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Molecular insights into induced pluripotency mediated by the OCT4, SOX2, KLF and c-MYC gene regulatory network

ABSTRACT: Human somatic fibroblasts can be reprogrammed to induced pluripotent stem (iPS) cells by exogenic expression of the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) after about 1 month. To gain some insight into the early processes operative in fibroblast reprogramming, we profiled genome-wide transcription levels using Illumina microarrays in the starting donor cells-human foreskin fibroblast (HFF1) cells and at three time points after OSKM transduction (24 h, 48 h, 72 h), as well as two iPS cell lines (iPS2, iPS4) and hES cell lines (H1, H9). We show that within the context of the viral transduction reprogramming protocol, the donor cell response to viral transfection perturbs redox homeostasis, which induces oxidative damage on the donor cells' protein and DNA. This leads to activation of p53, senescence, and apoptosis, greatly reducing the efficiency of reprogramming.

ORGANISM(S): Homo sapiens

PROVIDER: GSE28688 | GEO | 2011/10/25

SECONDARY ACCESSION(S): PRJNA139019

REPOSITORIES: GEO

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Molecular insights into induced pluripotency mediated by the OCT4, SOX2, KLF and c-MYC gene regulatory network

Project description:Human somatic fibroblasts can be reprogrammed to induced pluripotent stem (iPS) cells by exogenic expression of the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) after about 1 month. To gain some insight into the early processes operative in fibroblast reprogramming, we profiled genome-wide transcription levels using Illumina microarrays in the starting donor cells-human foreskin fibroblast (HFF1) cells and at three time points after OSKM transduction (24 h, 48 h, 72 h), as well as two iPS cell lines (iPS2, iPS4) and hES cell lines (H1, H9). We show that within the context of the viral transduction reprogramming protocol, the donor cell response to viral transfection perturbs redox homeostasis, which induces oxidative damage on the donor cells' protein and DNA. This leads to activation of p53, senescence, and apoptosis, greatly reducing the efficiency of reprogramming. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, OSKM-transduced HFF1 cells after 24h, 48h, 72h, undifferentiated hESCs, iPSCs.

2011-10-24 | E-GEOD-28688 | biostudies-arrayexpress

Aberrant DNA methylation reprogramming during iPS cell generation is dependent on the choice of reprogramming factors

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

Human Induced Pluripotent Stem Cells Harbor Homoplasmic and Heteroplasmic Mitochondrial DNA Mutations While Maintaining human embryonic stem cells-like Metabolic Reprogramming

Project description:Gene expression analyis of two hESCs, two human neonatal fibroblasts, and four human iPSCs generated with retroviral transduction using the OSKM cocktail. Total mRNA obtained from two fibroblasts (BJ and HFF1), two hESCs (H1 and H9), two BJ-derived iPSCs (iB4 and iB5), and two HFF1-derived iPSCs (iPS2 and iPS4).

2011-07-08 | E-GEOD-26575 | biostudies-arrayexpress

Gene expression profiles of murine fibroblasts and iPS

Project description:We reprogrammed adult murine fibroblasts obtained from p14f/f (wt version of conditional loxP-flanked p14 knockout (KO); C57/Bl6) by transduction of a lentiviral vector (pRRL.PPT.SF.hOKSMco.idTom.PRE) overexpressing the four Yamanaka factors (c-Myc, Klf-4, Oct-4, Sox-2). After successful reprogramming and establishment of stable iPS clones we performed gene expression profiling of three different murine iPS clones (#2, #2EX, #3) and their parental fibroblasts.

2018-03-01 | GSE97152 | GEO

Expression data from OSKM-mediated 2nd reprogramming cells and the corresponding iPS cell line

Project description:Forced expression of four transcription factors Oct4,Sox2, Klf4 and Myc (OSKM) induces somatic cell reprogramming towards pluripotency. Major efforts have been made to characterize the molecular events involved in this process. Yet, it remains elusive how gene expression change, epigenetic landscape remodelling and cell fate conversion are triggered by expression of these Yamanaka factors.To address this gap,we utilized a secondary inducible reprogramming system and performed genome-wide profilings of Oct4 binding, histone modification(H3K4me3/H3K27me3/H3K4me1/H3K27ac), and gene expression analysis during this process. Through integrative analysis, we revealed stage-specific Oct4 binding and enhancer signatures in consistence with gene expression changes,in which the initial regression of somatic program is followed by the gradual acquisition of pluripotent program. Oct4 preferatially binds to H3K4me1 marked enhancer regions and Oct4 binding is positively correlated with active mark H3K27ac. Moreover,we observed significant enhancer activation of epigenetic related genes, especially acetylation associated genes, prior to pluripotency network activation, suggesting a pivotal role of epigenetic remodelling in the process of pluripotency acquisition and maintenance. We used microarrays to explore the global changes of gene expression during OSKM-mediated somatic cell reprogramming. time series design with bulk population samples collected at different time point of 2nd reprogramming as well as a corresponding iPS cell line. Each sample include two replicates. 2nd mouse embryonic fibroblasts(sample day0) were treated with ES medium supplemented with 1Âµg/ml Doxcycline and 50Âµg/ml Vitamin C for 15 days (sample day1-day15); 72 hours after dox and Vc withdrawl, dox-independent iPS cells are collected(sample day18).

