The developmental potential of iPSCs is greatly influenced by the selection of the reprogramming factors
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Richard Young
PROVIDER: E-GEOD-59569 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM), in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog, Oct4 and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the vast majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation were attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecular trajectories assumed during different cell reprogramming methods. poly RNA-Seq and Chromatin accesibility (ATAC-seq) were measured during conversion of mouse embryonic fibroblasts to neural stem cells using OSKM trans-differentiation method, as well as in mouse emrbyonic fibroblasts, iPSCs and mouse ESCs.
2015-04-30 | E-GEOD-67298 | biostudies-arrayexpress
Project description:Reprogramming cells from one fate to another, using transcription factors, generates cells for research and potential therapy, yet little is known about the initial engagement of reprogramming factors with the genome. We mapped the interactions between Oct4, Sox2, Klf4, and c-Myc (OSKM) and the human genome during the first 48 hours of cellular reprogramming to pluripotency. Unlike that reported in ES/iPS cells, we find extensive overlap in the initial binding of OSKM, demonstrating that the initial regulatory network differs markedly from that in pluripotency. OSK act as pioneer factors for c-Myc, and c-Myc enhances the engagement of OSK, including at many genes that are required for conversion to pluripotency. Distal enhancer sites in closed chromatin dominate the initial OSKM distribution. Hierarchical chromatin binding during reprogramming resembles that employed during development. Four chIP-seq data sets (Oct4, Sox2, Klf4, and c-Myc) are included, one lane per factor, no replicates. Also included is an input lane from the same conditions and two mock lentiviral controls (no exogenous OSKM factors) treated with Oct4 IP and c-Myc IP.
2012-11-16 | E-GEOD-36570 | biostudies-arrayexpress
Project description:Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of endogenous retroviruses, thus facilitating the transition through reprogramming. Gene expression profiling using high through put sequencing at day 7 of Oct4, Sox2, Klf4 and cMyc (OSKM) expression in mouse embryonic fibroblasts with or without Trim28 / Setdb1 knockdown
2016-06-28 | E-GEOD-80550 | biostudies-arrayexpress
Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.
2015-11-06 | E-GEOD-69237 | 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:Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM), in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog, Oct4 and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the vast majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation were attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecular trajectories assumed during different cell reprogramming methods. WGBS (Whole-Genome-Bisulfite-sequencing) were measured during conversion of mouse embryonic fibroblasts to neural stem cells using OSKM trans-differentiation method, as well as in mouse emrbyonic fibroblasts, and mouse ESCs.
2015-05-07 | E-GEOD-68601 | biostudies-arrayexpress
Project description:It remains unclear how the ectopic expression of defined transcription factors induces dynamic changes in gene expression profiles that establish a pluripotent state during direct cell reprogramming. In the present study, we first identified a temporal gene expression program during the reprogramming process. Promoter analyses then predicted the role of two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of the gene expression program. Knockdown of Foxd1 or Foxo1 reduced the number of induced pluripotent stem cells (iPSCs). The knockout of Foxd1 prevented the downstream transcription program, including the expression of reprogramming marker genes. Interestingly, the expression level of Foxd1 was also transiently increased in a small population of cells in the middle stage of reprogramming. The presence or absence of Foxd1 expression in this stage was correlated with a future cell fate as iPSCs or non-reprogrammed cells. These results suggest that Foxd1 is a mediator and indicator of the successful progression of the gene expression program in cell reprogramming. Mouse embryonic fibroblasts (MEFs) were infected with retroviruses encoding either the four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or GFP (control) at day 0 and sampled on the indicated days. Two replicates each.
2014-01-11 | E-GEOD-38509 | biostudies-arrayexpress
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 1µg/ml Doxcycline and 50µg/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
Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line. 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)
2014-01-01 | E-GEOD-53212 | biostudies-arrayexpress
Project description:We performed 5hmC DNA Immunoprecipitation followed high-throughput sequencing using R1 ESCs, OT ESCs, TSKM ESCs, Day3 TSKM and OSKM induction cells. We compared the profiling of 5-hydroxymethylcytosine modifications in these different cell lines. We found that: the TSKM and OT iPSCs shared similar 5hmC modification pattern while apart from that of R1 ESCs. However the comparison based on the gene promoter (-1000/+500 of gene’s TSS) showed less differences. The TSKM 2nd induction samples were quite like each other and have shared the basic pattern with traditional OSKM 2nd sample. However comparison based on the gene promoter showed sharp increase. Moreover higher enrichment of 5hmC on the distal enhancer of Oct4 was specifically detected in Day 3 TSKM 2nd cells as well as TSKM and OT iPSCs, which might be helpful for us to study the Tet1-mediated somatic reprogramming. Examination of 5-hydroxymethylcytosine modifications in 5 different cell lines.
2012-11-17 | E-GEOD-33781 | biostudies-arrayexpress