FGF10 Cardiac Differentiation Figure 6. FGF-10 promotes cardiomyocyte differentiation from embryonic stem cells in vivo. A, Schematic illustration of the intramyocardial administration of embryonic stem (ES) cells and peptide nanofibers (NF). B, Statistical summary of EGFP-cTnT double positive cells per total cell count from images such as in C (n = 4; **p,0.01). C, Photomicrographs of ES cells from hearts injected with cells and NF only (upper panel), along with FGF-10 (middle panel) and with FGF-10 plus PD173074 (lower panel). Also shown are cardiomyocytes stained for EGFP (green), cardiac Troponin-T (cTnT, red) and DAPI (blue). Note the co-localization of EGFP and cTnT (yellow). doi:10.1371/journal.pone.0014414.g006 Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Culture of mouse ES-aMHC-EGFP cells and cardiomyocyte differentiation induction Mouse ES-aMHC-EGFP cells were obtained by transfection of the a-myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP) reporter into the CGR8 ES cell line [8]. For normal maintenance, ES cells were cultured in Glasgow Minimum Essential Medium (Sigma, St. Louis, MO) supplemented with 15% knockout serum replacement (Invitrogen, Carlsbad, CA), 1% non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 1% penicillin/streptomycin (Invi- trogen), 0.1 mM -mercaptoethanol (Sigma) and 1000 U/mL leukemia inhibitory factor (Chemicon, Temecula, CA). To initiate cardiomyocyte differentiation (day 0), ES cells were cultured in hanging drops of 400 cells per 20 mL of differentiation medium (composition identical to maintenance medium except without leukemia inhibitory factor and 10% fetal bovine serum replacing knockout serum replacement) to form EBs. On day two, EBs were transferred into fresh differentiation medium for suspension cultivation in non-adhering petri dishes. To screen the effects of FGF members on cardiomyocyte differentiation, FGF-2, FGF-3, FGF-10 (R&D Systems, Minneapolis, MN), FGF-11, FGF-13 (both from Abnova, Taipei, Taiwan) and FGF-7, FGF-8, FGF-15 (all from PeproTech, Rocky Hill, NJ) recombinant proteins were applied on day 2 at 100 ng/ml. Culture of mouse iPS cells Mouse iPS-MEF-Ng-20D-17 cells were obtained from the Cell Bank, Riken BioResource Center (Tsukuba, Japan). These cells contained a Nanog promoter-driven EGFP reporter [31]. Mouse iPS cells were cultured with or without mouse embryonic fibroblasts as a feeder layer. The procedures of maintenance and cardiomyocyte differentiation initiation of iPS cells were otherwise identical to ES cells as described above. PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e14414