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| Status |
Public on Aug 10, 2012 |
| Title |
Distinct developmental ground states of different epiblast stem cell lines determine pluripotency features |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
Epiblast stem cells (EpiSC) are pluripotent stem cells derived from mouse postimplantation embryos between E5.5 to E7.5, a time window in which gastrulation commences. Therefore, EpiSC represent a valuable tool for studying mammalian postimplantation development in vitro. Beyond their pluripotent features, EpiSC can also be reprogrammed into a mouse embryonic stem cell-like (mESC) state. Published reports showed that EpiSC reversion requires transcription factor overexpression, while others suggest that applying stringent mESC culture conditions alone was sufficient to revert EpiSC to mESC-like cells. In an effort to clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. Indeed, the different lines shared a number of defining EpiSC characteristics such as teratoma formation ability, regardless of the day of isolation from the embryo. However, despite being grown in identical, self-renewal-promoting conditions, some lines displayed strong expression of genes associated with mesendodermal differentiation. Strikingly, these same lines were not revertable to a mESC-like state by applying stringent mESC culture conditions. Moreover, expression of mesendodermal marker genes correlated in a negative manner with the ability to efficiently undergo neural induction. Overall, our data suggest that different EpiSC lines may self-renew in distinct developmental ground states, which has important consequences for functional readouts such as revert ability to a mESC-like state and directed differentiation.
For EpiSC lines derivation E5.5 embryos were plated onto irradiated mouse embryonic feeder cells (MEF cells) seeded at low density. Outgrowths were picked and expanded manually. Basal medium consisted of Knock-Out-DMEM, 20% Knock-Out Serum Replacement, Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco). For cultivation on feeders 5ng/ml bFGF (Peprotech) was added to the medium to yield EpiSC medium. For preparation of mouse embryonic fibroblast-conditioned medium, EpiSC medium incubated on inactivated MEFs, followed by addition of another 5ng/ml bFGF (CM+). For feeder free culture, EpiSCs were seeded onto FCS-coated dishes. EpiSC were routinely passaged using 1mg/ml Collagenase IV (Invitrogen). Mouse ESC were cultured in standard conditions - Knock-Out-DMEM, 15% Knock-Out Serum Replacement (both Gibco), 5% fetal calf serum (Biowest), Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco), supplemented with either homemade or commercially available LIF (ESGRO) at 1000U/ml. Some experiments were carried out in chemically defined N2B27 medium 11 in the presence of 10ng/ml bFGF. For reversion experiments, EpiSC were harvested using Accutase (Invitrogen) and seeded as single cells at approximately 16.000 cells / well of a 6-well plate (Sarstedt) in CM+. After 24h medium was changed to PCL medium (basal medium, LIF (1000U/ml), PD0325901 (Axon Medchem 1µM), and CHIR99021 (Axon Medchem 3µM). After 48h treatment, reversion medium was further supplemented with 10µM SB431542 (Sigma) to eliminate remaining EpiSC.
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| Overall design |
7 samples were analyzed:
OG2.1: OG2.1 EpiSC, 1 biological rep
OG2.2: OG2.2 EpiSC, 1 biological rep
E3: E3 EpiSC, 1 biological rep
E5: E5 EpiSC, 1 biological rep
T9: T9 EpiSC, 1 biological rep
C1a1: C1a1 EpiSC, 1 biological rep
mESC: R1 ESC, 1 biological rep
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| Contributor(s) |
Bernemann C, Greber B, Ko K, Sterneckert J, Han DW, Araúzo-Bravo MJ, Schöler HR |
| Citation(s) |
21898681 |
| Submission date |
Mar 30, 2011 |
| Last update date |
Jan 15, 2022 |
| Contact name |
Marcos J. Araúzo-Bravo |
| E-mail(s) |
mararabra@yahoo.co.uk
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| Phone |
+34 943 00 6108
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| Organization name |
Max Planck Institute for Molecular Biomedicine
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| Department |
Cell and Developmental Biology
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| Lab |
Computational Biology and Bionformatics
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| Street address |
Rogentstrasse
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| City |
Muenster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
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| Platforms (1) |
| GPL6885 |
Illumina MouseRef-8 v2.0 expression beadchip |
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| Samples (7)
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| Relations |
| BioProject |
PRJNA139269 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE28270_RAW.tar |
3.1 Mb |
(http)(custom) |
TAR |
| GSE28270_non-normalized.txt.gz |
1.2 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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