differentiation into the epicardial cell lineage, and FGF-10 and FGF receptor (FGFR)-2-III mice have abnormal heart positioning [12]. Nevertheless, the function of FGFs in ES/iPS cell cardiomyocyte differentiation remains largely unknown. In this study, we used mouse ES cells stably transfected with a cardiac-specific a-myosin heavy chain (aMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. We focused on the expression pattern of FGFs during ES/iPS cell differentiation and carried out experiments to explore the role of FGFs in cardiomyocyte differentiation. Results Temporal expression profile of FGF members during ES cell cardiomyocyte differentiation Cardiomyocyte differentiation of ES cells underwent several stages denoted by the temporal expression of specific markers (Figure 1A, and 1B). Undifferentiated ES cells (day 0) highly expressed Oct4, which gradually diminished after differentiation into ectodermal, mesodermal, and endodermal lineages. Meso- dermal cells expressed Brachyury from day 2 to day 4, and cardiovascular progenitors, the offspring of mesodermal cells, expressed Flk-1 from day 4 to day 6. Cardiac precursors started to appear on day 4, indicated by the expression of Gata4. Cardiomyocytes, as denoted by the expression of the cardiac structural markers aMHC and cardiac troponin-T, can only be detected by flow cytometry after day 6 of treatment. However, the more mature cardiomyocyte marker, cardiac troponin-I, was not detected until day 8. One logical approach to increase the proportion of ES cell- derived cardiomyocytes is to take action at an early stage in the differentiation process by augmenting the ratio of ES cells committed to the mesodermal and cardiovascular progenitor lineages. Therefore, members of the FGF family that have a high potential to generate cardiomyocytes may be expressed early in the differentiation process when cells are at the stages of mesodermal and cardiovascular progenitors, that is, from day 2 to day 4. Screening of the temporal expression profile of FGF genes revealed that FGF-3, FGF-8, FGF-10, FGF-11, FGF-13 and FGF-15 were mostly expressed from day 2 to day 4 (Figure 1C). Among them, the exogenous FGF-10 recombinant protein was the most effective in increasing cardiomyocyte differentiation from ES cells by two-fold (Figure 1D). FGF-10 is crucial to cardiomyocyte differentiation from ES cells Certain growth factors, Wnt-3a being a notable example, are well known to act both stimulatory and inhibitory to cardiomy- ocyte differentiation depending on the application time during the differentiation process [13]. We found that FGF-10 only enhanced cardiomyocyte differentiation when applied at day 2 or day 4 (Figure 2A, and 2B), which coincided with the expression pattern of the FGF-10 gene (Figure 1C). Also, the stimulatory effect of FGF-10 was concentration-dependent, and immunoblotting confirmed the increased expression of three structural proteins cardiac tropomyosin (Tpm), cardiac troponin-T (cTnT), and cardiac troponin-I (cTnI) (Figure 2B, 2C and 2D). EGFP-positive cells were observed to have more synchronized beating in the FGF-10 treatment group through real-time imaging of the contractile embryoid bodies (Video S1, S2, S3, S4) as well as a more obvious sarcomeric structure through confocal imaging using antibodies against cTnI, cTnT, and Tpm (cTnI data shown in Figure 2G, other data not shown). Our data showed that the FGF10 Cardiac Differentiation cardiomyocyte differentiation ratio increased over time and reached ,14% in the FGF-10 treated group on day 8 and ,20% if we prolonged the differentiation time to 14 days (Figure S1A). The group treated with 100 ng/ml of FGF-10 had an optimal cardiomyocyte differentiation ratio that was significantly higher than the group treated with 10 ng/ml of FGF-10 (Figure 2B). Therefore, 100 ng/ml of FGF-10 was used for all subsequent treatments. Not only does FGF-10 promote cardiomyocyte differentiation, it is crucial during normal cardiomyocyte differentiation process in ES cells. The importance of FGF-10 was demonstrated by the finding that both FGF-10 neutralizing antibody, which inhibited the activation of Akt by FGF-10, and FGF-10 short hairpin RNA (shRNA) blocked cardiomyocyte differentiation without affecting the cell number (Figure 2E, 2F and Figure S2). Moreover, FGF-10 did not influence the ES cell cycle (data not shown), indicating that the FGF-10 response was specific to cardiomyocyte differentiation and not a consequence of ES cell proliferation increase or ES cell apoptosis decrease. To gain more insight on the effect of FGF-10 in the cardiomyocyte differentiation process, we examined the expression pattern of several markers using cells treated with FGF-10 recombinant protein and shRNA for knockdown (Figure S3). From the semi-quantitative PCR results, FGF-10 increased the expression of Brachyury, Flk-1 and mature cardiac markers aMHC, cTnI and cTnT. Interestingly, FGF-10 knockdown resulted in downregulation of Flk1, Gata4, aMHC, cTnT, and cTnI, but not Brachyury. Together, these results demonstrate that FGF-10 may induce the mesodermal and cardiogenic gene expression. However, the increased mesoderm differentiation by FGF-10 is sufficient but may not be required for cardiac differentiation. In addition to mesoderm induction, FGF-10 also promoted expression of the ectoderm genes at early stages (day 3 to day 5) and that of the endoderm genes in later differentiation process (day 5 to day 7) (Figure S1B, S1C, and S1D) Response to FGF-10 is mediated via FGF receptors Members of the FGF family, with the exception of FGF-11, -12, -13 and -14, exert their functions via FGF receptors (FGFRs) [14]. Genes encoding all seven isoforms of FGFR RNA, as well as FGFR-1 and FGFR-2 proteins were expressed during the cardiomyocyte differentiation process of ES cells (Figure 3A, 3B and 3C). The small molecule FGFR-specific inhibitor, PD173074, diminished the degree of cardiomyocyte differentiation with or without FGF-10 stimulation, and without altering the cell number (Figure 3D, 3E and data not shown). FGFR inhibition also downregulated the expression of the cardiomyocyte-specific genes Gata4, Mlc1a, Mlc2v, cTnT and cTnI (Figure 3F). FGF-10 is known to primarily activate FGFR-2 with little stimulatory action on FGFR-1 [15]. To determine which FGFR isoforms contribute to the FGF-10-mediated cardiomyocyte differentiation response, shRNA interference was used. FGFR-1 and FGFR-2 shRNA reduced cardiomyocyte differentiation from both non-stimulated and FGF-10-stimulated ES cells, while the scramble control vector had no effect (Figure 3G). Reduced cardiomyocyte differentiation was more obvious in the FGFR-2 knockdown group than that in FGFR-1 knockdown. Therefore, FGFR-2 may play a more important role in FGF-10 promoted cardiomyocyte differentiation from ES cells. To prove that the increased percentage of cardiomyocyte differentiation was specifically due to FGFR-2 activation by FGF-10 rather than by other FGFR-2 activators, ES cells were treated with FGF-7 and evaluation of cardiomyocyte differentiation showed no significant difference from that of the control (Figure 1D). PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e14414