Fibroblasts reprogramming was performed following a previously described protocol (Yang et al. 2017; doi:10.1038/nature24052). Briefly, fibroblasts were electroporated with reprogramming vectors expressing Oct4, c-Myc, Klf4, Sox2 (OCKS 4F, 5µg) and Rarg, Lfh1 (RL 2F, 5.0 µg) using Amaxa Nucleofector (Lonza, Germany) then plated on SNL feeders with M15 media (Knockout DMEM Invitrogen, 15% Fetal Bovine Serum Hyclone, 1X Glutatamin-Penicillin-Streptomycin Invitrogen, 1X non-essential amino acids Invitrogen). Upon the appearance of colonies, media was replaced with EPSCM (DMEM/F12 Invitrogen, 20% Knockout Serum Replacement Invitrogen, 1X Glutamin-Penicillin-Streptomycin, 1X non-essential amino acids Invitrogen, 0.1 mM β Mercapto-ethanol Sigma, 106 U/ml hLIF Millipore supplemented with the following inhibitors: CHI99021 Tocris 1µM, JNK Inhibitor VIII Tocris 4 µM, SB203580 Tocris 10µM, A-419259 Santa Cruz 1µM and XAV939 Stratech 1µM). Colonies were picked and plated in 24 well SNL feeders’ plates for expansion and characterization. For embryoid bodies (EB) generation, EPSC lines were harvested and feeders removal was performed in T25 flasks for 30 minutes at 37 degrees to allow feeders to attach to the bottom and floating EPSC harvested were transferred in ultra-low attachment 96 well plates at different densities (60K, 45K, 35K and 20K) in EB media (DMEM/Knockout Invitrogen #10829-018, Fetal Bovine serum Gibco #16141061, non-essential amino acid Invitrogen #11140, glut-pen-strep Invitrogen #10378 and β mercapto-ethanol) changed every other days. After 7 days, they were transferred in 24 well plates on gelatine coated glass coverslips and after 10 to 14 days, cells were fixed with PFA 4%, 30 minutes at room temperature and immunocytochemistry was performed. For iNSC induction, a commercially available kit was used following the manufacturer’s protocol (Gibco, #A1647801). Briefly, iPSC colonies were harvested, feeders removal was performed, as previously described for EB formation, and plated at density from 0.25 to 0.75 x106 per well of 6 well geltrex coated plates in EPSCM and Rock inhibitor 10μM (Stem cell technology #Y27632). The next day, the media was replaced with Gibco neural induction media (Neurobasal media #211030, pen-strep 1X and neural induction supplement 1X #A1647801, rock inhibitor 10μM), then changed every 2 days until day 7, cells were passaged at the density of 1x105/cm2 in Gibco neural expansion media (Neurobasal 0.5X, Advanced DMEMF/12 0.5X #12634, pen-strep 1X and neural induction supplement 1X, rock inhibitor 10μM), then media was changed every 2 days and 2-3 passages were performed with accutase (Millipore # SCR005) to establish the iNSC lines. Cells were frozen in synth-a-Freeze cryopreservation medium (Gibco #A12542). GIBCO® Human Neural Stem Cells (H9 hESC-Derived, #N7800-100) were used as control, cultured in StemPro® NSC SFM (Cat. no. A10509-01).
Growth protocol
Patient consent and ethical approval was available for the study (08/H0716/16 Amendment 1 17/10/2014). GIC were isolated from bulk tumor following a previously described protocol (Pollard et al 2009; doi:10.1016/j.stem.2009.03.014). Fresh GBM tissue was sliced and triturated with razor blade, dissociated with Accumax (Sigma, A7089) at 37˚C for 10 mins then filtered through a 70μm cell strainer. Dissociated cells were plated on laminin-coated 6-well plate in NeuroCult NS-A Proliferation kit media (STEMCELL, 05751), heparin (2 μg/ml; Gibco 12587-010), mEGF (20ng/ml, Prepro Tech, 315-09) and hFGF (10ng/ml; Prepro tech, AF-100-18B). Established cells were passaged when 70% confluent, frozen in Stem Cell Banker (Ambsio ZENOAQ, 11890) and stored in liquid nitrogen. Cell lines used in this study are primary lines derived from human tumors, they have been characterised by transcriptomic profiling and cultured according to current practice, including contamination assessment. Fibroblasts cultures were established from small strips of dura mater, collected at surgery. Tissue was minced with a scalpel then spun down and resuspended in trypsin for 5 minutes at 37 degrees. To stop the reaction fresh fibroblast media (DMEM, Glutamax, 10% Foetal calf serum, 2% L-Glutamin and 1% penicillin-streptomycin) was added. Samples were centrifuged and resuspended in fresh media, then plated in 6 well plates (Corning #BC010). Media was topped up frequently during the first week then changed every other day.
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted from cell pellets using the RNA/DNA/Protein Purification Plus kit (NORGEN, #47700), following the manufactured protocol.
Label
Cy3, Cy5
Label protocol
Standard Illumina Protocol
Hybridization protocol
Bisulphite converted DNA was amplified, fragmented and hybridized to Illumina Infinium HumanMethylation850 BeadChip
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
Description
GBM052_P4n5
Data processing
Raw array data were first pre-processed using the ChAMP package in R to remove failed detections and probes with known design flaws, before normalization using the SWAN algorithm
Comparative analysis of glioblastoma initiating cells and patient-matched EPSC-derived neural stem cells as a discovery tool and drug matching strategy [array]
Comparative analysis of glioblastoma initiating cells and patient-matched EPSC-derived neural stem cells as a discovery tool and drug matching strategy
