Expression analysis of stepwise induction of iPS cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Jianxin Bao
PROVIDER: E-GEOD-28197 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We were able to achieve an initial stable intermediate phase by the transduction of Oct4, Klf4, and c-Myc. Furthermore, over-expression of Sox2 in these intermediate stage cells leads to final iPS cell phase. After examining the gene expression profiles from the initial to final iPS cell phases, we have identified Sox2 downstream genes important for iPS cell induction.
2011-12-29 | GSE28197 | GEO
Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).
2015-01-31 | E-GEOD-62957 | biostudies-arrayexpress
Project description:Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Considering that ectopic expression of c-Myc causes tumourigenicity in offspring and retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here, we show that adult mouse neural stem cells (NSCs) express higher endogenous levels of Sox2 and c-Myc than ES cells and that exogenous Oct4 together with either Klf4 or c-Myc are sufficient to generate iPS cells from NSCs. These two-factor (2F) iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germline, and form chimeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Experiment Overall Design: 8 hybridizations in total. Experiment Overall Design: NSC derived iPS cells by 2 factors (Oct4 and Klf4) in triplicate: Experiment Overall Design: - iPS cell_2F_1 Experiment Overall Design: - iPS cell_2F_2 Experiment Overall Design: - iPS cell_2F_3 Experiment Overall Design: Embryonic Stem cells (ESC) in triplicate: Experiment Overall Design: - ESC_1 Experiment Overall Design: - ESC_2 Experiment Overall Design: - ESC_3 Experiment Overall Design: NSC cultures in duplicates: Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: NSC derived iPS cells by 4 factors (Oct4, Sox2, c-Myc and Klf4) in triplicate: Experiment Overall Design: - iPS cell_4F_1 Experiment Overall Design: - iPS cell_4F_2 Experiment Overall Design: - iPS cell_4F_3
2008-10-20 | E-GEOD-10806 | biostudies-arrayexpress
Project description:Increased genomic integrity an improved protein-based iPS cell method compared to current viral induced strategies We used microarrays to detail the global gene expression of protein-based iPS cells Total RNAs were prepared from mH, mES, miPS cells usingTRIzol reagent (Invitrogen, Carlsbad, CA) and their cDNAs were allowed to hybridize to Affymetrix Mouse Expression Array 430 containing over 43,000 mouse transcripts at the Harvard Partners Center for Genomics and Genetics.
2014-03-08 | E-GEOD-53299 | biostudies-arrayexpress
Project description:Pluripotency can be induced in murine and human fibroblast by transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Previously we reported that two factors (Oct4 and Klf4) are sufficient for reprogramming adult mouse neural stem cells (NSCs) to a pluripotent state. However, although NSCs endogenously express the factors Sox2, c-Myc, and Klf4, our previous report does not elucidate why exogenous expression of either Klf4 or c-Myc is still required for reprogramming. Here we report that exogenous expression of Oct4 is sufficient to generate one-factor induced pluripotent stem (1F iPS) cells without any oncogenic factors, such as c-Myc and Klf4, from mouse adult NSCs, which endogenously express Sox2, c-Myc, and Klf4, and also intermediate reprogramming markers alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1). These results extend our previous report proposing that somatic cells can be reprogrammed to a pluripotent state with a reducing number of reprogramming factors when the complementing factors are endogenously expressed in the somatic cells. Experiment Overall Design: 10 hybridizations in total. Experiment Overall Design: NSC-derived iPS cells by one-factor (Oct4) in triplicate: Experiment Overall Design: - NSC_1F_iPS_1 Experiment Overall Design: - NSC_1F_iPS_2 Experiment Overall Design: - NSC_1F_iPS_3 Experiment Overall Design: One-factor (Oct4) iPS cell-derived NSC in triplicate: Experiment Overall Design: - 1F_iPS_NSC_1 Experiment Overall Design: - 1F_iPS_NSC_2 Experiment Overall Design: - 1F_iPS_NSC_3 Experiment Overall Design: Neural stem cell (NSC) derived from brain of OG2/Rosa26 mice: Experiment Overall Design: - NSC_1 Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: - NSC_4
2009-02-08 | E-GEOD-12499 | biostudies-arrayexpress
