Identification and Characterization of a Novel + Multipotent Sub-Population of Sca-1 Cardiac Progenitor Cells for Myocardial Regeneration Michitaka Takamiya, Khawaja H. Haider, Muhammad Ashraf* Department of Pathology, University of Cincinnati, Cincinnati, Ohio, United States of America Abstract Methods and Results: The cardiac stem/progenitor cells from adult mice were seeded at low density in serum-free medium. The colonies thus obtained were expanded separately and assessed for expression of stem cell antigen-1 (Sca-1). Two colonies each with high Sca-1 (CSH1; 95.9%; CSH2; 90.6%) and low Sca-1 (CSL1; 37.1%; CSL2; 17.4%) expressing cells were selected for further studies. Sca-1+ cells (98.4%) isolated using Magnetic Cell Sorting System (MACS) from the hearts were used as a control. Although the selected populations were similar in surface marker expression (low in c-kit, CD45, CD34, CD31 and high in CD29), these cells exhibited diverse differentiation potential. Unlike CSH1, CSH2 expressed Nanog, TERT, Bcrp1, Nestin, Musashi1 and Isl-1, and also showed differentiation into osteogenic, chondrogenic, smooth muscle, endothelial and cardiac lineages. MACS sorted cells exhibited similar tendency albeit with relatively weaker differentiation potential. Transplantation of CSH2 cells into infarcted heart showed attenuated infarction size, significantly preserved left ventricular function and anterior wall thickness, and increased capillary density. We also observed direct differentiation of transplanted cells into endothelium and cardiomyocytes. Conclusions: The cardiac stem/progenitor cells isolated by a combined clonal selection and surface marker approach possessed multiple stem cell features important for cardiac regeneration. Citation: Takamiya M, Haider KH, Ashraf M (2011) Identification and Characterization of a Novel Multipotent Sub-Population of Sca-1+ Cardiac Progenitor Cells for Myocardial Regeneration. PLoS ONE 6(9): e25265. doi:10.1371/journal.pone.0025265 Editor: Gangjian Qin, Northwestern University, United States of America Received August 12, 2011; Accepted August 30, 2011; Published September 28, 2011 Copyright: 2011 Takamiya 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 National Institutes of Health (NIH) Grants # R37-HL074272, HL-080686, HL-087246 to MA and HL-087288, HL-089535 to KHH. 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: muhammad.ashraf@uc.edu Introduction The pioneering work of Anversa et al. has provided an intriguing evidence of increased mitotic activity in the cardiomyocytes in patients with myocardial infarction (MI) besides the existence of resident population of cardiac stem/progenitor cells [1]. These results are supported by other research groups to show that resident stem/progenitor cells have the ability to differentiate into all cell types required for myocardial regeneration including cardiomyocytes, smooth muscle cells and endothelial cells [2]. Despite their immense differentiation potential, use of cardiac stem/progenitor cells is limited due to lack of markers for selection of a specific stem cell population [3]. Stem cell antigen-1+ (Sca-1+) [3,4], Islet-1+ (Isl-1+) [5], c-kit+1, side population (SP) cells [6,7] and cardiosphere-derived cells [8] have been extracted from heart tissue. Nevertheless, there is no distinct relationship between these reported cardiac stem/progenitor cell populations due to differ- ence in isolation and characterization methods. Moreover, it remains to be determined whether these individual stem/ progenitor cell populations represent different intermediate developmental stages of the same stem/progenitor cells or these are independent cell lineages which have originated from different stem/progenitor cell populations. Similarly, their relationship with the cardiac precursor cells observed during embryonic develop- ment has not been established [9,10]. In this study, we isolated several sub-populations purified from adult mouse heart based on clonogenicity and surface marker expression and investigated the important features of cardiac stem/progenitor cell population for cardiac differentiation. Materials and Methods Ethics statement and the Animals used C57BL6 mice (Harlan) were used for cell isolation and cell transplantation models. All animal experiments conformed to the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and all protocols of animal experiments were approved by the Institutional Animal Care and Use Committee, University of Cincinnati, Protocol #06-03-13-03. Cell culture The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells. Aorta, pulmonary artery, and pericardium were removed carefully. The hearts were minced and digested for 20-minutes at 37uC with 0.1% type-II collagenase (Invitrogen) and 0.01% DNAse-I (Worthington Biochemical Corporation). The cells were passed through 40 mm filter, fractionated with 70%Percoll (Fluka) and cultured in maintenance medium PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25265