FGF10 Cardiac Differentiation Figure 5. FGF-10 modulates the expression of cardiac related genes and pathways. A, Gene ontology process network analysis showing predicted functions of FGF-10 mediated upregulated genes. (p-value x0.01). B, Gene validation using quantitative real-time PCR, *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0014414.g005 Goosecoid, Rspo3, and early cardiac marker Mesp1 [27–30] were upregulated following FGF-10 treatment. Gene ontology analysis also indicated that FGF-10 may act through BMP, TGF-b, Wnt- b-catenin, Notch, and VEGF signaling to induce cardiomyocyte differentiation, although the specific mechanisms have yet to be determined. FGF-10 not only promoted cardiomyocyte differen- tiation via activating mesodermal lineages at early stage of differentiation but also increased the expression of cTnT, and cTnI, two mature cardiac markers, in ES cell-derived cardiomy- ocytes at later differentiation stage. To explore the in vivo role of FGF-10 in cardiomyocyte differentiation, we injected FGF-10 incubated with peptide nanofibers into the myocardium. Interestingly, similar to in vitro results, FGF-10 increased the ratio of EGFP-positive cells. However, while these EGFP-positive cells co-expressed cTnT, the indication that these cells turned into mature cardiomyocytes is yet to be confirmed because the sarcomeric structure is not obvious. Our present two-week study falls short in answering some pending concerns, such as teratoma formation and immune response to FGF-10-stimulated ES cell-based therapy. A long term in vivo study is required to verify the roles of FGF-10-guided ES cell differentiation in aiding cardiac repair after myocardial infarction. In conclusion, we discover that FGF-10 is a novel regulator of cardiomyocyte differentiation from ES cells and iPS cells. Our results not only help in understanding the molecular mechanisms mediating ES/iPS cell cardiac differentiation, but also have the potential for translation into clinical applications. Materials and Methods Ethics Statement All animal research procedures were approved by the National Cheng Kung University Animal Care and Use Committee. The investigation conformed to the Guide for the Care and Use of Laboratory PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e14414