Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome of mouse pluripotent embryonic stem cells (mESC) cultured in R2i, 2i, PD and SB conditions

ABSTRACT: In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells. Whole gene expression study of 4 different mouse embryonic stem cell lines maintained under 4 different chemically defined conditions: R2i, 2i, PD and SB

ORGANISM(S): Mus musculus

SUBMITTER: Ali Sharifi-Zarchi

PROVIDER: E-GEOD-43682 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Inhibition of TGFβ signaling promotes ground state pluripotency.

Hassani Seyedeh-Nafiseh SN Totonchi Mehdi M Sharifi-Zarchi Ali A Mollamohammadi Sepideh S Pakzad Mohammad M Moradi Sharif S Samadian Azam A Masoudi Najmehsadat N Mirshahvaladi Shahab S Farrokhi Ali A Greber Boris B Araúzo-Bravo Marcos J MJ Sabour Davood D Sadeghi Mehdi M Salekdeh Ghasem Hosseini GH Gourabi Hamid H Schöler Hans R HR Baharvand Hossein H

Stem cell reviews and reports 20140201 1

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. ...[more]

PMID: 24036899

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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

Dataset Information

Transcriptome of mouse pluripotent embryonic stem cells (mESC) cultured in R2i, 2i, PD and SB conditions

Publications

Inhibition of TGFβ signaling promotes ground state pluripotency.

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