Cell culture and differentiation of NT2 cells

Ntera2/D1 (NT2) (ATCC, LCG standards) and HEK293T cells (human embryonic kidney cells, provided by Dr Alexander Deutsch, Division of Haematology, Medical University Graz) were maintained in high glucose Dulbecco’s modified Eagle Medium (DMEM, Sigma-Aldrich) containing 10% Fetal bovine serum (FBS, Sigma-Aldrich), 1% Non-Essential Amino Acids (NEAA, Sigma-Aldrich) and 1% Penicillin-Streptomycin (Sigma-Aldrich) at 5% CO₂ and 37°C.

Differentiation of NT2 cells was performed as described before [14]. Briefly, 5x10⁶ undifferentiated NT2 cells were seeded into ultra-low attachment (ULA) flasks (VWR International) with 10 μM retinoic acid (RA, Sigma-Aldrich). Medium was changed every 2 days until day 15 and 10 μM RA was added. On day 15, medium was changed without adding RA. On day 17, neurospheres were plated onto flasks coated with a reduced growth factor basement membrane extract (Geltrex, Life Technologies) and cultivated in the presence of a mitosis inhibitory mixture (10 μM 5-Fluoro-2-Desoxyuridine, 1 μM Cytosine-β-D-Arabinofuranoside and 10 μM Uridine, Sigma-Aldrich) until day 28 with medium changes on alternating days.