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| Status |
Public on Nov 02, 2018 |
| Title |
Activin A in combination with ERK1/2 MAPK pathway inhibition sustains propagation of mouse embryonic stem cells |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
The combination of Wnt pathway activation by the GSK3 inhibitor and ERK pathway inhibition by the MEK inhibitor, which is known as 2i is a well-established method to maintain mouse embryonic stem cell (mESC) self-renewal. Here we show that Activin A also has the ability to promote naive pluripotency of mESCs when combined with the MEK inhibitor PD0325901. mESCs were efficiently propagated in a medium containing both Activin A and the MEK inhibitor (PD0325901). mESCs cultured in Activin+PD retained a pluripotency state that expresses high levels of naive pluripotency-related transcription factors and is able to differentiate into three germ layers under appropriate conditions. They also showed naive pluripotency features, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding provides another way to maintain the naive pluripotency state and 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 naive and primed pluripotency states and their responses to Wnt and ERK1/2 MAPK pathway, we performed genome-wide gene expression analysis of mESCs and EpiLCs, and those treated with Wnt pathway activator alone or Wnt pathway activator combined with ERK1/2 MAPK pathway inhibitior.
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| Overall design |
EpiLCs were induced from mESCs by culturing them in N2B27 supplemented with 20 ng/ml ActivinA and 12 ng/ml bFGF on dishes coated with serum for 2 days. We prepared three samples for both mESCs and EpiLCs : non-treated cells, cells treated with CHIR99021, and cells treated with a combination of CHIR99021 and PD0325901. Total RNA was isolated with RNeasy Mini kit (Invitrogen). During the isolation, on-column DNaseI digestion was performed. Biotinylated sense-strand DNA was synthesized with GeneChip WT PLUS Reagent kit (Affymetrix), hybridized to Mouse Gene 2.0 ST Array (Affymetrix) and analyzed by GeneChip Scanner 7G System (Affymetrix). The data were analyzed by GeneSpringGX11.5 software (Agilent).
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| Contributor(s) |
Ashida Y, Nakajima-Koyama M, Hirota A, Yamamoto T, Nishida E |
| Citation(s) |
28097777 |
| |
| Submission date |
Nov 12, 2015 |
| Last update date |
Nov 04, 2018 |
| Contact name |
Eisuke Nishida |
| E-mail(s) |
nishida@lif.kyoto-u.ac.jp
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| Phone |
+81-75-753-4230
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| Organization name |
Graduate School of Biostudies, Kyoto University
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| Department |
Department of Cell and Developmental Biology
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| Street address |
Kitashirakawa, Sakyo-ku
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| City |
Kyoto |
| ZIP/Postal code |
606-8502 |
| Country |
Japan |
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| Platforms (1) |
| GPL16570 |
[MoGene-2_0-st] Affymetrix Mouse Gene 2.0 ST Array [transcript (gene) version] |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA302013 |
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