2015-12-31 | E-GEOD-67462 | biostudies-arrayexpress

Genome-wide analysis of pluripotency marker expression of ESKM-iPS cells

Project description:iPS cells, produced in in presence of exogeneously expressed E-cadherin and abcence of viral Oct4 were compared with conventional produced OSKM-iPS cells and murine embryonic stem cells (mESCs) or MEFs

2011-04-14 | GSE28594 | GEO

Deciphering in vivo reprogramming using single cell RNA sequencing in the pancreas

Project description:The manipulation of cell fates through reprogramming is one of the most exciting advances in recent years. The most striking breakthrough in the field occurred when Yamanaka first illustrated the possibility to convert differentiated cells into pluripotent stem cells by the ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc or OSKM. Our laboratory has demonstrated that transient expression of these four Yamanaka factors leads to teratoma formation in mice indicative of in vivo reprogramming. We were interested in deciphering the intermediate events of de-differentiation induced by OSKM in vivo before reaching pluripotency. For this purpose, we focused on the pancreas which we found to be the organ with the highest reprogramming efficiency in vivo. To gain an insight into the cellular heterogeneity generated by OSKM activation in vivo, we performed single cell RNA sequencing of partially reprogrammed pancreas. We found that in vivo reprogramming leads to the loss of pancreatic acinar identity and the acquisition of an atypical tubular morphology different from normal pancreatic ducts or from the metaplastic ducts produced by pancreatitis. We were able to capture cells that have lost their acinar identity and they have acquired alternative cell fates. This analysis provide us with a list of markers characteristic of the intermediate stages of OSKM reprogramming.

2022-04-06 | GSE188819 | GEO

The developmental potential of iPSCs is greatly influenced by the selection of the reprogramming factors (SNEL iPSC and H2A.X deposition pattern)

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 selection of the reprogramming factors combination. 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 OSKM-iPSCs. For high-quality iPSCs, H2A.X pattern of SNEL is most similar to that of ESC, OSK and OSKM have more devoid regions than SNEL iPSCs. Compare H2A.X deposition pattern of the OSKM 4-factor iPS cell lines (4N-), SNEL 4-factor iPS cell lines (4N+) with ChIP-Seq. The same background ES cell line as the control line.

2014-09-07 | E-GEOD-55731 | biostudies-arrayexpress

miR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse somatic cells

Project description:To reprogram mouse embryonic fibroblasts (MEFs) to induced Pluripotent Stem Cells (iPSCs), we constructed the PiggyBac (PB) transposon carrying the four Yamanaka factor cDNAs controlled by a CAG promoter (PB-CAG-OCKS, Oct4, cMyc, Klf4 and Sox2). As the baseline reprogramming control, we transfected the PB-CAG-OCKS transposon into Oct4-reporter MEFs and plated the cells on STO feeder cells (4F-iPS). To examine the effects of miR-25 on reprogramming, in addition to the Yamanaka factors, we co-transfected the PB-CAG-OCKS plasmid with the PB-CAG-miR-25 plasmid and selected for puromycin resistance (2.0 mg/ml) (25-iPS). We then performed genome-wide gene expression microarray analysis on the iPS cells generated and compared the expression profiles to those of Oct4-reporter MEFs and wildtype ES cells.

2011-10-10 | E-MTAB-409 | biostudies-arrayexpress

Expression data from iPS and human ES cells

Project description:Assays to assess the quality of the reprogramming of human Induced pluripotent stem cells are needed. We have previously shown that hESC can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that some of these iPS are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs based and low and high-throughput expression analysis and detailed expression of globin expression patterns suggesting that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with hematopoietic differentiation regardless of the age or the tissue type of the donor cells. We also report that reprogramming can also lead to abnormal iPS that are undistinguishable from hES cells by morphology, and by the expression of their endogenous genes but that are grossly abnormal in their differentiation potential. The most likely cause of abnormal reprogramming is failure to silence the virally transduced reprogramming factors. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications the gene expression in different source of iPS and standard ES cells H1

2011-01-29 | E-GEOD-26946 | biostudies-arrayexpress

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