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Cell line dependent gene expression profiles of two mouse embryonic stem cells

ABSTRACT: Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ x 129S1) blastocyst (Nagy et al. 1993 PNAS 90: 8424-8428) and HM-1 ESC established from inbred strain (Selfridge et al. 1992 Somat Cell Mol Genet 18: 325-336), as our earlier study showed performance variations between these cells. ES cells were cultured on mitomycin C treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (Gibco-Invitrogen, Carlsbad, CA) supplemented with Na-pyruvate (0.11% w/w; Gibco-Invitrogen), 0.1mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), fetal bovine serum (15% v/v; HyClone; Logan,UT), 1000 U/ml murine-LIF (CHEMICON International, Temecula, CA) and antibiotics (penicillin: 50 U/ml, streptomycin: 50 μg/ml (Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, Düsseldorf, Germany) procedures. 15 µg of total RNA each from the contrasting samples were used for reverse transcription and labeling either with Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer and added to the cDNA arrays (custom produced at GSF) containing over 21,000 sequences and hybridized for 17 hours at 42°C. The microarray results of four independent hybridizations were analyzed and differentially regulated genes were identified. Finally, the results of four randomly selected genes were verified by real time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (p < 0.01) between the two ES cell lines. Of these, 8 transcripts were up regulated and the rest down regulated in the HM-1 ES cells. Most of these genes were overrepresented in important biological processes like growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the two ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the two ES cell lines. Keywords: ES cell lines, murine, nuclear embryo derived, Nocodazol,

ORGANISM(S): Mus musculus

PROVIDER: GSE7173 | GEO | 2008/01/01

SECONDARY ACCESSION(S): PRJNA98245

REPOSITORIES: GEO

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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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Cell line dependent gene expression profiles of two mouse embryonic stem cells

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