Laminin

PIGPCs were isolated as previously described [39 (link)] and grown in DMEM (Life Technologies) with 10% fetal bovine serum (FBS) and 1% PenStrep solution (Corning). U3046MG, U3035MG, U3082MG, U3084MG, and U3065MG cells were obtained from the Human Glioblastoma Cell Culture Resource (HGCC) and cultured as described previously [23 (link)] in Neurobasal (GIBCO) and DMEM/F12 with Glutamax media (Life Technologies), 1:1 mix, with 1% PenStrep, N2 and B27 (Life Technologies), 10 ng/mL epidermal growth factor (EGF), and 10 ng/mL fibroblast growth factor (FGF) (Peprotech). Cells were dissociated by Accutase (ThermoFisher) treatment, and grown as a monolayer on plastic dishes coated with polyornithine (Sigma) and laminin (Biolamina). U251MG and T98G cells were obtained from ATCC and grown in DMEM with 10% FBS and 1% PenStrep solution. Cells were used within ten passages. Mycoplasma contamination was tested every 3 months.
Hypoxia (1%O₂) was generated in a Whitley H35 Hypoxystation (Don Whitley Scientific, Bingley, UK).
Reagents: TAPI-2 (Sigma), MI1 (Tocris Bioscience), LY294002 (Sigma), added 24 h pre-hypoxia exposure.

The trigeminal ganglions (TGs) were dissected from the mice and placed in ice-cold Hanks’ balanced salt solution (HBSS). After cutting up the TGs, we added them to HBSS containing 67 mg/mL cysteine, 0.2% saturated sodium bicarbonate solution, and 2 mg/mL papain at 37°C. Then, HBSS containing type II collagenase was used to digest trigeminal ganglion neuron (TGN) cells. Percoll density gradient centrifugation was performed on the cell suspension. The cells were planted in Leibovitz's L-15 medium (Gibco) containing 10% FBS and 1% penicillin–streptomycin in a 48-well plate already coated with laminin (BioLamina, Sundbyberg, Sweden). This was transferred to a cell incubator, and we added Neurobasal-A medium (Gibco) with 2% B27 (Gibco) after 3 hours.

Human paediatric glioblastoma (GBM) cell lines SF188 and KNS42 have been extensively characterised previously [1 (link)]. KNS42 harbours a histone H3.3 (H3F3A) G34V mutation whilst SF188 is H3 wild-type [4 (link)]. These cell lines were grown as monolayers in DMEM/F12 Ham’s medium (Life Technologies) supplemented with 10% fetal calf serum (GIBCO) and antibiotics at 37 °C and 5% CO₂. Paediatric pHGG cell lines HSJD-DIPG-012 and HSJD-GBM-002 were grown according to the in house protocol from the Montero lab. Cells were cultured as monolayers in flasks pre-treated with laminin (10 μg/ml, BioLamina). Tumour Stem Medium (TSM) base was prepared with 50% Neurobasal-A Medium, 50% D-MEM/F-12, 1% HEPES buffer solution (1 M), 1% sodium pyruvate MEM Life 100MM(CE), 1% MEM non-essential amino acids solution 10 mM (100×), 1% GlutaMAX-I supplement and 1% antibiotic-antimycotic (100×) (LifeTechnologies). 10% FBS was added fresh to TSM base media for preparing a differentiation TSM media. Cells were grown as monolayers in this media at 37 °C with 5% CO₂.

Mechanical dissociation with Accutase (ThermoFisher) was used to prepare single cell suspensions. Cells were counted using a hemocytometer. For colony formation assay, 350 cells were seeded in 5 cm dishes coated with polyornithine (Sigma) and laminin (Biolamina). U3082MG, U3084MG and U3065MG cells were cultured for 14 days while PIGPCs for 8 days, under the indicated conditions, then washed in PBS and fixed using 4% paraformaldehyde. Cells were stained using 0,01% crystal violet/H2O. Wells were washed gently in water, then air-dried for 24 hours. Images were acquired with a Fujifilm LAS 3000 Imager.
Sphere formation assay was performed with the hanging-drop method. 10 cells in 35 µL drops were seeded on the lid of a 48 well plate and grown under the indicated conditions for 2 weeks. For secondary sphere assay, primary spheres were pooled, pelleted, dissociated with Accutase and reseeded at the indicated conditions. Wells with spheres were manually counted and images were acquired with a Zeiss AX10 inverted microscope.