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Global Transcriptional Analysis of Nuclear Reprogramming in the Transition from MEFs to iPSCs

ABSTRACT: Here, we focused on the intermediate stages of SCR by comparing the somatic cell line induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs). Transcriptional profiles of these four cell lines were analyzed by microarray, and we found that the transition process from day 7 to the formation of iPSCs is crucial for SCR and that the reverse expression patterns can provide more candidate markers to distinguish ESCs and somatic cells iPSC. Data confirmed that the viral infection results in defense innate immunity, DNA damage, and apoptosis in MEFs, which slows down cell proliferation and immortalization to inhibit SCR. Although SCR is initiated by OSK, the p53 signaling pathway can affect the transcriptional regulatory networks through cell cycle and genomic instability as a powerful core node. MEFs were derived from embryonic day 13.5 C57BL6 mice embryos. Cell line day7 is MEF induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs).

ORGANISM(S): Mus musculus

SUBMITTER: zhenwei song

PROVIDER: E-GEOD-37397 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Global Transcriptional Analysis of Nuclear Reprogramming in the Transition from MEFs to iPSCs

Project description:Here, we focused on the intermediate stages of SCR by comparing the somatic cell line induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs). Transcriptional profiles of these four cell lines were analyzed by microarray, and we found that the transition process from day 7 to the formation of iPSCs is crucial for SCR and that the reverse expression patterns can provide more candidate markers to distinguish ESCs and somatic cells iPSC. Data confirmed that the viral infection results in defense innate immunity, DNA damage, and apoptosis in MEFs, which slows down cell proliferation and immortalization to inhibit SCR. Although SCR is initiated by OSK, the p53 signaling pathway can affect the transcriptional regulatory networks through cell cycle and genomic instability as a powerful core node.

2012-10-01 | GSE37397 | GEO

Extensive protein group SUMO modification of repressive chromatin factors distinguishes pluripotent from somatic cells (total proteome)

Project description:Post-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development.

2020-09-16 | PXD020287 | Pride

Extensive protein group SUMO modification of repressive chromatin factors distinguishes pluripotent from somatic cells

2020-09-16 | PXD017697 | Pride

mRNA expression data from iPSCs, ntESCs and iPSC-nt-ESCs

Project description:We generated three kinds of genetically identical mouse reprogrammed cells: induced pluripotent stem cells (iPSCs), nuclear transfer embryonic stem cells (ntESCs) and iPSC-nt-ESCs that are established after successively reprogramming of iPSCs by nuclear transfer (NT). NtESCs show better developmental potential than iPSCs, whereas iPSC-nt-ESCs display worse developmental potential than iPSCs. We used microarrays to distinguish the gene expression differences among three pluriptoent stem cells and identified that imprinted genes had a similar expression pattern in iPSCs and iPSC-nt-ESCs. We sought to obtain genetic identical pluripotent stem cell lines in order to minimize genetic variations among different reprogrammed cells. To that end, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4 and c-Myc were isolated and used as donors for different reprogramming experiments. We generated iPSCs after plating MEFs in the presence of dox in ES culture conditions, and then derived ntESCs after transplantation of the nucleus from the same MEFs into enucleated oocyte. Furthermore, we successively reprogrammed iPSCs by means of NT and established a set of nt-iPSC lines. Pluripotent stem cells generated from different reprogramming strategies were for RNA extraction and hybridization on Affymetrix microarrays.

2011-05-31 | E-GEOD-24705 | 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

The developmental potential of iPSCs is greatly influenced by the selection of the reprogramming factors

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 M-bM-^@M-^\all-iPSCM-bM-^@M-^] 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. ChIP-Seq for Med1 in reprogrammed and partially reprogrammed mouse embryonic stem cell lines and corresponding controls

2014-07-30 | E-GEOD-59569 | biostudies-arrayexpress

The effect of miR-294 and miR-181 on the transcriptome during OSK reprogramming of MEFs

2013-09-15 | GSE49556 | GEO

The effect of miR-294 and miR-181 on the transcriptome during OSK reprogramming of MEFs

Project description:Study designed to determine the immediate effects of supplementing OSK reprogramming with miR-294 or miR-181. MEFs were infected on day 0, and transfected with miR-294, miR-181 or control mimic on day 1. On day 3 RNA was extracted. OSK infected MEFs samples were compared to non-infected MEFs and to fully reprogrammed iPSCs. Performed in biological triplicate, with each sample containing different OSK viral stocks, different preparations of MEFs and independent transfections. Biological triplicate iPSCs represent three independent and clonally expanded iPSC lines. Total RNA collected with Trizol. All processing conducted at the UCLA Neuroscience Genomics Core. MouseRef-8 v2.0 Expression BeadChips.

2013-09-15 | E-GEOD-49556 | biostudies-arrayexpress

Induced pluripotent stem cells can be generated in bulk culture

Project description:Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). However, successfully reprogrammed cells are highly proliferative and escape from cellular senescence - it is therefore conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency and size was higher when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs had indistinguishable in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs M-bM-^@M-^S this is of relevance for standardization and automation of cell culture procedures Gene expression profiles of induced fibroblasts (M-bM-^@M-^\iFM-bM-^@M-^]) from three different donors which were either clonally derived (M-bM-^@M-^\CM-bM-^@M-^]) or bulk-cultured (M-bM-^@M-^\BM-bM-^@M-^]) were compared

2013-06-03 | E-GEOD-42807 | biostudies-arrayexpress

CRY1-ChIP-seq in MEFs, PreiPSCs, iPSCs, and ESCs

Project description:Genome-wide occupancy of the circadian clock Cryptochrome1 (CRY1) was compared between somatic differentiated mouse embryonic fibroblasts (MEFs) and pluripotent stem cells including precursor-induced pluripotent stem cells (iPSCs), iPSCs, and embryonic stem cells (ESCs)

2023-06-01 | GSE230321 | GEO

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