Transcriptional analysis of iPSCs during cellular reprogramming using RNA FACS
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Sandy Klemm
PROVIDER: E-GEOD-55671 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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: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 generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. We used microarrays tc ompare the gene expression pattern among CML-iPSCs and normal cord blood (CB) iPSCs. Two CML derived iPS cells and one CB derived iPS cells were selected for RNA extraction and hybridization on Affymetrix microarrays.
2012-05-15 | E-GEOD-37982 | 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. 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: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
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
Project description:The reprogramming of differentiated cells to an embryonic stem cell-like state provides a powerful system to explore fundamental mechanisms of development, including how mammalian cells establish and maintain pluripotency and long-term self-renewal capability. Based on the similarities between embryonic stem cells and cancer cells, we investigated the potential role of the retinoblastoma tumor suppressor and cell cycle regulator RB in the reprogramming of fibroblasts into induced pluripotent stem cells (iPS cells). Herein we demonstrate that loss of RB function leads to both an acceleration of the reprogramming process and the generation of more iPS clones from fibroblasts. This effect is largely due to a restrictive role for RB at the early stages of reprogramming. Surprisingly, however, RB inactivation does not enhance the formation of iPS clones by accelerating the proliferation of cells undergoing reprogramming. Rather, a genome-wide investigation of RB targets indicates that RB binds to regulatory regions of pluripotency genes such as Sox2 and Oct4 and contributes to their full repression in differentiated cells. This effect correlates with the maintenance of a repressive chromatin structure at these loci. Accordingly, Rb-deficient fibroblasts can be reprogrammed into iPS cells even in the absence of exogenous Sox2, which is normally required to initiate reprogramming from fibroblasts. These experiments identify a novel barrier in the reprogramming process, mainly the repression of certain pluripotency genes such as Sox2 by RB, which provides a new link between tumor suppressor mechanisms and cellular reprogramming. RNAseq from MEFs with 2 biological replicates (save CP), Rb ChIPseq from MEFs with 2 biological replicates, Histone H3 modification ChIPseq from MEFs with 1 biological replicate
2014-11-13 | E-GEOD-40594 | biostudies-arrayexpress
Project description:We used heterokaryon cell fusion based reprogramming and identified the cytokine IL6 as a potential regulator of reprogramming to pluripotency. We generated iPS clones using the four reprogramming factors (4F) Oct4, Klf4, Sox2, and c-Myc. In addition, iPS clones were generated using only three factors (3F: Oct4, Klf4, amd Sox2) with the addition of the cytokine IL6 to reprogramming culture conditions. Global RNA-Seq of the 3F + IL6 derived iPS clones was done for comparison with 4F-derived iPS clones, mouse embryonic stem cells and mouse embryonic fibroblasts. This study includes 8 samples: 2 independently derived 3F + IL6 iPS clones, 2 independently derived 4F iPS clones, 2 biological replicates of mouse D3-GFP ES cells, and 2 biological replicates of mouse embryonic fibroblasts (MEFs). The latter 6 samples are provided as references for the 3F + IL6 iPS clones. Poly-A RNA was isolated and prepared for sequencing using the Illumina TruSeq RNA kit (v2) to generate 50bp reads. Reads were aligned to mm10.
2013-08-28 | E-GEOD-46104 | biostudies-arrayexpress
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
Project description:Induced pluripotent stem cells (iPSCs) offer opportunity for insight into the genetic requirements of the X chromosome for somatic and germline development. Turner syndrome is caused by complete or partial loss of the second sex chromosome; while more than 90% of Turner cases result in spontaneous fetal loss, survivors display an array of somatic and germline clinical characteristics. Here, we derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We analyzed gene expression profiles of derived iPSCs and in vitro differentiated cells by single cell qRT-PCR and RNA-seq. We whoed that two X chromosomes are not necessary for reprogramming or pluripotency maintenance. Genes that escape X chromosome inactivation (XCI) between control iPSCs and those with X chromosome aneuploidies revealed minimal expression differences relative to a female hESC line. Moreover, when we induced germ cell differentiation via murine xenotransplantation of iPSC lines into the seminiferous tubules of busulfan-treated mice, we observed that undifferentiated iPSCs, independent of X chromosome composition, when placed within the correct somatic environment, are capable of forming early germ cells in vivo. Results indicate that two intact X chromosomes are not required for germ cell formation; however, clinical data suggest that two sex chromosomes are required for maintenance of human germ cells. RNA-seq of H9 cells, iPSCs from Turner syndrome and control individuals and in vitro differentiated cells
2015-05-18 | E-GEOD-55939 | biostudies-arrayexpress