Trascriptome profiling of murine Embryonic Stem cells under different culture conditions
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ABSTRACT: Mouse Embryonic Stem (ES) cells have been cultured under chemically defined conditions in the absence of feeder cells on Gelatin coated dishes, for 5 passages under 4 different combinations of LIF and GSK3 or MEK inhibitors. Their trascriptome have been analysed by RNA sequencing. The ES cell line used, RexGFPd2, contains a destablised GFP protein in the Zfp42/Rex1 locus and was derived under 2i conditions, as described in Kalkan et al. 2017.
Project description:Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Differentiation of Rex1-GFPd2 ES cells was initiated by withdrawing 2i (Kalkan et al., 2016). Undifferentiated 2i-cells and post-2i withdrawal differentiating populations (16h, 25h-Rex1-High, 25h-Rex1-Low) were subjected to proteomic analysis by Mass Spectrometry.
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
Project description:Identification of transcripts in murine embryonic stem (ES) cell lines growing under 3 different self-renewing conditions; GMEM + 10% FCS, N2B27 + 2i (1 μM PD032 and 3 μM CHIR99021) and knockout serum replacement (KOSR) medium (80% Knockout DMEM, 20% Knockout Serum Replacement). Under all conditions, ES cells were grown on gelatin-coated dishes and passaged by trypsinisation. ES cells were cultured in each condition for at least 3 passages prior to sample collection. The aim of this array experiment is to identify significant differences in transcript levels of ES cells grown under different conditions. Differences of transcript levels from the different conditions should be consistent among the biological and technical replicates for each.
Project description:ES cell lines were established from mouse embryos, which were homozygous for the Trim33-flox allele and carried the UbcCreERT2 transgene. Cells were cultured without feeder cells in the presence of LIF and 2i. Embryoid bodies (EBs) were generated using the ATCC protocol on low attachment dishes under differentiating conditions. EBs were induced with Tamoxifen at day 4 and harvested at day 7.
Project description:ES cell lines were established from mouse embryos, which were homozygous for the Trim33-flox allele and carried the UbcCreERT2 transgene. Cells were cultured without feeder cells in the presence of LIF and 2i. Embryoid bodies (EBs) were generated using the ATCC protocol on low attachment dishes under differentiating conditions. EBs were induced with Tamoxifen at day 1 and harvested at days 2 and 2.5, respectively.
Project description:We report the application of single-cell and bulk RNA sequencing technology to examine the noncoding transcriptome of cells undergoing reprogramming to the pluripotent state. Examination of noncoding RNAs in reprogrammming cells. We examined iPS cells grown in standard ES cell culture conditions, as well as iPS cells grown in "2i" conditions (small molecule inhibition of Mek and Gsk3). We also compared our iPS samples to male and female ES cells (mES, fES).