H3149 50ku

HuH-7 cells were kindly provided by Dr. Jerome Laurence (University of Sydney). HEK293 cells were obtained from ATCC. All cells were tested for mycoplasma and were mycoplasma-free. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11965-092) supplemented with 100 U/mL penicillin/100 μg/mL streptomycin (Sigma-Aldrich, P4458) and 10% FBS (Sigma-Aldrich, F9423-500mL, lot no. 16K598). For HuH-7, media were also supplemented with non-essential amino acids (Gibco, 11140-050). Cells were passaged using TrypLE express enzyme (Gibco, 12604-21). For transduction studies, cells were plated into 24-well plates in complete DMEM at 1 × 10⁵ cells per well and incubated overnight in a tissue-culture incubator at 37°C/5% CO₂. 16 h later, the vector stock was added to cells (at the indicated vg copies [vgc]/cell). For the heparin competition assay (Figure S12), cells were seeded at 10⁵ per well into 24-well plates at day 0 and transduced at the indicated vgc/cell. When indicated, heparin sodium salt (Sigma, H3149-50KU, lot no. SLBW2119) was supplemented from a 100× stock at 100 μg/mL (Figures S12A and S12B) or at 400 μg/mL (Figures S12C and S12D). After 72 h, the cells were harvested using TrypLE express and analyzed for GFP using BD LSRFortessa cell analyzer. The data were analyzed using FlowJo 7.6.1.

Human NSCs were derived by differentiating human iPSC HEL24.3^{45 (link)}, and HEL46.11 lines using small molecule cocktail as described elsewhere^{46 (link)}, with minor adjustments. Briefly, iPSCs were detached with StemPro Accutase (Thermo Fisher Scientific) and dissociated gently into single cells suspension in hES-medium in the presence of 5 μM ROCK inhibitor (ROCKi; Y-27632, Selleckchem), 10 μM SB431542 (SB; S1067, Selleckchem), 1 μM dorsomorphin (DM; P5499-5MG, Sigma), 3 μM CHIR-99021 (CHIR; Tocris) and 0,5 μM purmorphamine (PMA; 04-0009, Stemgent) After 2 days, medium was changed to N2B27 medium (DMEM/F12:Neurobasal (1:1) supplemented with N2 and B27 without vitamin A, NEAA, PenStrep (all Thermo Fisher Scientific) and heparin (2 µg/ml; H3149-50KU, Sigma)) containing the same small molecule cocktail as above. On day 4, SB and DM were withdrawn and 150 µM ascorbic acid (AA) was added to N2B27. On day 6, the neurospheres were dissociated with 1 ml pipette and plated on Matrigel in N2B27 media containing AA, CHIR and PMA (growth media). First two passages were split at 1:3 ratio and cells were plated into growth media containing 5 μM ROCKi, which was removed next day. Later passages were split with 1:10 and 1:20 ratio using StemPro Accutase. Media were changed every other day.