Project description:To characterize the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed microarray analysis of Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
Project description:Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signalling control the formation of both the PSM and the somites, and show a graded distribution with highest levels in the posterior PSM. We have employed reporter for the PSM control gene Tbx6 to investigate the differentiation of mouse ESCs from the naĂŻve state to PSM state. Feeder-free ESCs were seeded and grown on fibronectin (3-5 ng/ml)-coated plates in ESC medium containing LIF, PD0325901 and CHIR99021 (2i medium) to bring them to a naive state of pluripotency. Following this, the cells were brought to an EpiSC state by treating them with Activin A and FGF2. The EpiSC-like cells were then differentiated to PSM using CHIR99021 either alone or along with FGF ligands, FGF2 or FGF8 (Low concentration: 10 ng/ml or High concentration: 250ng/ml).
Project description:To investigate the molecular mechanisms underlying the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
Project description:During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak (PS). However, it is unknown whether this restriction accompanies, at the single cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of Epiblast Stem Cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, EpiSCs express various early lineage-specific markers in self-renewing conditions. However, it is unknown whether cells that express these markers are pluripotent, spontaneously differentiated, or biased towards specific lineages. Here we show that EpiSC are inherently heterogeneous and contain two major and mutually exclusive subpopulations with PS and neural characteristics respectively. Using differentiation assays and embryo grafting we demonstrate that PS-like EpiSCs are biased towards mesoderm and endoderm differentiation but they still retain their pluripotent character. The acquisition of a PS character by undifferentiated EpiSC is mediated by paracrine Wnt signalling. Elevation of Wnt activity promotes further restriction into PS-associated cell fates which occurs via the generation of distinct clonal mesendodermal and neuromesodermal precursors. Collectively, our data suggest that primed pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula-stage epiblast. Total RNA obtained (3 biological replicates) from flow sorted dsRed2+, dsRed2-, and +CHIRON treated for 48h cells was isolated and labelled/amplified using the Illumina® TotalPrep™ RNA Amplification Kit (Life Technologies)
Project description:This study describes the transcriptome profiling of: 1) mouse ES cells and EpiSCs in LIF/serum-free (KSR) medium; 2) E14Tg2a (E14) ES cells in LIF/Serum with or without MM401 treatment; 3) rES reverted form EpiSC by MM401/LIF KSR treatment at P6, P30. RNA-Seq profiling on mouse pluripotent cells. Biological duplicates of each sample are labled as rep1/2.
Project description:The comparison of mouse ESCs and ESD-EpiSCs was done using three biological replicates for each cell type. ESC samples (ESR1 p10, p20, ESR1/E p15) and ESD-EpiSC samples (EpiSC-7 p20, EpiSC-5 p22, and EpiSC-7 p25) were used for microarray analysis. Total RNA was extracted using Trizol and labeled and hybridized to Agilent Whole Mouse Genome Oligo 4X44K Microarrays (one-color platform) according to the manufacturers protocols.
Project description:Epiblast stem cells (EpiSCs) are pluripotent cells that can be isolated and cultured from post implantation embryos. In contrast to embryonic stem cells (ESCs), systematic studies to investigate the genes that maintain pluripotency in EpiSCs have not been reported. Here we combine a genome-wide RNAi screen with genetic interaction, protein localization and protein-level dependency studies to delineate connectivity between factors that control Oct4 expression in EpiSCs and compare the role of these factors to their function in ES cells. We demonstrate the power of this integrative approach by the identification of Tox4 as an interactor of PP1 (Protein Phosphatase 1) and Paf1C, a complex that acts in multiple aspects of RNAPII regulation. Our results indicate that Tox4 cooperates with Paf1C and PP1 to influence the phosphorylation status of the RNAPII CTD tail during transcription and that this function is vital for maintenance of pluripotent cell identity. RNA-seq of Tox4 knockdown in mouse EpiSCs
Project description:Human pluripotent cell lines were derived from blastocyst-stage embryos and propagated in self-renewal conditions that maintain features of naive pluripotency characteristic of mouse embryonic stem cells. Genomic integrity of the HNES1 cell line was assessed with the Affymetrix CytoScan 750K array.
Project description:Epiblast stem cells (EpiSCs) were placed in the epiblastic cell maintenance condition (EpiSC) or in a neural plate developmental condition (Iwafuchi-Doi et al. Dev Biol 352, 354-366, 2011) for one (NPC1) or two (NPC2) days, and expression profiles of total mRNAs were compared. Duplicate EpiSC cultures (A and B) originating from the same seed culture and made in the above conditions were processed for total RNA extraction using TRI Reagent (Sigma-Aldrich), and analyzed
Project description:Here we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed RNA-sequencing experiments and examined the global gene expression profiles of EpiSCs, rsEpiSCs, in vivo isolated four regions of E6.5 mouse epiblasts: AP (anterior-proximal), AD (anterior-distal), PP (posterior-proximal) and PD (posterior-distal), human H1 ESCs, H1 rsESCs, H9 ESCs, H9 rsESCs, rhesus monkey ORMES23 rsESCs, and chimpanzee rsiPSCs. Examination of global gene expression profiles in 2 pluripotent stem cell types across multiple species.