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Gene expression in reprogramming MEF fractions FACS-sorted by Oct4, Klf4 and EpCAM expression

ABSTRACT: After 8 days of OKSM induction via doxycycline, Nanog-Neo secondary MEFs (Wernig et al. Nature Biotechnology 2008) were FACS sorted by KLF4, Oct4, and EpCAM expression. Four major subsets of MEFs have been sorted and analysed for gene expression.

ORGANISM(S): Mus musculus

PROVIDER: GSE56764 | GEO | 2014/08/01

SECONDARY ACCESSION(S): PRJNA244507

REPOSITORIES: GEO

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Gene expression in reprogramming MEF fractions FACS-sorted by Oct4, Klf4 and EpCAM expression

Project description:After 8 days of OKSM induction via doxycycline, Nanog-Neo secondary MEFs (Wernig et al. Nature Biotechnology 2008) were FACS sorted by KLF4, Oct4, and EpCAM expression. Four major subsets of MEFs have been sorted and analysed for gene expression. 4 cell types

2014-08-01 | E-GEOD-56764 | biostudies-arrayexpress

EpEX/EpCAM and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2α.

Project description:Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2α. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2α signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.

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Small molecules facilitate rapid and synchronous iPSC generation

Project description:The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations. 18 samples were analyzed in total, samples represent bulk cultures of reprogrammable-MEFs (Rep-MEFs) that express OKSM and were supplemented with ascorbic acid and GSK3i during iPS cell induction. Control samples represent similar bulk cultures of reprogrammable-MEFs that express OKSM but were not supplemented with ascorbic acid and GSK3i during iPS cell induction. GMP-iPSCs were generated using 48 hours of OKSM+AGi induction. Fibroblast-iPSCs were generated using 96 hours of OKSM+AGi induction.

2014-09-23 | E-GEOD-57774 | biostudies-arrayexpress

Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

Project description:Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO).

2010-12-31 | GSE21271 | GEO

Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

Project description:Histone profile for wild type MEF and secondary MEF with Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO). ChIP-Seq data for H3K4me3, H3K27me3 and H3K9me3

2010-12-31 | E-GEOD-21271 | biostudies-arrayexpress

Counteracting activities of OCT4 and KLF4 during reprogramming to pluripotency.

Project description:Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.

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PTIP-regulated genes in MEF

Project description:PPARg and C/EBPa cooperate to control preadipocyte differentiation (adipogenesis). However, the factors that regulate PPARg and C/EBPa expression during adipogenesis remain largely unclear. Here we show PTIP, a protein that associates with histone H3K4 methyltransferases, regulates PPARg and C/EBPa expression in mouse embryonic fibroblasts (MEFs) and during preadipocyte differentiation. PTIP deletion in MEFs leads to marked decreases of PPARg expression and PPARg-stimulated C/EBPα expression. Further, PTIP is essential for induction of PPARg and C/EBPa expression during preadipocyte differentiation. Deletion of PTIP impairs the enrichment of H3K4 trimethylation and RNA polymerase II on PPARg and C/EBPa promoters. Accordingly, PTIP-/- MEFs and preadipocytes all show striking defects in adipogenesis. Furthermore, rescue of the adipogenesis defect in PTIP-/- MEFs requires co-expression of PPARg and C/EBPa. Finally, deletion of PTIP in brown adipose tissue significantly reduces tissue weight in mice. Thus, by regulating PPARg and C/EBPa expression, PTIP plays a critical role in adipogenesis.

2009-05-29 | GSE16263 | GEO

An anti-inflammatory activation sequence governs macrophage transcriptional dynamics during tissue injury in zebrafish

Project description:Single cell RNA sequencing on FACS sorted cells from the zebrafish macrophage in 5df embryos in 11 different samples: homeo, 15min neomycin, 1hr post-neo, 3hr post-neo, 5hr post-neo, il10ra_sib_homeo, il10ra_sib_1hr, il10ra_sib_3hr, il10ra_mut_homeo, il10ra_mut_1hr and il10ra_mut_3hr

2022-07-27 | GSE209884 | GEO

Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.

Project description:The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

| S-EPMC4825772 | biostudies-literature

BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone.

Project description:Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ?1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells.

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