B27 with vitamin a

0.5X DMEM/F12 (ThermoFisher), 0.5X Neurobasal (ThermoFisher), 1X B27 with Vitamin A (ThermoFisher), 1X N2 (ThermoFisher), 5 μg/ml Cholesterol (Sigma-Aldrich), 1 mM Creatine (Sigma-Aldrich), 100 μM Ascorbic Acid, 0.5 mM cAMP (Sigma-Aldrich), 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 μg/ml Laminin, 0.5 mM Glutamax (ThermoFisher), 1X Normocin (InvivoGen), 50 U/ml Penicillin-Streptomycin (ThermoFisher).

Fibroblasts from neurotypical individuals were reprogrammed to induced pluripotent stem cells using lentiviral (9429) or non-integrating Sendai (Invitrogen) vectors^{12 (link)}. NPCs and neurons were generated following methods outlined by Brennand and Livesey^{91 (link),97 (link)}. Briefly, hiPSC-derived NPCs were plated on Matrigel (Fisher) or Poly-Ornithine (Sigma)/ Laminin (Invitrogen) and passaged 1:6 every 4 days. NPCs (< passage 14) were cultured in NPC medium (DMEM/F12 + Glutamax (Invitrogen), 1 × N2 (Invitrogen), 1X B27-Vitamin A (Invitrogen), 1ug/ml Laminin (Invitrogen), 20 ng/ml FGF-2 (Peprotech) and dissociated in Accutase (Fisher) for 5 min at 37 °C. hiPSC-derived neurons were cultured on Matrigel in Neuron Medium (DMEM/F12 + Glutamax, 1 × N2, 1X B27 with Vitamin A (Invitrogen), 20 ng/ml BDNF (Shenandoah Biotechnology), 20 ng/ml GDNF (Shenandoah Biotechnology), 1 mM dibutryl-cyclic AMP (Sigma), and 200 nM ascorbic acid (Stem Cell Technologies).

Dissociated cells (tumor-derived and normal NSCs) were plated at 50 000 cells/well in 6 well plates (Nunc, VWR Norge) coated with 15 μg/ml poly-L-ornithine (Sigma, St. Louis, MO). DMEM/F12 medium was augmented with fetal calf serum (3.75 %; PAA Laboratories, Pasching, Austria) and 25 μl/ml B-27 with vitamin A (Invitrogen), 1 μg/ml laminin and 10 μl/ml of Pen/Strep. All cultures were kept in an incubator at 5–6 % CO₂, pH 7.2-7.4 [40 (link)].

After 3 days of proliferative culture, the floating ENSdN (fresh or thawed) were harvested and utilized either for differentiation directly or dissociated in accutase (PAA Laboratories GmbH) for 20 min and triturated using consecutively 23 and 27 gauge needles, 3 times each. To avoid shear stress, aspiration of the tissues was performed slowly. After dissociation, the cells were centrifuged (120x g, 5 min), the supernatant removed and the pellet resuspended in 1 mL medium. The intact or dissociated ENSdN were plated on poly-L-lysine-laminin coated coverslips in 24 or 12 well plates (Becton Dickinson) in culture medium for differentiation (Neurobasal A (Gibco) supplemented with 2% B27+ (with vitamin A, Gibco), 1% albumin (Sigma-Aldrich), 0.25% 2-mercaptoethanol (50 mM, Invitrogen), 0.12% glutamine (200 mM, Sigma-Aldrich), GDNF (10 ng/mL, Tebue), neuturin (10 ng/mL Tebue), gentamycin and metronidazole (100 μg/mL) and 1% penicillin/streptomycin (PAA Laboratories GmbH)). In case of functional imaging, 10% of FCS (Gibco) was added to the medium in order to help cell attachment.

NB medium: neurobasal medium (Gibco, Waltham, MA, USA, 21103-049) supplemented with 2% B27 with vitamin A (Gibco, Waltham, MA, USA, 17504-044), 18 mM Hepes (Gibco, Waltham, MA, USA, 15630-056), 0.25× Glutamax (Gibco, Waltham, MA, USA, 35050-038), 1% Pen/Strep (Sigma-Aldrich, Saint Louis, MO, USA, P0781).
N2 medium: Dulbecco’s modified Eagle medium/nutrient mixture F12 medium (DMEM/F12) (Life Technologies, Waltham, MA, USA, #21331-046) supplemented with 1% N2 (Thermo Fisher Scientific, Waltham, MA, USA, #17502-048), 1× NEAA (Thermo Fisher Scientific, Waltham, MA, USA, #11140-035), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA, #25030-024), 2 μg/mL heparin (Sigma-Aldrich, Saint Louis, MO, USA, #H3393-50KU), 1% Pen/Strep.
N2B27—vit A: 1:1 DMEM/F12 with neurobasal medium supplemented with 0.5% N2, 1% B27 without vitamin A (Thermo Fisher Scientific, Waltham, MA, USA, #12587-010), 2.5 μg/mL insulin (Sigma-Aldrich, Saint Louis, MO, USA, #I9278), 0.5% L-glutamine, 0.5× NEAA, 50 μM beta-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA, #21985023), 1% Pen/Strep
N2B27 + vit A: 1:1 DMEM/F12 with neurobasal medium supplemented with 0.5% N2, 1% B27 with vitamin A, 0.25 mg/mL insulin, 0.5% L-glutamine, 0.5× NEAA, 50 μM beta-mercaptoethanol, 1% Pen/Strep