containing serum-free DMEM/F12 (Invitrogen) supplemented with B27 (Invitrogen), 20 ng/ml epidermal growth factor (EGF; Sigma), and 40 ng/ml basic fibroblast growth factor (bFGF; PeproTech). The cells were collected 1-week later and re-seeded on new culture dishes with serum-free maintenance medium at a low density (100cells/cm2) to initiate colony formation. Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 (Invitrogen) supplemented with 2%FBS, B27- Supplement (Invitrogen), 20 ng/ml EGF (Sigma), 40 ng/ml bFGF (PeproTech), and 10 ng/ml leukemia inhibitory factor (LIF; Chemicon). All colony-derived cells were re-seeded on new dishes after they reached 90–100% confluence and were maintained with expansion medium. Sca-1+ cell-enriched population was also purified from the heart-derived cells with Magnetic Cell Sorting system anti-Sca-1 MicroBead Kit (Miltenyi Biotec) per instructions of the manufac- turer and maintained in expansion medium similar to other colony-derived cell populations. Flow cytometry Flow cytometery of the purified cell populations was carried out as described in Text S1. Growth function assay Each cell population was seeded at a density of 16104 cells/cm2 in 1 ml expansion medium on 24-well plate and cultured at 37uC in humid air with 5% CO2. Cell number was counted on days 1,3,5, and 7 after initial seeding. The doubling time of each cell population was calculated with the cell numbers on day 1 and 3 [11]. RNA extraction and RT-PCR Total RNA was extracted from different treatment cell populations with RNeasy Mini Kit (Qiagen) and cDNA was generated with Omniscript-RT Kit (Qiagen). Each PCR reaction was performed with target gene specific primers. We also performed real-time PCR to determine the expression of VEGF gene under anoxic conditions using SYBR-Green Master Mix (BIO-RAD) in a BIO-RAD-iQ5 optical module. The mRNA level was standardized to inner control (GAPDH) and expressed as fold- increase versus normoxia condition. Sphere formation assay Sphere formation assay was performed as described in Text S1. Differentiation assay These cells were cultured in specific differentiation media for smooth muscle, endothelial cells, cardiomyocyte, osteogenic, chondrogenic, and adipogenic differentiation as detailed in Text S1 and detected by specific staining as described earlier [12]. Myocardial infarction model and cell transplantation Experimental MI was developed in young female C57BL6 mice (12-weeks old; 20–25 g body weight) by left anterior descending (LAD) coronary artery ligation [13]. Ten minutes later, 20 ml basal DMEM/F12 medium without cells (control group) or containing 26105cells (Cell Injection group) were injected into infarction and border zones as described in Text S1. Echocardiography The mouse heart function was assessed by transthoracic echocardiography before surgery (baseline), 1 and 4-weeks after Multi-Potent Sca-1+ CPCs surgery using 7–15 MHz probe on iE33 Echocardiography system (Philips) (Please see Text S1). Morphological analysis The hearts were prepared for measurement of infarction size, anterior wall thickness, and percent fibrosis as described earlier [14] and detailed in Text S1. Capillary density analysis Capillary density was determined as described earlier [15] and detailed in Text S1. Fate tracking of the transplanted cells and histological studies The cells were labeled with red fluorescent nanocrystals, QtrackerH625 Cell Labeling Kit (Invitrogen) before transplanta- tion according to manufacturer’s instructions. The hearts were harvested 1, 2 and 4-weeks after cell transplantation and perfused with ice-cold PBS. The extracted hearts were cut transversely and embedded into OCT compound (Sakura Finetek) and frozen with liquid nitrogen. Semi-thin cryosections (10 mm) were fixed in acetone at 220uC for 10 minutes and blocked with CAS BLOCKTM (Invitrogen). Sections were incubated with anti-von Willebrand Factor (Chemicon, dilution = 1:100) or anti-cardiac troponin-I (cTn-I) antibody (Santa Cruz; 1:100) for 1-hour at 37uC and subsequently incubated with secondary antibody conjugated to Alexa Fluor-488 (Invitrogen) for 1-hour at 37uC. Nuclei were stained with DAPI. Co-culture assay for myogenic differentiation Neonatal rat cardiomyocytes were isolated from 1–3days old rat pups using Neonatal Cardiomyocyte Isolation System (Worthing- ton Biochemical Corporation). For co-culture studies, cardiac cells were stained with QtrackerH625 Cell Labeling Kit (Invitrogen) and mixed with freshly isolated cardiomyocytes (1:4) and seeded on the fibronectin-coated chamber slides at a density of 2.56104/ cm2. For co-culture of the purified cardiac stem/progenitor cells with fixed neonatal cardiomyocytes, freshly isolated cardiomyo- cytes were seeded on fibronectin-coated chamber slides at a density of 26104/cm2. After 24-hours culture, cardiomyocytes were fixed with 0.5%PFA for 15 minutes at room temperature and washed with PBS [5]. Qtracker-labeled purified cardiac stem/ progenitor cells were seeded on fixed cardiomyocytes at a density of 56103/cm2. Co-culture of the purified cardiac stem/progenitor cells with live or fixed-cardiomyocytes were maintained for 6-days and, cells were fixed with 4%PFA and immunostained for anti a- sarcomeric actinin. Statistical analysis All data were expressed as mean6SD. Comparison between two mean values was calculated by an unpaired Student 2-tailed t- test. Comparison between multiple groups was performed by one- way ANOVA with post hoc analysis. A value of p,0.05 was considered statistically significant. Results Characterization of clonogenic cell populations The colony-derived cell populations were analyzed for Sca-1 and c-kit expression (Figure 1A). Sca-1 expression varied from 95.9% to 17.4% whereas c-kit expression was barely detected. The cell isolation procedure was successfully repeated three times to study the reproducibility of the procedure. We selected two high PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25265