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Co-expression networks in generation of induced pluripotent stem cells

ABSTRACT: We developed an adenoviral vector, in which Yamanaka’s four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-GFP-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as 3 days. These reprogrammed cells resembled human ES cells with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted Gene Co-Expression Network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ES cell marker genes, whereas the turquoise module contained cell-cycle related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all 4 RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE72676 | GEO | 2016/03/01

SECONDARY ACCESSION(S): PRJNA294632

REPOSITORIES: GEO

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Co-expression networks in generation of induced pluripotent stem cells

Project description:We developed an adenoviral vector, in which Yamanakaâ??s four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-GFP-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as 3 days. These reprogrammed cells resembled human ES cells with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted Gene Co-Expression Network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ES cell marker genes, whereas the turquoise module contained cell-cycle related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all 4 RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation. Cells were dedifferentiated and gene expression assessed at 13 timepoints

2016-03-01 | E-GEOD-72676 | biostudies-arrayexpress

Proteomic analysis of mouse oocytes identifies PRMT7 as reprogramming factor that replaces SOX2 in the induction of pluripotent stem cells

Project description:The inclusion of oocyte factors together with Yamanaka’s previously identified reprogramming factors (OCT4, SOX2, KLF4 with or without cMYC; OSK(M)) may facilitate the reprogramming process that leads to induced pluripotent stem cells (iPSCs). We previously applied label-free LC-MS/MS analysis to search for such facilitators of reprogramming (reprogrammome), resulting in a catalog of 28 candidates that are (i) able to robustly access the cell nucleus, and (ii) shared between mature mouse oocytes and pluripotent embryonic stem (ES) cells. In the present study we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature mouse oocytes, (iv) depleted after oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors during iPSC reprogramming. Using LC-MS/MS and isotopic labeling methods we found that the abundance profiles of the 28 proteins was below that of known oocyte-specific and housekeeping proteins. Out of the 28 proteins only arginine methyltransferase 7 (PRMT7) presented a substantially changing profile during mouse embryogenesis and impacted on the conversion of mouse fibroblasts into iPSCs. PRMT7 indeed could very efficiently replace SOX2 in a factor-substitution assay yielding iPSCs. These findings show that proteomics can be used to prioritize the functional analysis of reprogrammome candidates.

2016-05-31 | PXD003093 | Pride

Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [ChIP-Seq]

Project description:Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells. E14 mES cells were immunoprecipitated with Zfp322a antibody or IgG from rabbit.

2013-10-31 | E-GEOD-45138 | biostudies-arrayexpress

Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [Expression]

2013-10-31 | GSE45139 | GEO

Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [ChIP-Seq]

2013-10-31 | GSE45138 | GEO

Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [Expression]

Project description:Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells. Zfp322a depleted (RNAi) E14 mES cells were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding wild type (control RNAi cells).

2013-10-31 | E-GEOD-45139 | biostudies-arrayexpress

Expression profile of MEFs, iPS, and ES in different culture condition

2010-10-27 | GSE24861 | GEO

Expression profile of MEFs, iPS, and ES in different culture condition

Project description:MEFs can be reprograming to ES by defined reprogramming factors, the derived ES-like cells were termed iPSc. Completely reprogrammed iPSc have identifed gene expression profle with ES. Culture condition may influent the pluripotency of iPSCs.iPS/ES cultured in differient ES medium may have different gene expression profile. We preform an experiment to analyze the gene expression difference among MEFs cultured in FBS and iCD1,iPSc cultured in KSR medium and N2B27 supplementary with 2i medium , and ES cultured in N2B27 supplementary with 2i medium.

2010-10-27 | E-GEOD-24861 | biostudies-arrayexpress

SUMO safeguards somatic and pluripotent cell identities by enforcing distinct chromatin states

Project description:Purposes: (1) compare the SUMO chromatin landscape between mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESc) (2) Decipher how hyposumoylation enhances MEF to induced pluripotent stem cells (iPSc) reprogramming (3) Decipher how hyposumoylation enhances ESc to 2C-like cells conversion. Methods : (1) ChIP-seq in MEFs and ESc for SUMO isoforms and histone marks. (2) ChIP-seq for transcription factors and histone marks at day 4 of MEF to iPSc reprogramming comparing wild type (shCtrl) and hyposumoylated cells (shUbc9). ATAC-Seq at day 4 of MEF to iPSc reprogramming comparing wild type (shCtrl) and hyposumoylated cells (shUbc9). RNA-Seq in MEFs, iPSc, at day 4 and day 7 of MEF to iPSc reprogramming comparing wild type (shCtrl) and hyposumoylated cells (shUbc9). (3) RNA-Seq in ESc comparing wild-type (siCtrl) and hyposumoylated cells (siUbc9). Results: (1) SUMO chromatin landscape is highly different between MEFs and ESc and associates to distinct chromatin marks. (2) Hyposumoylation enhances MEFs to iPSc reprogramming by favoring the extinction of the MEF transcriptional program and redistributing pluripotency factors from MEF enhancers toward ES enhancers. (3) Hyposumoylation enhances ES to 2C-like conversion by destabilizing heterochromatin thus resulting in an activation of the 2-cell transcriptional program. Conclusion: SUMO at chromatin is gate-keeper of cell-identity.

2018-10-24 | GSE99009 | GEO

Nanog Independent Reprogramming to iPSCs with Canonical Factors

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

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