Unknown,Transcriptomics,Genomics,Proteomics

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Global expression profiling of newly derived EpiSC lines

ABSTRACT: To characterize molecular features of new EpiSC lines established by the Wnt inhibition method, global gene expression profiles of the EpiSC lines were determined by microarray, and compared to those of EpiSC lines established by other group using the conventional method. Epiblasts, the source of the EpiSCs, and mESCs were also analyzed. EpiSCs and mESC were maintained as undifferentiated state on feeder layers. The stem cells were then separated from feeders, and RNAs were extracted from the stem cell lines. Embryonic tissues were manually dissected out from mouse embryos of E5.5, E6.5 or E7.5, from which RNAs were extracted.

ORGANISM(S): Mus musculus

SUBMITTER: Kuniya Abe

PROVIDER: E-GEOD-58735 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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 10ÂµM SB431542 (Sigma) to eliminate remaining EpiSC.

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Global expression profiling of newly derived EpiSC lines

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