Genome-wide analysis of gene expression in LIF and LIF plus 2I in C57BL/6 mouse embryonic stem cells (B6 mESCs)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE46369 | GEO | 2013/04/26
SECONDARY ACCESSION(S): PRJNA198855
REPOSITORIES: GEO
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Project description:Analysis of genes induced by 2I condition 2i contains glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK) inhibitors: 3uM Chir99021 and 1uM PD0325901 Total RNA obtained from B6 mESCs treated with LIF or LIF/2I for 12 hours.
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Project description:Analysis of genes induced by CHIR99021 CHIR99021 is a inhibitor of glycogen synthase kinase 3 (GSK3) and can promote B6 mESC sef-renewal when combined with LIF in serum condition.
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Project description:Analysis of genes induced by CHIR99021 CHIR99021 is a inhibitor of glycogen synthase kinase 3 (GSK3) and can promote B6 mESC sef-renewal when combined with LIF in serum condition. Total RNA obtained from B6 mESCs treated with LIF or LIF/CHIR99021 for 12 hours.
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Project description:The objective of this study was to investigate the roles of GSK3 inhibitor CHIR99021 and MEK inhibitor PD0325901 on 2i-adapted mouse embryonic stem cells (ESCs) in serum-free conditions.Canonical Wnt signaling supports the pluripotency of mouse ESCs but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of mouse ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Further, canonical Wnt signaling up-regulates the activity of the Pou5f1 distal enhancer via the Sox motif in mouse ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest that Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a MEK/ERK inhibitor essential for mouse ESC culture suggests that MEK/ERK signaling and canonical Wnt signaling combine to mouse promote ESC differentiation. Triplicates of mouse embryonic stem cells cultured under the following conditions: 1) CHIR99021+PD0325901+LIF; 2) CHIR99021+PD0325901; 3) CHIR99021; 4) PD0325901; 5) DMSO
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Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
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Project description:Mouse embryonic stem cells (ESCs) grown in the 2i (an ERK inhibitor, PD0325901, and a GSK-3b inhibitor, CHIR99021) mimic pluripotent epiblasts in preimplantation blastocysts. Tfcp2l1 is a transcription factor highly expressed in ESCs grown in the 2i medium, suggesting that it plays in important role in ESC biology. To search for the putative role(s) of Tfcp2l1 in ESCs, we plan to use an integrated genomic approach--combing RNA-seq and ChIP-seq--to identify the direct targets of Tfcp2l1 in ESCs. ChIP-seq is used to identify the genomic bindings sites of Tfcp2l1 in ESCs grown in the 2i medium.
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Project description:Mouse embryonic stem cells (ESCs) grown in the 2i (an ERK inhibitor, PD0325901, and a GSK-3b inhibitor, CHIR99021) mimic pluripotent epiblasts in preimplantation blastocysts. ESCs grown in FBS containing medium are more heterogenous in morphology and expression of stem cell factors. Tfcp2l1 is a transcription factor highly expressed in ESCs grown in the 2i medium, suggesting that it plays in important role in ESC biology. To search for the putative role(s) of Tfcp2l1 in ESCs, we plan to use an integrated genomic approach--combing RNA-seq and ChIP-seq--to identify the direct targets of Tfcp2l1 in ESCs. RNA-seq is used to identify the transcripts regulated by Tfcp2l1 in ESCs grown in the 2i medium.
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Project description:In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells. Whole gene expression study of 4 different mouse embryonic stem cell lines maintained under 4 different chemically defined conditions: R2i, 2i, PD and SB
2013-10-21 | E-GEOD-43682 | biostudies-arrayexpress
Project description:In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells.
2013-10-21 | GSE43682 | GEO
Project description:Human pluripotent cells were reset to ground state pluripotency by transient overexpression of NANOG and KLF2 and subsequent inhibition of ERK and protein kinase C. Transcriptional profiling of reset cells and conventional pluripotent stem cell cultures was carried out by RNA-seq, in tandem with mouse embryonic stem cells propagated under similar conditions to assess the combinatorial effects of MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021 and PKC inhibitor Go6983.
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