Project description:Gliomas have been proposed to be driven by a population of neural stem-like cells. We isolated a panel of novel human glioma cell lines using adherent neural stem cell conditions. The normal human foetal (hf) NS cells and the tumorigenic glioma NS cell lines were expanded using growth factors EGF and FGF in adherent culture conditions. In these conditions apoptosis and differentiation are suppressed resulting in more homogeneous populations of stem cells than has been reported previously. We included parallel primary biopsies of non-malignant brain tissue ('Normal Brain').
Project description:Gliomas have been proposed to be driven by a population of neural stem-like cells. We isolated a panel of novel human glioma cell lines using adherent neural stem cell conditions. The normal human foetal (hf) NS cells and the tumorigenic glioma NS cell lines were expanded using growth factors EGF and FGF in adherent culture conditions. In these conditions apoptosis and differentiation are suppressed resulting in more homogeneous populations of stem cells than has been reported previously. We included parallel primary biopsies of non-malignant brain tissue ('Normal Brain'). Experiment Overall Design: Cell lines were expanded until 70-90% confluent and then harvested for RNA extraction. All cell lines were gorwn in identical culture media. We also include 'Normal Brain' samples which are regions of the adult human cortex.
Project description:The DNA methylation profiles of Glioma Stem Cell (GSC) lines were investigated in order to find the stem cell signature associated to glioblastoma (GBM). This goal was achieved through the comparison of GSC methylation data with FFPE-GBM biopsies and human foetal Neural Stem Cell (NSC) lines profiles.
Project description:The DNA methylation profiles of Glioma Stem Cell (GSC) lines were investigated in order to find the stem cell signature associated to glioblastoma (GBM). This goal was achieved through the comparison of GSC methylation data with FFPE-GBM biopsies and human foetal Neural Stem Cell (NSC) lines profiles. GSC lines: 3 (GBM2, G144, G166). FFPE-GBM biopsy pool: FFPE-GBM pool: 1 pool from 5 GBM biopsies. Human foetal NSC lines: 2 (CB660 from forebrain; CB660SP form spinal cord). Methylated DNA from each sample was enriched with the immunoprecipitation method using 5-methylcytosine antibody (Eurogentec). Immunoprecipitated DNA (IP-DNA) and total DNA were labeled and hybridized on Agilent Human CpG Island ChIP-on-Chip Microarray 244K. IP-DNA were labeled with Cy5 while the matching total DNA were labeled with Cy3.
Project description:To identify a novel miRNA that is aberrantly expressed in GICs, we analyzed differences in miRNA expression between the human GICs and glioma cell lines and neural stem cells by miRNA microarrays. We examined the miRNA expression profiles of five human GICs that were obtained from human glioma samples and two human glioma cell lines, U87 and U251, and NSC (neural stem cells) as a control.
Project description:In this study, we combined a genome-wide analysis of the Polycomb protein Bmi1 and an in vivo RNAi screening to identify critical targets whose repression in neural progenitor and Malignant Glioma cells enables normal and aberrant self-renewal. Bmi1 ChIP-sequencing generated in 2 primary adult mouse Neural Progenitor cell lines, 1 mouse astrocytic cell line, 1 mouse Glioma initiating cell line, and 1 mouse Glioma cell line. In human cell lines, Bmi1 ChIP-seq was performed for foetal Neural Progenitor Cells, and 2 Glioblastoma Stem-like cells. ChIP-seq for AP-1 in one Glioblastoma stem-like cell line. ChIP-sequencing were generated using either Illumina GxII or HiSeq. We have further performed RNA-seq of adult Ink4a/Arf -/-;BMI1or GFP dox-inducible shRNA mNPC with a doxycycline inducible shRNA targeting BMI1 or GFP (as control). These cells were either treated with doxyclyine for 48h to ablate BMI1 and GFP expression or further subjected to short-term differentiation using either BMP4 (10ng/ml or 50ng/ml for 3 or 6hrs), or FBS (1 or 10% for 3 or 6hrs). Additional mouse RNA-seq were performed in two biological replica of adult mouse brains (littermate) with either wild-type FVB background or BMI1 -/-. Finally, we performed RNA-seq in human Glioblastoma stem-like cells (most likely belonging to the mesenchymal GBM subtype) treated with BMP7, Arvanil and doxycycline or EtOH treated as ctrl.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)