flt3 Note that our EpiSC lines

Note that our EpiSC lines required continuous treatment with IWP-2 because removal of this Wnt inhibitor caused spontaneous differentiation. Since the original EpiSCs can be propagated without IWP-2, our EpiSCs must be distinguished from them by some intrinsic difference. There may be heterogeneities in Wnt signaling activity among epiblast cell populations, as previously observed for hESCs (Blauwkamp et al., 2012). Given that the inhibition of WNT secretion (or inhibition of Wnt signaling) promotes rapid EpiSC expansion, flt3 with lower Wnt activity or responsiveness can be selected and established preferentially as EpiSCs under the original derivation conditions. On the other hand, IWP-2 blocks WNT secretion, leading to a reduced level of Wnt signaling. Since the WNT ligands available in culture would be greatly decreased, almost all of the epiblast cells, including cells with high Wnt responsiveness, would expand as EpiSCs. Therefore, EpiSCs derived by the IWP-2 method retain Wnt responsiveness and can differentiate in response to the Wnt stimulus. The fact that the 129C1 EpiSC line established by the original protocol (Brons et al., 2007) showed reduced responsiveness to WNT3A supports the notion described above. Global gene-expression analysis revealed that addition of IWP-2 to 129C1 EpiSC cultures suppressed the expression of a specific cluster of genes. This tight cluster of genes was highly expressed in EpiSC lines obtained by the original protocol (i.e., 129C1 and Wt1 cells), whereas it was barely detectable in the EpiSCs derived by the IWP-2 method, in 129C1 cells cultured with IWP-2, or in epiblast tissues from E5.5–E7.5 embryos. This cluster of genes is involved in the regulation of development into the mesoderm, endoderm, or primitive streak. Interestingly, some of these genes (e.g., Cer1, Dkk1, and Sox17) have been considered to be characteristic markers of EpiSCs (Brons et al., 2007; Tesar et al., 2007; Kojima et al., 2014). However, we show here that the expression of these marker genes is not essential for the derivation and self-renewal of EpiSC lines. Our EpiSCs made by the IWP-2 method appear to represent authentic pluripotent EpiSCs, but with low levels of Wnt-induced spontaneous differentiation. In a study using XAV939 together with the Rho kinase inhibitor Y27632, Sumi et al. (2013) suggested that Wnt signaling inhibition plays a role in promoting less spontaneous differentiation. In the present study, we further demonstrated that genes suppressed by Wnt inhibition could be classified into at least two groups: one expressed in a partially differentiated subpopulation in EpiSC cultures (e.g., Gata4 or Sox17), and one expressed in SSEA1high undifferentiated EpiSCs, including Foxa2, Gsc, and Evx1. Expression of genes in both classes was dependent on Wnt signaling. Therefore, the Wnt inhibitor suppressed spontaneous, Wnt-induced differentiation to endoderm or mesoderm lineages, and also suppressed the expression of Wnt-dependent genes in SSEA1high pluripotent cells.
Experimental Procedures
Author Contributions
Acknowledgments