background We previously reported the generation of a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by the α-myosin heavy chain (αMHC) promoter. The GFP+/αMHC+ cells derived from this cell line behave as multipotent, human myocardial precursors (hMPs) in vitro. In this study, we evaluated the therapeutic effects of GFP+/αMHC+ cells isolated from the reporter line in a mouse model of myocardial infarction (MI). Received: March 5, 2015 Accepted: May 27, 2015 Published: July 31, 2015 Copyright: © 2015 Ye 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. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by a grant from the California Institute of Regenerative Medicine to HSB and YY (RC1-00104-1). Methods MI was generated in immunodeficient mice. hMPs were injected into murine infarcted hearts under ultrasound guidance at 3 days post-MI. Human fetal skin fibroblasts (hFFs) were injected as control. Cardiac function was evaluated by echocardiography. Infarct size, angiogenesis, apoptosis, cell fate, and teratoma formation were analyzed by immunohistochemical staining. Results Compared with control, hMPs resulted in improvement of cardiac function post-MI with smaller infarct size, induced endogenous angiogenesis, and reduced apoptosis of host cardiomyocytes at the peri-infarct zone at 28 days post-MI. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0131123 July 31, 2015 1 / 12 Myocardial Precursors Improve Cardiac Function Conclusion Intramyocardial injection of hMPs improved cardiac function post-MI. The engraftment rate of these cells in the myocardium post-MI was low, suggesting that the majority of effect occurs via paracrine mechanisms. Introduction We previously generated a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by an α-myosin heavy chain (αMHC) promoter.[1] GFP+/αMHC+ cells isolated from this line at the onset of GFP expression (day 8 of directed cardiac differentiation) by fluorescence-activated cell sorting (FACS) have been shown to give rise to atrial and ventricular cardiomyocytes (CMs) and Na/K hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4+) specialized conduction cells in vitro and in vivo.[1] Using this reporter line, we isolated early human myocardial precursors expressing Nkx2-5, but not expressing mature CM markers, such as cardiac troponin I (TnI) or myosin light chain, and we therefore refer to these cells as human myocardial precursors (hMPs).[1] Previous studies have shown that undifferentiated hESCs form teratomas,[2,3] while fully differentiated hESC-derived CMs provide limited functional benefit in vivo.[3,4] Therefore, it was our hypothesis that partially differentiated hMPs would demonstrate engraftment and functional benefit post-MI superior to what historically has been seen in studies with hESC-derived CMs including our own,[5] and be less likely to result in teratoma formation compared with undifferentiated hESCs. In this report, we used a myocardial infarction (MI) model in immunodeficient mice as previously reported,[5] injected hMPs into infarcted myocardium at 3 days post-MI by closedchest ultrasound-guided injection,[6] and compared the functional and tissue effects to human fetal skin fibroblasts (hFFs) as control. We found that hMPs improved cardiac function, limited infarct size, induced endogenous angiogenesis, and reduced apoptosis of host CMs at 28 days post-MI compared with hFFs. However, the engraftment rate of hMPs was not significantly different compared with hESC-derived CMs differentiated in culture for 21 days.[5] No teratoma was noted in injected hearts by hematoxylin and eosin staining at 28 days post-MI. Materials and Methods Preparation of GFP/αMHC-expressing hMPs and hFFs All procedures with hESCs were approved by the Human Gamete, Embryo and Stem Cell Research Committee of University of California San Francisco (Approval number: 10–04745). The parent H9 hESC line (WA09) was purchased from WiCell Research Institute (Wisconsin). A hESC line with eGFP expression driven by an αMHC promoter was generated and maintained as previously described.[1] Differentiation was initiated by human embryoid body (hEB) formation in suspension. Briefly, colonies of hESCs were dissociated into small clusters by exposure to Collagenase IV (Sigma-Aldrich), then allowed to differentiate in a medium comprised of Knockout DMEM (Invitrogen) supplemented with 20% Defined Fetal Bovine Serum (Hyclone), 2mM glutamine, 0.1mM non-essential amino acids, and 0.1mM β-mercaptoethanol. After 7 days in suspension, hEBs were attached to gelatin-coated 12-well culture plates and allowed to differentiate for an additional 7 days [4,7]. The differentiating reporter cells start expressing GFP on day 8 in suspension. On day 14, hEB were treated with 10μM PLOS ONE | DOI:10.1371/journal.pone.0131123 July 31, 2015 2 / 12 Myocardial Precursors Improve Cardiac Function ROCK inhibitor (Y-27632; Calbiochem) in differentiation medium overnight. hEBs were dissociated with TrypLE Express (Invitrogen) to generate single cell suspensions, stained with propidium iodide to distinguish between live and dead cells, and sorted on the basis of GFP expression using a FACSAria (Becton Dickinson). The sorted GFP+/αMHC+ cells were resuspended in MEF-conditioned medium at a concentration of 105/10μl for intramyoca (...truncated)