FGF10 Cardiac Differentiation Figure 2. FGF-10 enhances cardiomyocyte differentiation. A, Protocol to determine the effect of FGF-10 application. B, Cardiomyocyte differentiation of embryonic stem (ES) cells following FGF-10 administration at different times (n = 3). Control, no treatment. C, Representative results of flow cytometry for analysis of ES cell differentiation at different time points of FGF-10 treatment. Green boxes indicate EGFP-positive cardiomyocytes (percentages indicated on each panel, n§3). Control, no treatment. D, Immunoblot analysis of mature cardiac markers—cardiac tropomyosin (Tpm), cardiac troponin T (cTnT) and cardiac troponin I (cTnI) in embryoid bodies (n = 3). Control, no treatment. E, Differentiation and cell counts of ES cells with FGF-10 neutralizing antibody on day 6 (n = 3). Control, no treatment. *p,0.05, **p,0.01, ***p,0.001. F, Cardiomyocyte differentiation with treatment of FGF-10 short hairpin RNA (shRNA, n = 3). Control, cells treated with a scramble shRNA. ***p,0.001. G, Confocal imaging of disassociated embryoid bodies using the antibody against cardiac troponin I. Control, no treatment. doi:10.1371/journal.pone.0014414.g002 inhibitor PD173074 reduced iPS cell cardiomyocyte differentia- tion (Figure 4C, and 4D). Therefore, the FGF-10-FGFR axis is essential to activate iPS cells into the cardiomyocyte lineage, and FGF-10 is a promising candidate in augmenting this process. FGF-10 modulates the expression of genes that regulate cardiogenesis FGF-10 significantly upregulated cardiomyocyte differentiation on day 2 rather than day 4 when FGF-10 was highly expressed (Figure 2B). We anticipated the effect of FGF-10 on cardiomyo- cyte differentiation is to activate differentiation of mesodermal lineages. To shed light on how FGF-10 governs the course of cardiomyocyte differentiation, the global gene expression was profiled by Affymetrix Mouse Genome 430 2.0 Array. As predicted, the mesodermal markers Brachyury, Goosecoid, and surprisingly, the early cardiac marker, Mesp1 were upregulated upon FGF-10 treatment (Figure 5B and Table S1). Results from Gene Ontology analysis suggested that the genes involved in cardiac developmental pathways were induced by FGF-10. In addition, the effect of FGF-10 was not limited to the cardiac lineage. Gene Ontology also revealed the role of FGF-10 in cell adhesion and tissue development such as in neurogenesis, blood vessel morphogenesis, and spermatogenesis (Figure 5A). FGF-10 enhances cardiomyocyte differentiation from ES cells in vivo As a proof of concept, the cardiomyocyte differentiation response to FGF-10 was examined in an in vivo setting (Figure 6A). Self- assembling peptide nanofiber hydrogels were used to form a defined intramyocardial microenvironment where FGF-10 and embryoid bodies (EBs) were injected into [19]. FGF-10 significantly enhanced EGFP-cTnT double positive cells per total cell count at the injection area (Figure 6B and 6C). The number of the double positive EGFP- cTnT cells was decreased in the FGF-10 mixed with PD173074 compound group. This indicates that FGF-10 can promote cell differentiation into cardiomyocytes which expressed late stage cardiac markers such as cTnT though clear sarcomeric structure cannot be observed, possibly due to the limitation of cell spreading in the injected hydrogel microenvironment. Therefore, FGF-10 increases cardiomyocyte differentiation of ES cells in vivo. Discussion The major challenge of using ES cell-based cardiomyocyte replacement therapy is the inefficiency of the conventional protocol to generate cardiomyocytes [8,9]. In the present study, we identified FGF-10 as a key factor in ES/iPS cell differentiation into cardiomyo- cytes. FGF-10 doubled the proportion of ES/iPS cells entering the cardiomyocyte lineage. The FGFR-1 and FGFR-2 were required for the FGF-10 effect. These findings suggest that FGF-10 may serve as a target in developing novel therapies for cardiac regeneration. To explore the molecular mechanisms controlling ES cell differentiation into cardiomyocytes, we used an aMHC-based reporter system to profile the temporal gene expression of cardiac markers during ES cell differentiation. Previously, other groups used the Nkx2.5 reporter system to investigate stem cell differentiation into cardiomyocytes [20–22]. Because Nkx2.5 is an earlier marker or cardiac precursor cells than aMHC [20], a reporter system using Nkx2.5 may not be as representative as aMHC. Cells expressing Nkx2.5 may differentiate into lineages other than cardiomyocytes, such as conduction system cells, endothelial cells, and vascular smooth muscle cells [21,22]. We focused on the FGF family members and identified the expression pattern of several FGFs at the mesodermal and cardiovascular progenitor stages, around two to three days after EB formation. Among the candidates, only FGF-10 consistently enhanced ES cell and iPS cell differentiation into cardiomyocytes. Interestingly, the time of FGF-10 addition was critical for cardiac induction; there was no effect on cardiomyocyte differentiation when we applied FGF-10 at day 0, but there was a ,2-fold increase of cardiomyocyte differentiation when applying FGF-10 at day 2 or day 4. This suggests that FGF-10 interacts with mesodermal cells and commits them into precursors of the cardiomyocyte lineage. While we showed that FGF-10 may affect the cardiomyocyte lineage, its effect on other cardiovascular lineages is unclear and requires further investigation. Because of the low efficiency of ES cell-derived cardiomyocytes, the difficulty of purification is another obstacle that must be overcome. FGF signaling is triggered through FGFR activation [14]. In our study, FGFR-1 and FGFR-2 were expressed during ES cell cardiomyocyte differentiation. PD173074, an inhibitor of FGFRs, and FGFR-1, FGFR-2 shRNA reduced ES cell cardiomyocyte differentiation, thus demonstrating that the FGF-FGFR signaling pathway is critical in the control of ES/iPS cell differentiation into cardiomyocytes [12,23]. Reduced cardiomyocyte differentiation was much more significant in the FGFR-2 knockdown group than that in FGFR-1 knockdown. Thus, our findings suggest that FGFR-2 may be a potential biomarker to identify cells committed to cardiac lineages. Therefore, in the future we may pursue a genetic fate-mapping approach to establish that FGF-10-FGFR-2 signaling plays a pivotal role in ES/iPS cell cardiac differentiation. iPS cells are known to differentiate into cardiomyocytes [17,24,25]. The main advantages of iPS cells are the absence of an immune response and minimal ethical concerns, making these cells a powerful tool for stem cell therapy. Unfortunately, iPS cell cardiomyocyte differentiation ratio is extremely inefficient for therapies, much like ES cells [26]. Therefore, we also set out to test whether FGF-10 augments the ratio of iPS cell differentiation into cardiomyocytes. Interestingly, our results showed that FGF-10 and its inhibition had similar effects on iPS cells and ES cells. Our data revealed that the function of FGF-10 in regulating stem cell cardiac differentiation is similar in both ES cells and iPS cells. We performed microarray in search for a possible mechanism of how FGF-10 promotes cardiomyocyte differentiation from ES cells. The enhancement of FGF-10 on cardiomyocyte differenti- ation applied on day 2 better than on day 4 when the endogenous FGF-10 highly expressed indicated the possibilities of direct or indirect interaction of regulators involved in mesodermal lineages. Array data revealed mesodermal markers such as Brachyury, PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e14414