213 WNT inhibition by dickkopf WNT signalling pathway inhibitor 1 (DKK1) cannot replace IWR-1 during derivation of bovine embryonic stem cells

Abstract

Understanding the signalling pathways involved with derivation of embryonic stem cells could enhance our understanding of pluripotency in pre-implantation embryos. Recently, the small molecule IWR-1 has been shown to promote derivation of mouse epiblast stem cells and pluripotent bovine and porcine embryonic stem cells (ESC). IWR-1 blocks WNT signalling mediated by β-catenin-targeted gene expression through stabilisation of Axin2, a member of the destruction complex that induces β-catenin degradation. Here, we evaluated whether dickkopf WNT signalling pathway inhibitor 1 (DKK1) can replace IWR-1 for establishment of bovine pluripotent ESC. If so, it is likely that the actions of IWR-1 to promote pluripotency involve inhibition of WNT signalling. Treatment of bovine embryos with 100 ng mL⁻¹ recombinant human DKK1 beginning at Day 5 of development decreased (P = 0.02) immunofluorescent labelling of β-catenin in the resulting blastocysts (n = 41-45/group), indicating that bovine embryos are responsive to DKK1 treatment. For ESC derivation, blastocysts were plated on top of feeder cells and cultured in ESC medium supplemented with 2.5 µM IWR-1 (n = 21), 100 ng mL⁻¹ DKK1 (n = 34), or vehicle (n = 23). Cells were passaged every 5 to 7 days in their respective treatment medium. Seven days after plating, 57.9 ± 14.7% of blastocysts in IWR-1 ESC medium developed outgrowth, which was lower (P = 0.02) than the proportion of blastocysts with outgrowth in DKK1 medium (92.4 ± 5.2%) or vehicle (81.9 ± 10.0%). Outgrowth size did not differ among treatments. Labelling with CDX2 indicated that the majority of cells in outgrowths were trophectoderm cells. Thus, IWR-1 inhibits competence of blastocysts to form trophectoderm outgrowths during derivation of ESC. The percent of blastocysts from which cell lines were derived after 4 passages were 48% (10/21) for IWR-1, 41% (14/34) for DKK1, and 48% (11/23) for vehicle. Immunolabelling for the pluripotency marker SOX2 showed that only cells grown in IWR-1 medium were positive, whereas most of the cells derived in the other two media were not. Thus, IWR-1 could not be replaced by DKK1 for maintaining pluripotency. Immunoreactive β-catenin was abundantly distributed on the membrane of cells cultured with IWR-1 but not with DKK1 or vehicle-treated cells. Thus, β-catenin distribution to the cell membrane is linked with bovine pluripotency. Overall, results indicate that maintenance of pluripotency by IWR-1 may involve mechanisms other than WNT inhibition, and may be related to the localization of β-catenin to the plasma membrane.