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: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: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:<p>We used massively parallel, paired-end sequencing of expressed transcripts (RNA-seq) to detect novel gene fusions in short-term cultures of glioma stem-like cells freshly isolated from nine patients carrying primary glioblastoma multiforme (GBM). The culture of primary GBM tumors under serum-free conditions selects cells that retain phenotypes and genotypes closely mirroring primary tumor profiles as compared to serum-cultured glioma cell lines that have largely lost their developmental identities.</p>
Project description:We cultured tumor cells from 22 GBM under medium conditions favoring the growth of neural stem cells. 11 out of 15 primary GBM contained a significant CD133+ subpopulation that comprised cells showing all hallmarks of neural stem cells. Cell lines derived from these CD133+ GBM showed a neurosphere-like, non-adherent growth pattern. In contrast, 4 out of 15 cell lines derived from primary GBM grew adherent in vitro and were driven by CD133- tumor cells that fulfilled stem cell criteria. In vivo, these GBM were characterized by a significantly lower proliferation index but similar GFAP staining as compared to CD133+ GBM. Gene arrays from 2x3 representative cells lines are given. Experiment Overall Design: Human glioblastoma cells cultured in DMEM supplemented with EGF, FGF, LIF, B27.
Project description:In this study, we explored the transcriptomic consequences of strong activation of the Notch pathway in embryonic human neural stem cells and in gliomas. For this we used a forced expression of the Notch intracellular domain (NICD). Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signalling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration which was accompanied by the sharp reduction of neural stem cell transcription factor expression (ASCL1, OLIG2, SOX2) while HEY1/2, KLF9, SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRb and a-smooth muscle actin (aSMA)) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (HB-EGF, IL8, PLGF), metalloprotease (MMP9) and adhesion proteins (VCAM-1, ICAM-1, ITGA9). In xenotransplantation experiments, contrasting with the infiltrative and poorly-vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly-disseminating but highly-vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. Embryonic human neural progenitors obtained from Lonza, the U87 glioma cell line, and glioma cancer stem cells (Gb4 and Gb7) characterized in Guichet et al. (Glia, 2013, 61(2), 225-39) were infected with lentiviruses expressing YFP or YFP-IRES-NICD (activated form of Notch receptor). After 48h, RNA were extracted for Affymetrix microarray analysis.
Project description:We cultured tumor cells from 22 GBM under medium conditions favoring the growth of neural stem cells. 11 out of 15 primary GBM contained a significant CD133+ subpopulation that comprised cells showing all hallmarks of neural stem cells. Cell lines derived from these CD133+ GBM showed a neurosphere-like, non-adherent growth pattern. In contrast, 4 out of 15 cell lines derived from primary GBM grew adherent in vitro and were driven by CD133- tumor cells that fulfilled stem cell criteria. In vivo, these GBM were characterized by a significantly lower proliferation index but similar GFAP staining as compared to CD133+ GBM. Gene arrays from 2x3 representative cells lines are given. Keywords: Cancer stem cell, CD133, glioblastoma