TGFb signaling hyperactivation-induced germ cell-like tumorigenicity during ESC-derived neural progenitors
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA414401 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6176410_1.fastq.gz | Fastqsanger.gz | |||
| SRR6176410_2.fastq.gz | Fastqsanger.gz | |||
| SRR6176411_1.fastq.gz | Fastqsanger.gz | |||
| SRR6176411_2.fastq.gz | Fastqsanger.gz | |||
| SRR6176402_1.fastq.gz | Fastqsanger.gz |
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Project description:Despite the therapeutic promise for pluripotent stem cells (PSCs)-based transplantation, a crucial challenge for PSC implementation is the frequent development of teratomas or tumors in animal models or clinical patients. Therefore, clarifying the mechanism of cell therapy-derived carcinogenesis is of great importance to reduce the risk of tumorigenicity in stem cell therapy. Here we differentiate Oct4-GFP mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) or neurons and find that a minority of Oct4+ cells are sustained at an undifferentiated state. These cells are enriched and proliferated in standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, so defined as differentiation-resistant ESCs (DR-ESCs). Transcriptome and epigenomic analysis shows that DR-ESCs are characterized by primordial germ cell-like gene signatures (Dazl, Rec8, Stra8, Blimp1, etc) and specific histone modification patterns distinct from mESCs. Moreover, the DR-ESCs possess the germ cell potential to generate Sycp3+ haploid cells and are able to reside in sperm-free spermaduct induced by busulfan. Finally, we find that TGFβ signaling is over-activated in DR-ESCs, and inhibition of TGFβ signaling induces the fully differentiation of DR-ESCs and eliminates the tumorigenicity of mESC-derived NPCs. These data demonstrate that these TGFβ hyperactivated germ cell-like DR-ESCs are the main contributor for the tumorigenicity of ESCs-derived target cell therapy and that inhibition of TGFβ signaling in ESC-derived NPC transplantation can help to drastically reduce the risk of tumor development.
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Project description:We performed gene expression profiling on in vitro derived PGCs, undifferentiated ESCs, and somatic cells from the EB to examine germ cell expression in ESC-derived cells
2011-12-15 | GSE33121 | GEO
Project description:We performed gene expression profiling on in vitro derived PGCs, undifferentiated ESCs, and somatic cells from the EB to examine germ cell expression in ESC-derived cells All cells were collected using fluorescence activated cell sorting to isolate SSEA1+/cKit+ ESCs, SSEA1+/cKitbright PGCs, Oct4-gfp+/cKitbright PGCs, and SSEA1-/cKit- somatic cells and Oct4-gfp-/cKit- somatic cells.
2011-12-15 | E-GEOD-33121 | biostudies-arrayexpress
Project description:Identification of in vivo binding targets of Met4 and Met32 by conducting chromatin immunoprecipitation studies using genomic tiling arrays (ChIP-on-chip 4X44K whole yeast genome arrays by Agilent) following 90 minutes Met4 hyperactivation.
2012-02-21 | E-MEXP-3302 | biostudies-arrayexpress
Project description:The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale ChIP-Seq method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac and H2BK20ac), facilitating the identification of active and repressed cis-regulatory elements in germ cells in vivo. Comparison of active chromatin state between somatic, embryonic stem cells (ESC) and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. The nuclear receptor Nr5a2 motif is enriched at a subset of cis-regulatory regions and we confirm its role in germ cell differentiation. Interestingly, germ cells have comparatively more H3K27me3-marked sites that are absent in ESC and other somatic cell types. These repressed regions are enriched for retrotransposons and MHC genes and this indicates that these loci are specifically silenced in germ cells. Together, our study provides the first genome-wide histone modification maps of in vivo germ cells and revealed the molecular chromatin signatures unique to germ cells.
2013-01-25 | GSE38164 | GEO
Project description:The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale ChIP-Seq method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac and H2BK20ac), facilitating the identification of active and repressed cis-regulatory elements in germ cells in vivo. Comparison of active chromatin state between somatic, embryonic stem cells (ESC) and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. The nuclear receptor Nr5a2 motif is enriched at a subset of cis-regulatory regions and we confirm its role in germ cell differentiation. Interestingly, germ cells have comparatively more H3K27me3-marked sites that are absent in ESC and other somatic cell types. These repressed regions are enriched for retrotransposons and MHC genes and this indicates that these loci are specifically silenced in germ cells. Together, our study provides the first genome-wide histone modification maps of in vivo germ cells and revealed the molecular chromatin signatures unique to germ cells. Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. ChIP-seq of Histone modification was done for two timepoints in this study: E11.5 (male/female), E13.5 (male). For E13.5 timepoint, two biological replicates were analyzed. In order to validate small scale ChIP-seq method limited number of ES cells were used to check consistency of ChIP-seq data.
2013-01-25 | E-GEOD-38164 | biostudies-arrayexpress
Project description:Transcriptomics of Tgfb signaling
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Project description:Expression of germ cell nuclear factor (GCNF, Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using the RNeasy micro and mini kits with on-column DNase digestion as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software. Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. 8 samples were analyzed: ESC++, ESC control Oct4-GFP, 1 replicate ESC--, ESC Gcnf -/- Oct4-GFP, 1 replicate D15++, Day 15 in vitro derived PGCs from GCNF +/+ Oct4-GFP ESC, 1 replicate D15--, Day 15 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate D12++, Day 12 in vitro derived PGC from GCNF +/+ Oct4-GFP ESC, 1 replicate D12--, Day 12 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate EpiSC, EpiESC, 2 replicates
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