FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling.

Fibroblast growth factor-2 (FGF-2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF-2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF-2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co-localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase pathways (PI3-K) are activated following FGF-2 stimulation. Notably, inhibition of MAPK and PI3-K signaling using specific kinase inhibitors revealed that activated PI3-K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3-K activation, activation of Wnt/beta-catenin by FGF-2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF-2 stimulation, GSK3beta is phosphorylated following which nuclear translocation of beta-catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf-1 (DKK-1) resulted in only partial suppression of the FGF-2 induced TCF/LEF activity. Prolonged culture of hESC with DKK-1 did not affect pluripotent marker expression. These results suggest that FGF-2 mediated PI3-K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC.