mesp1, forward: 59 -TGTACGCAGAAACAGCATCC-39, reverse: 59- TTGTCCCCTCCACTCTTCAG-39; nestin, forward: 59 - GCTGGCTGTGGAAGCCCTGG -39, reverse: 59- CTGGGCACTGTGGCCTCAGC -39; gata3, forward: 59- CCTGAGCAGCCACCACACCG -39, reverse: 59- GCAGGCTGCTGGGTGGGAAG -39; sox17, forward: 59 - TCGACGGCTACCCTCTGCCC -39, reverse: 59- CGTGCGGTCCACCTCCCCTA -39; transferrin, forward: 59 - TAAGCTGTCGGAGCCCCGCA - 39, reverse: 59- AAAGGCCACATCCCCACCGC -39; Cardiomyocyte differentiation quantification by flow cytometry analysis Cardiomyocyte differentiation from ES cells or iPS cells was quantified by flow cytometry (BD FACSCalibur, BD Bioscience, Bedford, MA) of cardiac specific markers (aMHC promoter-driven EGFP or cTnT). EBs were collected on day 6 (or as specified otherwise) and dispersed into single cells by trypsin (Invitrogen). Cells of ES cell origin were subsequently examined for EGFP signals. For cells of iPS cell origin, they were first stained with anti- cTnT antibody (1:100, clone CT3; Developmental Studies Hybridoma Bank, Iowa City, IA) and Alexa Fluor 568 secondary antibody (1:1000; Invitrogen) before flow cytometry analysis. Cardiomyocyte differentiation was quantified by the number of EGFP positive cells (ES cell-derived cardiomyocytes) or cTnT positive cells (iPS cell-derived cardiomyocytes). A total of 10000 cells were analyzed at a time and the assay was repeated at least three times to average the percentage of cardiomyocyte differen- tiation. FGF-10 neutralization and FGFR inhibition For loss-of-function studies, day 2 EBs were cultivated with FGF-10 neutralizing antibody (BioVision, Mountain View, CA) or a small-molecule FGFR inhibitor, PD173074 (Sigma, St. Louis, MO). Gene knockdown assay Bacteria transformed with FGF-10, FGFR-1, FGFR-2 shRNA in pLKO.1-puro lentiviral backbone were obtained from National RNAi Core Facility (Academia Sinica, Taipei, Taiwan) and scramble control vector was obtained from Addgene. Further culture and expansion were conducted according to the manu- facturer’s protocol. Purified FGF-10, FGFR-1, FGFR-2 shRNA plasmid was then transfected into 293T cells with Lipofectamine (Invitrogen) together with an envelope expression plasmid (pMD2.G) and a packaging vector (psPAX2) to generate the shRNA-containing lentivirus. Each lentivirus was then infected into ES cells according to the National RNAi Core Facility protocol. Immunohistochemical staining EBs collected on day 2 to day 7 were fixed, paraffin-embedded, and subjected to immunohistochemical staining with anti-FGFR-1 antibody (1:100, clone C-15; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-FGFR-2 antibody (1:100, clone 133730; R&D Systems), and then with 3,39-diaminobenzidine (DAB) for visualization (Vector Laboratories, Burlingame, CA). FGF10 Cardiac Differentiation Cell cycle analysis ES cells were treated with FGF-10 for 24 h, fixed and subjected to propidium iodide (1 mg/ml; Sigma) staining and flow cytometry analysis as previously described [9]. Preparation of a EBs-peptide nanofiber mixture for transplantation A mixture of EBs and peptide nanofibers was prepared by suspending twenty ES cells-derived day 2 EBs into 20 ml of 0.5% peptide nanofiber (RAD16-II peptide: AcN-RARADADARAR- ADADA-CNH2, Synpep, Dublin, CA) in phosphate buffered saline (pH 7.4). FGF-10 (100 ng/ml) or 30 nM PD173074 was added into the mixture as needed. In vivo evaluation of FGF-10 on cardiomyocyte differentiation C57BL/6 mice (20–25 g, acquired from the National Laboratory Center, Taipei, Taiwan) were anesthetized with Zoletil (50 mg/kg, Virbac, France) and Rompun (0.2 ml/kg, Bayer Healthcare, Germany), artificially ventilated, and had their hearts exposed. Three groups: 0.5% nanofibers only, 0.5% nanofibers +100 ng/ml FGF-10, 0.5% nanofibers +100 ng/ml FGF-10+30 nM PD173074, mixed with day 2 EBs were subsequently injected into the free wall of left ventricle myocardium by three injections (n = 4, in each group) [32]. The chest was then closed, and the mice were allowed to recover. Two week later, mice were sacrificed, and the hearts were harvested, paraformaldehyde-fixed and paraffin-embedded. Immunohistochemistry was subsequently performed using antibod- ies against EGFP (1:100, clone 1E4; MBL, Woburn, MA), cTnT (1:100) and then incubated with Alexa Fluor-conjugated secondary antibodies (1:200, Molecular probes, Invitrogen). After counter- staining with DAPI (Sigma-Aldrich), sections were mounted and observed under a fluorescence microscope. Six sections per heart were evaluated for ES cell incorporation and EGFP-cTnT double positive cells were counted and divided by the total cell count. Microarray analysis Two-day EBs were treated with or without 100 ng/ml FGF10 and RNA was obtained 24 hours later for microarray analysis, which were performed by the Affymetrix Gene Expression Service Lab (http://ipmb.sinica.edu.tw/affy/), supported by the Acade- mia Sinica. The full normalized data are in Supplementary Data 1. A total of 172 upregulated genes (FGF-10 treatment/without treatment, p-value ,0.001) were analyzed in MetaCoreTM of GeneGo Inc. for enrichment of gene ontology terms. Terms with P values adjusted for multiple testing # 0.01 were considered enriched. Microarray report conformed to the MIAME guidelines, and the data was submitted to Gene Expression Omnibus. Statistical analysis Results were expressed as mean 6 S.E.M. for at least three independent experiments. Data were analyzed by Student t-test or one-way or two-way analysis of variance with Newman-Keuls post-hoc test where appropriate. Statistical significance was set as p,0.05 (two-tailed). Supporting Information Table S1 Microarray results of gene symbol, accession number, fold change and predicted functions of the up- and down- regulated genes in ES cells treated with FGF-10. Found at: doi:10.1371/journal.pone.0014414.s001 (0.47 MB XLS) PLoS ONE | www.plosone.org 11 December 2010 | Volume 5 | Issue 12 | e14414