A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.
Bottom Line: Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion.Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas.The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a "primed" pluripotent state, and directed differentiation from the primed state.
Affiliation: Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.Show MeSH
Related in: MedlinePlus
Mentions: Principal-component analysis (PCA) of the expression profile data revealed that mESC lines, EpiSC lines, and epiblast/embryonic ectoderm were clustered at distinct positions on the PC1-PC2 plane (Figure 4A). Moreover, differences in the expression profiles of each group were revealed along the PC3 axis. For example, mESCs cultured in 2i medium were distantly located from mESCs cultured in KSR/FBS-containing medium, and EpiSC lines made by the IWP-2 method were located separately from the EpiSCs established by the original method. Hierarchical cluster analysis also revealed that three groups—mESCs, epiblast/ectoderm, and EpiSCs—displayed globally different expression profiles (Figure 4B; Table S2). Although the EpiSC lines generated by the IWP-2 method exhibited profiles similar to those of EpiSCs made by the original method, they were classified into different clusters. To examine differences among the EpiSC lines, we subjected 17,819 probes that showed differential expression among EpiSCs and epiblast/ectoderm to a k-means cluster analysis, which revealed nine clusters (Figure S4A). Clusters 2, 3, 6, and 8 represent probes that exhibited differences between EpiSCs and epiblast/ectoderm. Clusters 1 and 5 represent genes that were upregulated in 129C1 and Wt1 cells (the EpiSC lines established without IWP-2), and cluster 9 includes genes that were upregulated in the EpiSCs made by the IWP-2 method. Cluster 1 genes are particularly interesting because they were highly expressed in 129C1 cells but were repressed when 129C1 cells were cultured in the IWP-2 medium. These genes were also suppressed in 129Ba1, 129Ba2, and BNa17 cells cultured in the IWP-2 medium, suggesting that expression of the cluster 1 genes depends on WNT secretion, and therefore these genes function downstream of Wnt signaling.
Affiliation: Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.