Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/β-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/β-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/β-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

We acknowledge the support from Spanish Ministry of Economy and Competitiveness and FEDER funds (SAF2011-28580, BFU2014-54717-P, BFU2017-86760-P and BFU2015-71984-ERC to M.P.C.), as well as ‘Centro de Excelencia Severo Ochoa 2013–2017’, Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya grant (2014 SGR1137 to M.P.C.), AGAUR (SGR 2017-2019 to M.P.C) and the CERCA Programme/Generalitat de Catalunya (to M.P.C.), the European Union’s Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to M.P.C.), People Programme Marie Curie Actions of the European Union’s Seventh Framework Programme (FP7/2007–2013/, n° 290123 to I.T.), La Caixa international PhD fellowship (to F.S.) and Ministerio de Ciencia e Innovació FPI (to F.A.) and People Program (Marie Curie Actions) FP7/2007–2013 under REA grant (608959 to M.V.N.).