The Ultraconserved Long Noncoding RNA, T-UCstem1, controls proliferation and safeguards self-renewal of embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE108662 | GEO | 2018/03/15
REPOSITORIES: GEO
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Project description:Pluripotent mouse embryonic stem cells (ESCs) were originally derived and stably maintained on feeder cells such as inactivated mouse embryo fibroblasts, and can generate complete ESC-pups by tetraploid embryo complementation (TEC), the most stringent functional test of naive pluripotency. Remarkably, 2i (inhibitors of Mek and Gsk3β signaling) medium with LIF in the absence of serum and feeders was developed to achieve ground state of mouse ESCs, and also has been successfully used for derivation of germline competent ESCs in other species such as rat. Notably, 2i-culture gives rise to transcriptional profiles and epigenetic landscapes quite distinct from serum-based ESCs. To better understand transcriptional landscape and signal transductions, we performed RNA-seq analysis of ESCs cultured in four different conditions: serum without feeder, serum with feeder, serum with feeder and 2i, and N2B27+2i.
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Project description:DAX1 (also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell lines could be established in 2i/LIF and their pluripotency was confirmed. In contrast, Dax1-null ESCs could not be continuously passaged in LIF/serum due to severe differentiation and apoptosis. In LIF/serum, the activities of the Core module and Myc module were significantly reduced, while the PRC2 module was activated after Dax1KO. The expression of most pro-apoptotic genes and lineage-commitment genes were drastically increased, while the down-regulated expression of anti-apoptotic genes and many pluripotency genes was observed. Our research on the pluripotent state-dependent role of Dax1 provides clues to understand the molecular regulation mechanism at different stages of early embryonic development.
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Project description:Co-suppression of Gsk3 and Mek1/2 signaling (“2i culture”) in mouse embryonic stem cells (ESCs) induces a naïve state that more closely resembles the inner cell mass (ICM) of the pre-implantation embryo, including global DNA hypomethylation. While the ICM exists only transiently in vivo, it remains unclear how continuous suppression of Mek1/2 and Gsk3 signaling and associated hypomethylation affect the stability and functionality of ESCs in vitro. Here we show that, compared to ESCs maintained in conventional serum/LIF media, culturing male ESCs in 2i/LIF for >6 passages results in the erosion of genomic imprints and a concomitant impairment in the ability to generate adult, entirely ESC- derived mice (all-ESC mice). We further demonstrate that female ESCs cultured in serum/LIF phenocopy male ESCs cultured in 2i/LIF, including global hypomethylation, imprint erosion and impaired developmental potential. Unexpectedly, culture of female ESCs in serum/LIF and male ESCs in 2i/LIF for >16 passages causes recurrent chromosomal aberration that have been associated with a selective growth advantage. We provide evidence that inhibition of Mek1/2, but not Gsk3, is responsible for hypomethylation, imprint loss and chromosomal instability in ESCs, and identify alternative conditions that preserve epigenetic integrity. Taken together, our data suggest that while short-term suppression of Mek1/2 in ESCs captures an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise developmental potential.
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Project description:The pluripotency of mouse embryonic stem cells (ESC) and induced-pluripotent stem cells (iPSC) can be maintained by feeder cells, which secrete Leukemia Inhibitory Factor (LIF). We found that feeder cells provide a relatively low concentration (25 unit/ml) of LIF, which is insufficient to maintain the ESC/iPSC pluripotency in feeder free conditions. In order to identify additional factors involved in the maintenance of pluripotency, we carried out a global transcript expression profiling of mouse iPSC cultured on feeder cells and in feeder-free (LIF-treated) conditions. This identified 17 significantly differentially expressed genes (adjusted p-value<0.05) including 7 chemokines over-expressed in iPSC grown on feeder cells. Ectopic expression of these chemokines in iPSC revealed that CC chemokine ligand 2 (Ccl2) induced the key transcription factor genes for pluripotency, Klf4, Nanog, Sox2 and Tbx3. Further, addition of recombinant Ccl2 protein drastically increased the number of Nanog-GFP-positive iPSC grown in low LIF feeder free conditions. Interestingly, this effect was not observed in the absence of LIF. We further revealed that pluripotency promotion by Ccl2 is mediated by activating the Stat3-pathway followed by Klf4 up-regulation. We demonstrated that Ccl2 mediated increased pluripotency is independent of PI3K and MAPK pathways and that Tbx3 may be directly up-regulated by Klf4. Overall, Ccl2 cooperatively activates the Stat3-pathway with LIF in feeder free condition to maintain pluripotency for ESC/iPSC.
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Project description:The pluripotency of mouse embryonic stem cells (ESC) and induced-pluripotent stem cells (iPSC) can be maintained by feeder cells, which secrete Leukemia Inhibitory Factor (LIF). We found that feeder cells provide a relatively low concentration (25 unit/ml) of LIF, which is insufficient to maintain the ESC/iPSC pluripotency in feeder free conditions. In order to identify additional factors involved in the maintenance of pluripotency, we carried out a global transcript expression profiling of mouse iPSC cultured on feeder cells and in feeder-free (LIF-treated) conditions. This identified 17 significantly differentially expressed genes (adjusted p-value<0.05) including 7 chemokines over-expressed in iPSC grown on feeder cells. Ectopic expression of these chemokines in iPSC revealed that CC chemokine ligand 2 (Ccl2) induced the key transcription factor genes for pluripotency, Klf4, Nanog, Sox2 and Tbx3. Further, addition of recombinant Ccl2 protein drastically increased the number of Nanog-GFP-positive iPSC grown in low LIF feeder free conditions. Interestingly, this effect was not observed in the absence of LIF. We further revealed that pluripotency promotion by Ccl2 is mediated by activating the Stat3-pathway followed by Klf4 up-regulation. We demonstrated that Ccl2 mediated increased pluripotency is independent of PI3K and MAPK pathways and that Tbx3 may be directly up-regulated by Klf4. Overall, Ccl2 cooperatively activates the Stat3-pathway with LIF in feeder free condition to maintain pluripotency for ESC/iPSC. Total RNAs were purified from feeder cells (as a reference sample), iPSC grown on feeder cells and iPSC grown in feeder-free condition in triplicates and applied to the arrays. iPSC grown on feeder cells were separated mechanically from the feeder layer to minimize feeder cell contamination.
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Project description:The purpose of the study was to identify transcriptomic changes between feeder-free ES and XEN cells. ES cells were cultured feeder-free on 0.1% gelatin in 2iLIF in N2B27 media. XEN cells were also cultured feeder-free on 0.1% gelaton in RPMI1640 supplemented with 15% FBS, 2mM GlutaMAX and BME.
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