Excluding Oct4 from Yamanaka cocktail unleashes the developmental potential of iPSCs
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PROVIDER: PRJNA564252 | ENA |
REPOSITORIES: ENA
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Project description:Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs, and thereby bear major implications for further development and application of iPSC technology.
2019-11-06 | GSE137001 | GEO
Project description: Not available
| S-EPMC6900749 | biostudies-literature
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
Project description:The success of Yamanaka factor reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state to facilitate factor binding to target genes. We asked how the Yamanaka factor-dependent chromatin opening occurs. We found Oct4, but neither Sox2 nor Klf4, is responsible for the loosening of the condensed state. Oct4 acts as a pioneer factor that facilitates the binding of Klf4 and the expression of epithelial genes in the early stage of reprogramming.
2021-03-09 | GSE129728 | GEO
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 (for endogenous Sox2) from a 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)
2014-03-20 | GSE55671 | GEO
Project description:we generate iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28) and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identify Yamanaka-iPSC (Y-iPSCs)-specific and Thomson-iPSCs (T-iPSC)-specific recurrent aberrations. Bisulphite converted DNA from 9 iPS cells derived using Yamanaka factors (OSKM), 6 iPS cells derived using Thompson factors (OSLN), 2 parental fibroblasts and one embrionic ES cell were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
2014-01-16 | E-GEOD-54115 | biostudies-arrayexpress
Project description:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, and grown in standard 2i/serum media conditions. Hybridized iPSCs were fixed with 4% paraformaldehyde; incubated for 12 hours at 30C in an RNA preserving hybridization buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C in with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM pH 8.0 Tris, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K). RNA extracted from live and hybridized iPSCs (1 sample each) was compared by microarray.
2014-03-20 | E-GEOD-55672 | biostudies-arrayexpress
Project description:we generate iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28) and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identify Yamanaka-iPSC (Y-iPSCs)-specific and Thomson-iPSCs (T-iPSC)-specific recurrent aberrations.
2014-01-16 | GSE54115 | GEO
Project description:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, and then grown in standard 2i/serum media conditions. Hybridized iPSCs were fixed with 4% paraformaldehyde; permeabilized in 70% ethanol for over 12 hours; incubated for 12 hours at 30C in an RNA preserving hybridization buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K).
2014-03-20 | GSE55672 | GEO
Project description:Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4 and Myc (OSKM) into somatic cells. Though iPSCs are pluripotent, they frequently exhibit high variation in their quality as measured by chimera contribution and tetraploid (4n) complementation. Thus, improving the quality of iPSCs is an indispensable prerequisite for future iPSC-based therapy. Here we show that one major determinant for iPSCs quality is the combination of reprograming factor selected. Ectopic expression of Sall4, Nanog, Esrrb and Lin28 (SNEL) in MEFs efficiently generated high quality iPSCs as compared to other combinations of factors. SNEL-iPSCs produced approximately 5 times more efficiently “all-iPSC” mice compared to OSKM-iPSCs. While differentially methylated regions, transcript number of master regulators, establishment of ESC-specific super enhancers, and global aneuploidy were comparable between the lines, aberrant expression of 1,765 genes, trisomy of chromosome 8 and abnormal H2A.X deposition were frequently observed in poor quality OSK-iPSCs and OSKM-iPSCs. Whole-genome single-base resolution MethylC sequencing collected from a variety of Mouse pluripotent cells To reveal the DNA methylation signature associated with developmental competence, we selected the following groups of iPSC lines for whole-genome bisulfite sequencing: i) "Poor quality" iPSCs: This group included the three OSKM-iPSC lines Nanog-GFP OSKM#2, Oct4-GFP OSKM#2 and KH2 OSKM(Stadtfeld et al., 2010), that either did not produce fully developed pups or produced very low number of pups; ii) "Good quality" iPSCs: This group included BC_2 OSKM (Carey et al., 2011) and Nanog-GFP SNEL#3, both of which gave rise to live, normal pups that survived only few hours; iii) "High quality" iPSCs consisting of Nanog-GFP SNEL#2 and Oct4-GFP SNEL#1, both of which generated live pups that survived postnatally (representative pups from each iPSC group are shown in Figure S3A). iv) As controls we used Nanog-GFP, Oct4-GFP and KH2 (Beard et al., 2006) ESCs. Note that all cells were cultured in 2i medium but not in mES medium.
2014-07-30 | E-GEOD-59696 | biostudies-arrayexpress