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Transcriptome analysis during somatic cell reprogramming

ABSTRACT: By combining deep sequencing with high-throughput quantitative RT-PCR, we reveal that somatic splicing profiles are reorganized to pluripotent splicing profiles during reprogramming from mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. The splicing pattern in pluripotent stem cells resembles that in testis, and the regulatory regions have specific characters in length and sequence. These results indicate that the dynamic alteration in splicing is an integral part of the molecular network involved in the reprogramming process. Sequencing data from MEF, two iPS cell lines and one ES cell line. RT-PCR data will be represented within Figures or Tables in published manuscripts.

ORGANISM(S): Mus musculus

SUBMITTER: Takuya Yamamoto

PROVIDER: E-GEOD-46149 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptome analysis during somatic cell reprogramming

Project description:By combining deep sequencing with high-throughput quantitative RT-PCR, we reveal that somatic splicing profiles are reorganized to pluripotent splicing profiles during reprogramming from mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. The splicing pattern in pluripotent stem cells resembles that in testis, and the regulatory regions have specific characters in length and sequence. These results indicate that the dynamic alteration in splicing is an integral part of the molecular network involved in the reprogramming process.

2013-10-17 | GSE46149 | GEO

How C/EBPa creates an elite cell state for reprogramming to pluripotency

Project description:Mouse B cells, upon ectopic expression of the transcription factor C/EBPalpha for 18h, can be reprogrammed to induced pluripotent stem (iPS) cells with extremely high efficiency. To understand the molecular control of this phenomenon we performed a time course proteomic analysis by label-free protein quantification of proteins during B cell reprogramming to iPS cells.

2016-02-24 | PXD002769 | Pride

Highly efficient reprogramming to pluripotency and directed differentiation using synthetic mRNA

Project description:Clinical application of induced pluripotent stem (iPS) cells is limited by low efficiency of iPS derivation, and protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-mutagenic strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Our method represents a safe, efficient strategy for somatic cell reprogramming and directing cell fates that has broad applicability for basic research, disease modeling and regenerative medicine. We isolated RNA from human RNA derived iPS cells, viral derived iPS cells, different human fibroblasts and human embryonic stem cells for hybridization to the Affymetrix gene expression microarrays.

2010-09-30 | E-GEOD-23583 | biostudies-arrayexpress

Genome-wide maps of Tbx3 binding sites in mouse ESCs

Project description:Induced pluripotent stem (iPS) cells can be obtained through the introduction of defined factors into somatic cells. The combination of Oct4, Sox2 and Klf4 (OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts (MEFs). Through the genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, we identified Tbx3 as a transcription factor that significantly improves the quality of iPS cells. Induced-PS cells generated with OSK + Tbx3 (OSKT) are superior in both germ cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide ChIP-sequencing analysis of Tbx3 binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. ChIP-seq of Tbx3 binding in mouse ESCs

2009-12-04 | E-GEOD-19219 | biostudies-arrayexpress

Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1 and TIA1

Project description:Background. Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results. We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enable the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNAbinding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions. Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.

2021-04-26 | GSE158633 | GEO

Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells [HG-U133_Plus_2]

Project description:Using global gene expression and proteomic analyses, we identified a molecular signature in human embryonic and induced pluripotent stem cells that suggested a central regulatory role for RNA splicing in self-renewal. Through genetic and biochemical approaches, we established reciprocal functional links between the master regulatory factor OCT4 and SFRS2, a member of the serine/arginine-rich family of splicing factors. SFRS2 regulates expression of two isoforms of the methyl-CpG-binding protein MBD2 that play opposing roles in human ESC and during the reprogramming of fibroblasts. Both the MBD2a isoform expressed in fibroblasts and the MBD2c isoform found in pluripotent cells bind OCT4 and NANOG promoters in human ESC, but only MBD2a interacts with NuRD chromatin remodeling factors. Members of the miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and the somatic specific MBD2a isoform. These data are consistent with a model in which OCT4, SFRS2, and MBD2 participate in a positive feedback loop to regulate proteome diversity in support of self-renewal in pluripotent cells. We isolated RNA from human iPS cells, different human fibroblasts and human embryonic stem cells for hybridization to the Affymetrix gene expression microarrays.

2014-04-18 | E-GEOD-55673 | biostudies-arrayexpress

Expression data from mouse embryonic fibroblasts and pluripotent stem cells

Project description:Splicing profiles in pluripotent stem cells are different from those in somatic cells. Generally, alternative splicing is regulated by RNA binding proteins. To identify the candidate RNA-binding protein-encoding genes, we performed gene expression profiling experiments. Expression levels were profiled in mouse embryonic fibroblasts (MEFs), 2 iPS cell lines (492B4, 178B5), and ES cells (v6.5).

2013-10-17 | E-GEOD-45916 | biostudies-arrayexpress

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Project description:We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells. Small RNA profiles of MEF, ES, iPS, NT-ES and NT-iPS cells were generated by Solexa sequencing. MEF and ES cells were performed in triplicate. iPS, NT-ES and NT-iPS cells were sequenced in duplicate.

2014-07-05 | E-GEOD-52950 | biostudies-arrayexpress

Transcription profiling of mouse differentiated, partially- and fully-reprogrammed MEFs/B-cells

Project description:Expression profiles generated during dissection of the molecular mechanisms underlying direct reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells, iPS). Experiment Overall Design: 2 technical replicates of B lymphocytes, partially reprogrammed (MCV8, MCV6, BIV1), MEF-iPS(Oct4) and B-iPS(Nanog) cell lines.

2008-06-18 | E-GEOD-10871 | biostudies-arrayexpress

A central role for TFIID in the pluripotent transcription circuitry [PP003]

Project description:Pluripotent stem cells can be isolated from early embryos or induced by cell fusion, somatic nuclear transfer, or expression of a selected set of transcription factors. Embryonic stem (ES) cells are characterized by an open chromatin configuration and high transcription levels achieved via autoregulatory and feed-forward transcription factor loops. How the general transcription machinery is involved in pluripotency is unclear. Here, we show that TFIID knockdown affected the pluripotent circuitry in ES cells and inhibited reprogramming of fibroblasts. TFIID and pluripotency factors form a feed-forward loop to induce and maintain a stable transcription state. Strikingly, transient expression of TFIID subunits greatly enhanced reprogramming by Oct4, Sox2, Klf4 and c-Myc reaching efficiencies upto 50%. These results show that TFIID is a critical and selective component for transcription factor-mediated reprogramming. We anticipate that by creating plasticity in gene expression programs, basal transcription complexes such as TFIID assist reprogramming into different cellular states. Three iPS lines, iPS#1, iPS#4, and iPS#5 were used in duplicate for microarray analysis against a pool of RNA from ES-cells.

2013-03-21 | E-GEOD-33645 | biostudies-arrayexpress

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