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
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Mentions: To test our hypothesis that Wnt inhibition would enhance the efficiency of EpiSC derivation, we separated epiblasts of embryonic day 5.5 (E5.5) mouse embryos obtained from C57BL/6 (B6) × 129S2/Sv (129) strain crosses from the VE and cultured them in EpiSC medium with or without the Wnt inhibitor IWP-2, as described in the Experimental Procedures (Figure 1A). Epiblast explants attached to the substratum on day 2 of culture, and epiblast cells formed flat colonies on day 3 irrespective of the presence or absence of IWP-2 (Figure S1). By day 5, only the epiblast explants cultured with IWP-2 still showed flat colonies of compact cells characteristic of EpiSCs (Figure 1B). In contrast, explants cultured without IWP-2 gave rise to colonies with heterogeneous morphologies, and differentiated cells emerged in most cases (Figure 1C). At 1 or 2 days after passage 3 or 4, we examined the morphologies of ten colonies, and if more than eight of the colonies showed “good” undifferentiated morphologies (see Figure S1), we judged that EpiSC lines had been established. Expression of alkaline phosphatase was also examined (Figure S1). EpiSC lines were derived from E5.5 epiblasts in the presence of IWP-2 with high efficiency (5/5), whereas EpiSC lines were established at a much lower rate in the absence of IWP-2 under our culture conditions (3/16 hybrid embryo explants; 19%) (Table 1). Using E6.5 embryos, we obtained essentially similar results (two lines of 24 E6.5 epiblasts (8%) in the absence of IWP-2, versus 6/6 in the presence of IWP-2; Table S1). These results suggest that inhibition of WNT secretion greatly enhanced the derivation of EpiSC lines.
Affiliation: Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.