BackgroundThe self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.Methodology/Principal FindingsIn this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.Conclusions/SignificanceOur study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

Epithelial-mesenchymal transition (EMT) has been thought to occur during early embryogenesis, and also the differentiation process of human embryonic stem (hES) cells. Spontaneous differentiation is sometimes observed at the peripheral of the hES cell colonies in conventional culture conditions, indicating that EMT occurs in hES cell culture. However, the triggering mechanism of EMT is not yet fully understood. The balance between self-renewal and differentiation of human pluripotent stem (hPS) cells is controlled by various signal pathways, including the fibroblast growth factor (FGF)-2. However, FGF-2 has a complex role for self-renewal of hES cells. FGF-2 activates phosphatidylinositol-3 kinase/AKT, mitogen-activated protein kinase/extracellular signal-regulated kinase-1/2 kinase, and also protein kinase C (PKC). Here, we showed that a PKC rapidly induced an early growth response protein-1 (EGR-1) in hES cells, which was followed by upregulation of EMTrelated genes. Before the induction of EMT-related genes, EGR-1 was translocated into the nucleus, and then bound directly to the promoter region of SNAIL, which is a master regulator of EMT. SNAIL expression was attenuated by knockdown of EGR-1, but upregulated by ectopic expression of EGR-1. EGR-1 as the downstream signal of PKC might play a key role in EMT initiation during early differentiation of hES cells. This study would lead to a more robust understanding of the mechanisms underlying the balance between selfrenewal and initiation of differentiation in hPS cells.