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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Stage-specific regulation of reprogramming to iPSCs by Wnt signaling and Tcf proteins

ABSTRACT: Wnt signaling is intrinsic to mouse embryonic stem cell self-renewal. Therefore it is surprising that reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is not strongly enhanced by Wnt signaling. Here, we demonstrate that active Wnt signaling inhibits the early stage of reprogramming to iPSCs, while it is required and even stimulating during the late stage. Mechanistically, this biphasic effect of Wnt signaling is accompanied by a change in the requirement of all four of its transcriptional effectors: Tcf1, Lef1, Tcf3, and Tcf4. For example, Tcf3 and Tcf4 are stimulatory early but inhibitory late in the reprogramming process. Accordingly, ectopic expression of Tcf3 early in reprogramming combined with its loss-of-function late enables efficient reprogramming in the absence of ectopic Sox2. Together, our data indicate that the step-wise process of reprogramming to iPSCs is critically dependent on the stage-specific control and action of all four Tcfs and Wnt signaling. For genome wide expression analysis: A. Tcf3 wt and ko OSK iPS cells were analyzed (ko was performed from 2 different cell lines). B. Tcf3 ko OCK iPS cells were analyzed (2 different cell lines) C. Tcf3 wt and ko OCK reprogramming timecourse was analyzed at the indicated days after the beginning of the reprogramming. D. OCK partial iPS cell clone treated with control siRNA or 2 independent Tcf3 siRNA and RNA were analyzed 48hr after siRNA treatment.

ORGANISM(S): Mus musculus

SUBMITTER: Kathrin Plath

PROVIDER: E-GEOD-46532 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Stage-specific regulation of reprogramming to iPSCs by Wnt signaling and Tcf proteins

Project description:Wnt signaling is intrinsic to mouse embryonic stem cell self-renewal. Therefore it is surprising that reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is not strongly enhanced by Wnt signaling. Here, we demonstrate that active Wnt signaling inhibits the early stage of reprogramming to iPSCs, while it is required and even stimulating during the late stage. Mechanistically, this biphasic effect of Wnt signaling is accompanied by a change in the requirement of all four of its transcriptional effectors: Tcf1, Lef1, Tcf3, and Tcf4. For example, Tcf3 and Tcf4 are stimulatory early but inhibitory late in the reprogramming process. Accordingly, ectopic expression of Tcf3 early in reprogramming combined with its loss-of-function late enables efficient reprogramming in the absence of ectopic Sox2. Together, our data indicate that the step-wise process of reprogramming to iPSCs is critically dependent on the stage-specific control and action of all four Tcfs and Wnt signaling.

2013-05-01 | GSE46532 | GEO

Role of the reprogramming factors in inducing pluripotency

Project description:Induced pluripotent stem (iPS) cells can be obtained from fibroblasts by expression of Oct4, Sox2, Klf4, and c-Myc. To determine how these factors induce this change in cell identity, we carried out genomewide promoter analysis of their binding in iPS and partially reprogrammed cells. Most targets in iPS cells are shared with ES cells and the factors cooperate to activate the ES-like expression program. In partially reprogrammed cells, genes bound by c-Myc have achieved a more ES-like binding and expression pattern. In contrast, genes that are co-bound by Oct4, Sox2, and Klf4 in ES cells and that encode pluripotency regulators show severe lack of both binding and transcriptional activation. Among the factors, c-Myc has a pivotal effect on the initiation of the ES transcription program, including the repression of fibroblast-specific genes. Our analysis begins to unravel how the four factors function together and suggests a temporal and separable order of their effects during reprogramming. For genome wide expression analysis: A. ES/iPS/partial iPS cells were analyzed in duplicates from 2 different cell lines (e.g four samples per cell type) B. tet inducible factors (tetO/tetS/tetC/tetK) were analyzed individually, a control was an uninduced tet line. C. OCK expression was performed in duplicate with 2 different clones. For genome wide location analysis (ChIP-chip): Oct4/Sox2/c-Myc/Klf4 were performed with biological duplicates (2 clonally isolated iPS/partial iPS lines and ES cells were performed with v6.5 and E14 cell lines)

2010-05-06 | E-GEOD-14012 | biostudies-arrayexpress

Transcriptome of iPSC-derived Cerebral Organoids with Heterozygous Knockout in CHD8

