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CC chemokine ligand 2 and LIF cooperatively promote pluripotency in mouse induced pluripotent cells

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE28262 | GEO | 2011/06/01

SECONDARY ACCESSION(S): PRJNA139791

REPOSITORIES: GEO

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CC chemokine ligand 2 and LIF cooperatively promote pluripotency in mouse induced pluripotent cells

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.

2011-06-01 | E-GEOD-28262 | biostudies-arrayexpress

Nanog Independent Reprogramming to iPSCs with Canonical Factors

Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line. 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)

2014-01-01 | E-GEOD-53212 | biostudies-arrayexpress

Genome-wide maps of Tbx3 binding sites in mouse ESCs

Project description:Induced pluripotent stem (iPS) cells can be obtained through the introduction of defined factors into somatic cells. The combination of Oct4, Sox2 and Klf4 (OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts (MEFs). Through the genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, we identified Tbx3 as a transcription factor that significantly improves the quality of iPS cells. Induced-PS cells generated with OSK + Tbx3 (OSKT) are superior in both germ cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide ChIP-sequencing analysis of Tbx3 binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. ChIP-seq of Tbx3 binding in mouse ESCs

2009-12-04 | E-GEOD-19219 | biostudies-arrayexpress

LIF signaling-dependent gene expression

Project description:Leukemia Inhibitory Factor (LIF) plays an essential role in the maintenance of pluripotency of mouse embryonic stem cells (mESCs). LIF withdrawal induces mESC differentiation. To define noval pluripotent factors downstream of LIF signaling, cDNA microarray was used and seveal well-known pluripotent genes were found to respond to LIF withdrawal, including Klf4, Esrrb, Tbx3, and Prdm14.

2015-06-23 | GSE70104 | GEO

Nanog Independent Reprogramming to iPSCs with Canonical Factors

2014-01-01 | GSE53212 | GEO

Genome-wide maps of Tbx3 binding sites in mouse ESCs

2009-12-04 | GSE19219 | GEO

LIF signaling-dependent gene expression

2015-06-23 | E-GEOD-70104 | biostudies-arrayexpress

Genome stability in different mouse embryonic stem cells [exome-seq]

Project description:Naïve mouse ESCs exhibit full term developmental competence thus hold great potential in regenerative medicine. Maintaining genome stability is essential for potential application of ESCs in stem cell therapy. While ESC potency fluctuate with retrovirus activity, it is unclear whether long-term cultures affect retrotransposition and genomic stability. We compared retrotransposons in naïve ESCs using various approaches. We show that feeder-serum based culture conditions and small molecule LCDM maintain appropriate expression of retrovirus activity following long-term cultures, whereas cultures under 2iLif and a2i result in aberrant upregulation of retrotransposons. This leads to high frequent retrotransposition. Moreover, naïve ESCs on feeder-serum based culture conditions and small molecule LCDM still generate complete ESC pups by TEC assay, functional test of pluripotency, following long-term cultures, despite that all culture conditions maintain high expression levels of pluripotent genes such as Oct4, Nanog, Klf4 and Sox2. Meanwhile, long telomeres are maintained in ESCs under cultures in Feeder-serum and LCDM conditions but telomeres shortened in other conditions with increasing passages. These data provide insights into retrotransposition, genomic stability and pluripotency of naïve ESCs.

2023-09-12 | GSE157875 | GEO

Genome stability in different mouse embryonic stem cells

2023-09-12 | GSE157874 | GEO

Genome stability in different mouse embryonic stem cells [ATAC-seq]

2023-09-12 | GSE157873 | GEO