Left Atrial Appendages from Adult Hearts Contain a Reservoir of Diverse Cardiac Progenitor Cells Jussi V. Leinonen . , Avishag K. Emanuelov . , Yardanna Platt, Yaron Helman, Yael Feinberg, Chaim Lotan, Ronen Beeri* Cardiovascular Research Center, Heart Institute, Hadassah Hebrew University Medical Center, Jerusalem, Israel Abstract Aims: There is strong evidence supporting the claim that endogenous cardiac progenitor cells (CPCs) are key players in cardiac regeneration, but the anatomic source and phenotype of the master cardiac progenitors remains uncertain. Our aim was to investigate the different cardiac stem cell populations in the left atrial appendage (LAA) and their fates. Methods and Results: We investigated the CPC content and profile of adult murine LAAs using immunohistochemistry and flow cytometry. We demonstrate that the LAA contains a large number of CPCs relative to other areas of the heart, representing over 20% of the total cell number. We grew two distinct CPC populations from the LAA by varying the degree of proteolysis. These differed by their histological location, surface marker profiles and growth dynamics. Specifically, CD45 pos cells grew with milder proteolysis, while CD45 neg cells grew mainly with more intense proteolysis. Both cell types could be induced to differentiate into cells with cardiomyocyte markers and organelles, albeit by different protocols. Many CD45 pos cells expressed CD45 initially and rapidly lost its expression while differentiating. Conclusions: Our results demonstrate that the left atrial appendage plays a role as a reservoir of multiple types of progenitor cells in murine adult hearts. Two different types of CPCs were isolated, differing in their epicardial-myocardial localization. Considering studies demonstrating layer-specific origins of different cardiac progenitor cells, our findings may shed light on possible pathways to study and utilize the diversity of endogenous progenitor cells in the adult heart. Citation: Leinonen JV, Emanuelov AK, Platt Y, Helman Y, Feinberg Y, et al. (2013) Left Atrial Appendages from Adult Hearts Contain a Reservoir of Diverse Cardiac Progenitor Cells. PLoS ONE 8(3): e59228. doi:10.1371/journal.pone.0059228 Editor: Toru Hosoda, Tokai University, Japan Received August 19, 2012; Accepted February 13, 2013; Published March 12, 2013 Copyright: ß 2013 Leinonen 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 grant 2005250, from the US–Israel Binational Science Foundation (www.BSF.org.il) and grant R01HL072265 from NIH/ NHLBI (to Dr Beeri- sub contract)(www.nhlbi.nih.gov). Dr Leinonen was supported by a grant from Finnish Foundation for Cardiovascular Research, Helsinki, Finland (www.sydantutkimussaatio.fi)and Foundation of Jorma and Martha Sihvola, Lahti, Finland (no website). Program for upgrading the Flow Cytometry laboratory in the Core Research Facility of the Hebrew University of Jerusalem was supported by a grant from USAID’s American Schools and Hospitals Abroad (ASHA)(www.usaid.gov). 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: rbeeri@hadassah.org.il . These authors contributed equally to this work. Introduction During the last decade there has been evidence supporting the regenerative capability of the adult heart, but the mechanism is still debated. There is strong evidence supporting the claim that endogenous cardiac progenitor cells (CPCs) are key players in cardiac regeneration, but the anatomic source and transcriptional phenotype of the master cardiac progenitors remains uncertain [1]. CPCs are a heterogenic group and are thought to be concentrated in specific areas of the heart, e.g. atria or epicardium [2,3].Non-myocyte cells are the predominant cell population of the heart in number: cardiomyocytes are estimated to constitute 75% of the normal myocardial tissue volume in murine hearts, but only 30–55% of the total cell number [4]. This enables great variability in the cellular composition between different cardiac structures. Embryonic cardiogenesis has been lately shown to be much more diverse than previously thought [5]. The heart contains multiple complex structures, which originate from distinct cell types. Mesodermal-derived first and secondary heart field cells contribute most of the structures, but a number of cells originate from the cardiac neural crest. The pro-epicardium, on the other hand, gives rise to the adult epicardium, but possibly also contributes to the cardiac chamber formation [6]. Epicardial cells retain, in adulthood, the potential to activate embryonic transcription factors in response to cardiac injury [7]. The atrial appendages (Figure 1A) have several unique features. They have a different embryonic origin compared to the atria, as the formation of the two appendages differentiates the morpho- logically right and left sides of the primary atrium [8]. At a later stage of the development the sinus venosus sprouts the pulmonary vein, which will eventually form the atria and the inter-atrial septum [9]. In addition, the atrial appendages have a distinctive anatomy. While the atria are smooth-walled, the appendages contain numerous trabeculae (pectinate muscles), resembling the ventricles. Interestingly, the epicardium on the surface of the atrial appendages is significantly thicker than over the ventricles [10]. In addition, the LAA lies in close epicardial contact to the left ventricle within the confines of the pericardium. The atrial appendages also function as storage for atrial natriuretic factor (ANF). In normal hearts, 30% of the ANF is contained in the LAA PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e59228