Project description:The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. However, the relative flaws in the understanding of the molecular mechanisms promoting or limiting reprogramming still hinder the efficient generation of high quality iPS cells. Whereas modulation of the initial Oct4, Sox2, Klf4 and c-Myc (OSKM) cocktail with new transcription factors has been extensively documented, comparatively little is known about soluble molecules promoting the process, even if such recombinant factors could be highly valuable for therapeutic applications. In this study we developed a large-scale identification method to uncover novel programmed cell death (PCD)-related mechanisms limiting somatic cell reprogramming to pluripotency (SCRP). We identified Netrin-1 and its dependence receptor Dcc (Deleted in Colorectal Carcinoma), previously described for their respective survival/death functions both in normal and oncogenic contexts, as novel key SCRP modulators. We show that the early phase of SCRP is accompanied with a strong Netrin-1 deficiency, due to the improper epigenetic regulation of the Ntn1 promoter by OSKM. Mechanistically, we demonstrate that such Netrin-1 imbalance induces apoptosis mediated by the dependence receptor Dcc in a p53-independent manner. Correction of the Netrin-1/Dcc equilibrium by gain-of-ligand and loss-of-receptor experiments constrains apoptosis and improves reprogramming. As a consequence, we propose a novel iPS derivation protocol including a sequential treatment with recombinant Netrin1 that greatly facilitates the generation of mouse and human iPS cells. RNA-sequencing of mouse embryonic fibroblasts (passage 2 and passage 4), mouse pre-iPS cells (passage 5 and passage 25), 1 clone of control iPS (passage 5 and passage 25) and 2 independent clones of "Netrin-1 derived" iPS cells (passage 5 and passage 25). For this analysis, mouse iPS cell lines were grown in KSR+LIF media.
2015-09-01 | E-GEOD-54107 | biostudies-arrayexpress
Project description:We tested a genome-scale artificial transcription factor (ATF) library in reprogramming mouse embryonic fibroblasts to induced pluripotent stem (iPS) cells. Three combinations of ATFs (C2, C3, and C4) could induce pluripotency when expressed with Sox2, Klf4, and c-Myc. The transcriptional profiles of ATF-induced iPS cells are similar to that of iPS cells induced with Oct4, Sox2, Klf4, and c-Myc and mouse embryonic stem cells, exhibiting up-regulation of pluripotency markers and down-regulation of fibroblast markers. We performed ChIP-seq on the active histone mark H3K27ac and the repressive histone mark H3K9me3 in C2+SKM iPS cells, C3+SKM iPS cells, in Oct4+SKM to validate the total transcriptome results. In addition, the ATFs from C2 were chromatin immunoprecipitated to determine where they are bound in the genome at an intermediate stage before being fully reprogrammed to iPS cells. This study provides a proof-of-principle that a gene-activating ATF library can be used to identify cell fate-defining transcriptional networks in an unbiased manner.
2016-12-05 | GSE89220 | GEO
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:To identify binding partners of Salmonella’s thioredoxin protein encoded by the trxA gene, we performed tandem-affinity purification (TAP) using a C-terminal fusion of thioredoxin with calmodulin-binding peptide and Protein A. TrxA-binding molecules were identified by mass spectrometry (MS) analysis
2023-03-11 | PXD037199 | Pride
Project description:E-cadherin upregulation is an early event of reprogramming of fibroblasts to induce pluripotent stem cells (iPS). Knocking down of E-cadherin by shRNA impairs iPS generation, though some colonies with great morphorlogical difference to shRNA control colonies remain. To illustrate the molecular and functional difference between shECAD iPS clones and shRNA control iPS clones, three respective iPS clones (shECAD 4,8,9 and Ctrl 2,3,4) were derived and DNA microarrays were run to analyze the transcriptional profile of these clones. OG2 MEFs were infected with Sox2, Klf4, Oct4 and c-Myc (SKOM) plus either Luciferase shRNA (shLUC) or E-cadherin shRNA (shECAD) retrovirus. At Day 6 post infection cells were split onto feeder cells. Several colonies from SKOM+shLuc and SKOM+shECAD were picked out at Day 14 post infection respectively and three cell lines were established, namely Ctrl 2,3,4 for SKOM+shLuc iPS and shECAD 4,8,9 for SKOM+shECAD iPS. All clones were maintained on feeder cells in mESC medium. RNA were extracted from these six cell lines and DNA microarrays were run to analyze the transcriptional profile.
2010-06-08 | E-GEOD-21062 | biostudies-arrayexpress