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MEK and GSK3β inhibition drives ES cells into a naive pluripotent state irrespective of their global methylation status

ABSTRACT: Mouse embryonic stem cells (mESCs) can convert to a ground state by dual inhibition of MEK and GSK3β signalling in defined media in the presence of LIF (2i). The cellular transition in 2i leads to uniform expression of pluripotency markers and global DNA hypomethylation. Whether hypomethylation is required for achieving ground state or is an outcome of the conversion process is still not clear. Here we show that J1 wild type and hypomethylated mESCs (dnmt3a-/-, dnmt3b-/-, dnmt1-/- [TKO]) lacking DNA methyltransferases undergo similar morphological and expression changes upon culturing in 2i that are consistent with conversion of both to a more naive state. Maintenance of global DNA methylation levels in 2i media by constitutive expression of de novo methyltransferases is also not a barrier to conversion. Hence, signalling pathways in mES cells regulate pluripotency networks and cell state independently of their global DNA methylation status. Indicated mESCs were cultured in 2i/LIF for 2 weeks and then harvested along with serum/LIF counterparts for microarray analysis. 1ug of total RNA was used to prepare Cy3 labelled aRNA using an Amino Allyl MessageAmp II aRNA kit (Ambion) following manufacturers protocol. The samples were hybridised to SurePrint G3 mouse GE 8x60k microarrays (Agilent) and were scanned using NimbleGen MS 200 (Roche). Results were analysed with custom-written scripts implemented in the statistical programming language R (http://www.R-project.org).

ORGANISM(S): Mus musculus

PROVIDER: GSE72302 | GEO | 2020/06/16

REPOSITORIES: GEO

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