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Heterokaryon RNA Sequencing

ABSTRACT: We report the application of bi-species RNAseq for investigating mechanisms of reprogramming towards pluripotency using heterokaryons (mouse embryonic stem cell X human fibroblast cell fusions). The use of mixed species allows one to monitor reprogramming of the human somatic nuclei independently of contributions from the mouse nuclei using nucleotide differences. We used RNAseq to monitor heterokaryon reprogramming over a 3-day timecourse, generating transcriptome-wide data for cell fusion based reprogramming of human fibroblasts towards pluripotency.

OTHER RELATED OMICS DATASETS IN: PRJNA197269PRJNA186683

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE43549 | GEO | 2013/08/28

SECONDARY ACCESSION(S): PRJNA186683

REPOSITORIES: GEO

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The nuclei of somatic cells fused to mouse embryonic stem cells express pluripotency markers similar to early reprogramming events

Project description:The mechanisms that direct nuclear reprogramming to a pluripotent state are still not fully understood. For species in which the derivation of true induced pluripotent stem (iPS) cells has not yet been completely successful, insights into the particular mechanisms that govern pluripotency can help in determining which are the appropriate conditions to induce stable reprogramming in somatic cells. Cell fusion of a somatic cell to a pluripotent cell is known to induce expression of pluripotency markers in the somatic nucleus. We hypothesized that fusion of bovine fetal fibroblasts (bFFs) to mouse embryonic stem (mES) cells would induce expression of pluripotency markers in the bFF nucleus. We have previously established a method to produce and specifically select multinucleated cells originated from both mES cell and bFF (bi-species heterokaryon), using indirect immunofluorescence on live cells and the combined use of imaging and sorting flow cytometry. With these tools in place, we analyzed 200 heterokaryons collected at 24, 48 and 72h after fusion, as well as unfused bFFs and mES cells, and analyzed using RNA-seq. We found significant changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was also upregulated in heterokaryons, although its role and mechanism of action is still unclear. The cell fusion model presented here can be used to characterize early changes in somatic nuclear reprogramming, and to study the effect of different conditions during reprogramming.

2019-03-02 | GSE127731 | GEO

Cell fusion-mediated pluripotent reprogramming triggers ordered changes in chromatin and gene expression across the human inactive X chromosome

Project description:Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we used cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show a rapid and widespread loss of Xi-associated H3K27me3 and XIST in fused cells that precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-Seq analysis confirmed that Xi reactivation remained partial and showed that induction of human pluripotency-specific XACT transcripts occurred, but was rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation, and suggest a hierarchy where early events such as XIST-delocalisation, are required but are insufficient to establish stable human X reactivation. We performed RNA-sequencing of human fibroblast clones derived from TERT-immortalised NHDF17914 (Lonza) before and at 5 days after fusion with mouse ESC (E14Tg2a:puroR). Two biological replicates were performed and sequenced.

2016-07-01 | E-GEOD-77443 | biostudies-arrayexpress

Cell fusion-mediated pluripotent reprogramming triggers ordered changes in chromatin and gene expression across the human inactive X chromosome

2016-07-01 | GSE77443 | GEO

RNA sequencing analyses of fused heterokaryons between mouse embryonic stem cells and human B lymphocytes collected at early timepoints after fusion

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2020-11-12 | GSE114238 | GEO

Global transcriptome profiling of Oct4/Klf4/Sox2 (3Factor, 3F) + IL6 iPS clones derived from mouse embryonic fibroblasts.

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Oct4 binding and Histone modification profiling during OSKM-mediated 2nd reprogramming

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 ChIP-seq to explore the global changes of Oct4 binding profiling during OSKM-mediated somatic cell reprogramming. The exogenous Oct4 was tagged with 3XFlag, thus we used anti-flag antibody to monitor exogenous Oct4 changes; anti-Oct4 antibody which recognizes both endogenous and exogenous Oct4 was used to monitor total Oct4 changes during iPSC induction. In addition, we also examined genome-wide H3K4me1/H3K27ac/H3K4me3/H3K27me3/RNAPII profiling during 3Flag-OSKM 2' reprogramming.

2015-12-31 | E-GEOD-67520 | biostudies-arrayexpress

Human cortical nuclei RNAseq

Project description:Human cortical nuclei RNAseq

| PRJNA631228 | ENA

Mus musculus

Project description:A Myc-dependent role for cohesin in the heterokaryon-mediated reprogramming of somatic nuclei

| PRJNA263831 | ENA

Global transcriptome profiling of Oct4/Klf4/Sox2 (3Factor, 3F) + IL6 iPS clones derived from mouse embryonic fibroblasts.

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

Transcriptional reprogramming of somatic cells after nuclear transfer to mouse 4-cell embryos

Project description:We have developed a nuclear transfer (NT) system in which somatic nuclei are transplanted into mouse embryos arrested at the 4-cell stage. The transplanted somatic nuclei show swelling and epigenetic reprogramming towards 4-cell-like nuclei. To assess genome-wide transcriptional reprogramming of the injected nuclei, the newly transcribed genes in NT embryos were examined by RNA-seq analyses. As a control, NT was also performed using mouse embryos at the 2-cell stage.

2021-11-03 | GSE162345 | GEO

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