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Comparisons between fully and partially reprogrammed iPS cells induced by pMX-Klf4, pMX-Oct4 and pMX-Sox2 retroviruses

ABSTRACT: Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing heterochromatin marks. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative Electron Spectroscopic Imaging (ESI). In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocenter structures of densely packed 10 nm chromatin fibers. In contrast, chromocenter boundaries are poorly defined in pluripotent ES and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibers in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin reorganization serves as a novel biomarker with retroviral silencing for identifying iPS cells in the very late stages of reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE21595 | GEO | 2011/09/22

SECONDARY ACCESSION(S): PRJNA127129

REPOSITORIES: GEO

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Comparisons between fully and partially reprogrammed iPS cells induced by pMX-Klf4, pMX-Oct4 and pMX-Sox2 retroviruses

Project description:Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing heterochromatin marks. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative Electron Spectroscopic Imaging (ESI). In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocenter structures of densely packed 10 nm chromatin fibers. In contrast, chromocenter boundaries are poorly defined in pluripotent ES and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibers in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin reorganization serves as a novel biomarker with retroviral silencing for identifying iPS cells in the very late stages of reprogramming. We compared the expression profiles of partially and fully reprogrammed iPS cell lines derived from CD1 mouse embryonic fibroblasts (MEFSs) by retroviral reprogramming (pMX-Oct4, pMX-Klf4 and pMX-Sox2). to the differentiated MEFS and the J1 embryonic stem cell line. We also studied the effect of a 2i cocktail treatment in partially reprogrammed iPS cells.

2011-09-21 | E-GEOD-21595 | biostudies-arrayexpress

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Project description:Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. Profiling the chromatin-bound proteome (chromatome) by genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos lead to developmental arrest, reduction of H3K9me3 and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

2023-04-18 | PXD019703 | Pride

Viral and transgene-free human induced pluripotent stem (iPS) cells using non-integrating Sendai virus (SeV) vectors.

Project description:Whole-genome expression analysis of non-integrating Sendai virus (SeV) vector and retroviral vector-generated human iPS cells. Parental fibroblast and induced pluripotent stem (iPS) cells.

2010-09-22 | E-GEOD-24240 | biostudies-arrayexpress

Generation of germ-line competent induced pluripotent stem cells

2007-06-07 | GSE7841 | GEO

The distribution of genomic variations in human iPSCs is related to replication timing reorganization during reprogramming [Affymetrix]

Project description:Cell fate change involves significant genome reorganization, including change in replication timing, but how these changes are related to genetic variation has not been examined. To study how change in replication timing that occurs during reprogramming impacts the copy number variation (CNV) landscape, we generated genome-wide replication timing profiles of induced pluripotent stem cells (iPSCs) and their parental fibroblasts. A significant portion of the genome changes replication timing as a result of reprogramming, indicative of overall genome reorganization. We found that early and late replicating domains in iPSCs are differentially affected by copy number gains and losses, and that in particular CNV gains accumulate in regions of the genome that change to earlier replication during the reprogramming process. This differential relationship was present irrespective of reprogramming method. Overall, our findings reveal a functional association between reorganization of replication timing and the CNV landscape that emerges during reprogramming. Keywords: Expression profiling by array [Gene expression] We isolated RNA from normal human fibroblast derived iPS cells, normal human fibroblasts, and human embryonic stem cells for hybridization to the Affymetrix gene expression microarrays. [CNV] We isolated genomic DNA from iPSCs derived with retroviral and episomal vectors and their parental fibroblast cells for hybridization to the Affymetrix Genome-Wide Human SNP 6.0 Array.

2014-03-28 | E-GEOD-55395 | biostudies-arrayexpress

Single-cell transcriptome profiling of Sox2-low iPS cells

Project description:Single-cell RNA-seq was performed on homozygous Sox2 knockout induced pluripotent stem (iPS) cells, where residual Sox2 expression is observed from incompletely silenced retroviral transgenes. These were compared to Sox2-low iPS cells rescued by exogenous Sox2 transgenic expression, and wild-type iPS cells to assess the lineage expression profile of Sox2-low cells and degree of transcriptional heterogeneity in each population.

2020-12-28 | E-MTAB-9931 | biostudies-arrayexpress

Transcription profiling of mouse induced pluripotent stem cells

Project description:We have previously shown that pluripotent stem cells are induced from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 expression. These iPS (induced pluripotent stem) cells are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation. Fbx15-iPS cells, however, are different in gene expression and DNA methylation patterns and fail to produce adult chimeras. Here we show that selection for Nanog results in germ-line competent iPS cells, with more ES cell-like gene expression and DNA methylation patterns. The four transgenes were strongly silenced. We obtained adult chimeras from seven Nanog-iPS clones, with one clone transmitted through the germ-line to the next generation. Approximately 20% of the offspring developed tumors, where c-Myc transgene was reactivated. Thus iPS cells competent for germ-line chimeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application. Experiment Overall Design: Total RNA from Fbx15-null MEF (duplicate), Fbx15-null ES cells (duplicate), Fbx15-iPS cells (clone MEF4-7, duplicate), or Nanog-iPS cells (clones 20D -2, 16, 17, and 18) were labeled with Cy3. Samples were hybridized to a Mouse Oligo Microarray (Agilent) according to the manufacturerâs protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent). Data were analyzed using GeneSprings GX software (Agilent). We excluded genes whose value fluctuated more than 2-fold between duplicated analyses.

2008-06-15 | E-GEOD-7841 | biostudies-arrayexpress

Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

Project description:In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR, centromeric protein dCenpA and heterochromatic protein HP1a to generate a molecular map of HMR, dCenpA and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.

2020-03-23 | PXD012551 | Pride

Expression data of induced pluripotent stem cell

Project description:We present a robust serum-free system for the rapid and efficient reprogramming of mouse somatic cells by Oct4, Sox2 and Klf4. The elimination of fetal bovine serum and oncogene c-Myc allowed reprogramming cells to be detected as early as Day 2 and reached greater than 10% of the population at Day 7 post retroviral transduction. The resulting iPS colonies were isolated with high efficiency to establish pluripotent cell lines. Based on this method, we further developed iPS-SF1 as a dedicated reprogramming medium for chemical screening and mechanistic investigations. Comparasion of iPS cell lines derived from serum-free culture condition, MEF cells cultured in serum and serum-free condition and ES cell lines was shown.

2009-03-18 | GSE15267 | GEO

Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells

Project description:Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cellM-bM-^@M-^Slike state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. The generation of reprogrammed pluripotent cells using a somatic cell donor source such as bone marrow (BM) or peripheral blood is of particular therapeutic interest because of the relative ease of harvesting these cell types. Here we show that mouse adult BM mononuclear cellsM-oM-<M-^HBM MNCsM-oM-<M-^Iare competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and embryonic fibroblasts (MEFs) from Oct4-EGFP transgenic mice, fused them with ES cells and infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM cells formed more ES-like colonies than did MEFs. Infected BM cells gave rise to iPS cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed embryonic stem cell markers, formed teratomas, and contributed to chimera mice with germline development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Here we demonstrate, for the first time, the induction of pluripotent cells directly from hematopoietic tissue. Gene expression profiling was performed in mouse BMMNCs, ES and BMMNC derived iPS cell lines.

2009-07-02 | E-GEOD-15775 | biostudies-arrayexpress

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