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Activin A in Combination with ERK1/2 MAPK Pathway Inhibition Sustains Propagation of Mouse Embryonic Stem Cells

ABSTRACT: Activin/Nodal/TGF-β signaling pathway plays a major role in maintaining mouse epiblast stem cells (mEpiSCs). The mEpiSC medium which contains Activin A and bFGF induces differentiation of mouse embryonic stem cells (mESCs) to mEpiSC. Here we show that Activin A also has an ability to efficiently propagate mESCs without differentiation to mEpiSCs when combined with a MEK inhibitor PD0325901. mESCs cultured in Activin+PD retained high-level expression of naive pluripotency-related transcription factors. Genome-wide analysis revealed that the gene expression profile of mESCs cultured in Activin+PD resembles that of mESCs cultured in 2i. mESCs cultured in Activin+PD also showed features which are related to naive pluripotency of mESCs, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding reveals a role of Activin/Nodal/TGF-β signaling in stabilizing self-renewal gene regulatory networks in mESCs. To compare the gene expression patterns of mESCs cultured in Activin+PD, 2i and LIF+BMP4 and mEpiSCs, we performed genome-wide gene expression analysis by using Affymetrix GeneChip oligonucleotide microarrays

ORGANISM(S): Mus musculus

PROVIDER: GSE84679 | GEO | 2019/07/11

REPOSITORIES: GEO

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Project description:Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse ES cells leads to their exit from self renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2 the primary downstream transcriptional factor of the Nodal/Activin pathway followed by massively parallel sequencing and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression. Four biological replicates consisting of 4 different passages of E14TG2a ES cells at P20, P21, P23 and P24

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Activin A in Combination with ERK1/2 MAPK Pathway Inhibition Sustains Propagation of Mouse Embryonic Stem Cells

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