Project description:CHD8 (chromodomain helicase DNA binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is the most commonly mutated gene in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities, and affects cancer cell proliferation. To better understanding the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout (KO) one copy of CHD8 in induced pluripotent stem cells (iPSCs) and build cerebral organoids, a model for the developing telencephalon. RNA-seq was carried out on KO organoids (CHD8+/-) and isogenic controls (CHD8+/+). Differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, forebrain development, Wnt/β-catenin signaling and axonal guidance. The SZ and bipolar disorder (BD) candidate gene TCF4 was significantly upregulated. Our CHD8 KO DEGs were significantly overlapped with those found in a transcriptome analysis using cerebral organoids derived from a family with idiopathic ASD and another transcriptome study using iPS cell-derived neurons from patients with BD, a condition characterized in a subgroup of patients by dysregulated WNT/β-catenin signaling. Overall, the findings show that distinct ASD, SZ and BD candidate genes converge on common molecular targets - an important consideration for developing novel therapeutics in genetically heterogeneous complex traits.

2017-02-16 | GSE85417 | GEO

Transcriptional responses to Wnt pathway stimulation in mouse embryonic stem cells cultured in serum+LIF condition

Project description:The objective of this study was to identify genes regulated by canonical Wnt signaling in mouse embryonic stem cells (ESCs).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 ?-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 ?-catenin mutants, our findings suggest that Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by ?-catenin entry into this complex. Wnt pathway stimulation also triggers ?-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 promote mouse ESC differentiation. Triplicates of mouse embryonic stem cells cultured with GSK3 inhibitor CHIR99021 or with Wnt pathway inhibitor XAV939.

2013-03-01 | E-GEOD-43420 | biostudies-arrayexpress

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Project description:We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells. Small RNA profiles of MEF, ES, iPS, NT-ES and NT-iPS cells were generated by Solexa sequencing. MEF and ES cells were performed in triplicate. iPS, NT-ES and NT-iPS cells were sequenced in duplicate.

2014-07-05 | E-GEOD-52950 | biostudies-arrayexpress

Gene expression transcriptome of porcine induced pluripotent stem cell

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2013-11-21 | E-GEOD-48434 | biostudies-arrayexpress

RNA sequencing analyses of fused heterokaryons between mouse embryonic stem cells and human B lymphocytes collected at early timepoints after fusion

Project description:Tcf3 is an effector of Wnt canonical pathway and deletion of Tcf3 in mouse embryonic stem cells (mESCs) strongly enhances reprogramming efficiency after fusion. We fused murine Tcf3-/- mESCs with human B-lymphocytes using the Polyethylene glycol (PEG) reagent and isolated heterokaryons at different time points to analyze early reprogramming events starting from 4 hours after fusion. We performed a genome-wide expression profile of the heterokaryons by RNA-sequencing at a high resolution.

2020-11-12 | GSE114238 | GEO

ChIP-seq analysis of M-NM-2-catenin in mouse embryonic stem cells treated with GSK3 inhibitor CHIR99021 under serum+LIF standard condition.

Project description:The objective of this study was to identify the direct target genes of M-NM-2-catenin acting downstream of canonical Wnt signaling in mouse embryonic stem cells (ESCs).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 M-NM-2-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 M-NM-2-catenin mutants, our findings suggest that Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by M-NM-2-catenin entry into this complex. Wnt pathway stimulation also triggers M-NM-2-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 promote mouse ESC differentiation. bCatenin ChIP-seq using anti-FLAG antibody and Streptavidin were otained from embryonic stem cells treated by CHIR99021 for 24 hours. Input without IP process were used as the control.

2013-03-01 | E-GEOD-43565 | biostudies-arrayexpress

Transcriptional responses to GSK3 inhibitor and MEK inhibitor on 2i-adapted mouse embryonic stem cells

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

2013-03-01 | E-GEOD-43421 | biostudies-arrayexpress

Transcriptional analysis of iPSCs during cellular reprogramming using RNA FACS

Project description:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc for 32 consecutive days. iPSCs were isolated by RNA FACS (endogenous Sox2) from the heterogeneous reprogramming culture and transcriptionally compared with (1) iPSCs stabilized in 2i medium (iPS-2i) and (2) iPSCs stabilized in co-culture with mouse embryonic fibroblasts (iPS-MEF) n = 2 replicates for each sample/condition

2014-03-20 | E-GEOD-55671 | biostudies-arrayexpress

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