Stempro nsc sfm

NSCs were cultured using Gibco PSC neural induction medium and StemPro NSC SFM (Life Technologies, USA), and neural differentiation was performed according to the manufacturer's instructions. Briefly, about 24 h after hESCs and iPS cells were split into six-well plates, the culture medium was switched to Gibco PSC neural induction medium containing neurobasal medium and Gibco PSC neural induction supplement. The neural induction medium was changed every other day from the beginning of neural induction. The neural induction medium was changed every day after 4 days of neural induction. At day 7, primitive NSCs were dissociated using Accutase (Life Technologies) and plated on Geltrex-coated dishes in an NSC expansion medium containing 50% neurobasal medium, 50% Advanced DMEM/F12, and neural induction supplement. The NSC expansion medium was changed every other day until NSCs reached confluence at day 5 after plating of primitive NSCs [17 (link)]. For neural differentiation, NSCs were plated onto laminin (10 μg/mL; Life Technologies) coated six-well chamber slides at a density of 5 × 10⁴ cells/cm² in a neuronal differentiation medium consisting of neurobasal medium, B-27, and GlutaMAX (Life Technologies). The culture medium was changed every 2-3 days, according to the manufacturer's instructions (Life Technologies).

A172 and U251 cells were obtained from the European Collection of Cell Cultures (EACC, UK) in 2014 and authenticated in 2020 by Cell Bank Australia using short tandem profiling. Cells were cultured in DMEM supplemented with 10% FBS and Antibiotic-Antimycotic solution (Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO₂. Patient-derived HW1, RN1, MMK1 and RKI1 glioblastoma stem cells^{34 (link)} were cultured in KnockOut DMEM/F-12 supplemented with StemPro NSC SFM, 2 mM GlutaMAX-ICTS, 20 ng/mL EGF, 10 ng/mL FGF-β and Antibiotic-Antimycotic solution (all Life Technologies) as adherent cells on flasks coated with MatriGel (Corning, NY, USA). The protocols were approved by the Human Ethics Committee of the Royal Brisbane & Women’s Hospital (RBWH 2004/161). Cell cultures were routinely tested for mycoplasma infection and culturing did not exceed 15 passages.

For preparation of cortical neural stem cells [54 (link)], cells were plated into Nunc 90 mm petri dishes, previously coated with Cell Start (Gibco), at a seeding density of 500 x10⁵ cells/ml. Neural stem cells were cultured in StemPro NSC SFM composed of: Knockout D-MEM / F12; Glutamax (2mM); bFGF (20ng/ml); EGF (20ng/ml); StemPro Neural Supplement (2%); all from (Gibco). Under these growth conditions at 7 DIV cells were proliferative and were Nestin and Ki67 positive.

Twelve patient-derived glioblastoma cell lines were kindly provided by the QIMR Berghofer Medical Research Institute (Brisbane, Australia). The cell lines are fully characterised with publicly available molecular and patient data, published by Stringer et al. [13 (link)]. Cells were grown as adherent monolayers in Matrigel^® (Corning^®, Corning, NY, USA)-coated tissue culture flasks in StemPro^® NSC SFM (Gibco^TM, Waltham, MA, USA) containing 100 I.U./mL penicillin and 100 µg/mL streptomycin (Gibco^TM, Waltham, MA, USA), and incubated at 37 °C in 5% CO₂/95% humified air. Cells were passaged using StemPro^® Accutase^® solution (Gibco^TM, Waltham, MA, USA) for detachment of adherent cells. TMZ was purchased from Sigma-Aldrich (Burlington, MA, USA), aliquoted in dimethyl sulfoxide (DMSO) (100 mM) and stored between 2 and 8 °C. RT was delivered using a medical linear accelerator (LINAC) at GenesisCare, Gateshead NSW (Australia) or RS-2000 Small Animal Irradiator (Rad Source, Buford, GA, USA).

Media (DMEM/F12, MEM, Neurobasal medium), GlutaMAX^TM, StemPro^® NSC SFM, B27 supplement, N2 supplement, poly-L-ornithine (PLO), and Lipofectamine 2000 were purchased from Invitrogen. Horse serum (HS) was obtained from HyClone Laboratories. Apotransferrin, biotin, bovine serum albumin (BSA), 5-bromo-2′-deoxyuridine (BrdU), cytosine arabinoside (Ara-C), diethylpyrocarbonate (DEPC), hydrocortisone, insulin, N-acetyl-cysteine (NAC), poly-D-lysine (PDL), sodium ampicillin, sodium pyruvate, sodium selenite, triiodothyronine (T3), and lysolecithin were obtained from Sigma. Ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and platelet derived growth factor-AA (PDGF-AA) were obtained from ProSpec. The antibodies used in this study are listed in Table 1.