Cerebral organoids were generated from iPSC as previously described (18 (link)). For control lines, EBs were generated from 9000 cells each. For patient lines, EBs were generated from 9000 and 18 000 cells with no visible difference in size or development. Two control- and two SPG11-iPSC lines (SPG11-1 and SPG11-2) were analyzed. Controls were grouped. Cerebral organoids were cultured for 40 to 61 days (6 and 9 weeks) at 37°C under 5% CO2 and atmospheric oxygen. For the rescue experiment, control- and SPG11 organoids were treated with 1 μM of the GSK3 blockers CHIR99021 and tideglusib and kept in culture for 40 to 60 days (6 and 9 weeks). Compounds were replaced every 2–3 days (Supplementary Material, Fig. S4D ).
For the neurosphere assay (3 (link)), NPCs were kept in suspension in 96 well ultra-low attachment plates (Corning, Amsterdam, the Netherlands) for 72 h at a density of 5 × 104 cells/cm2 under proliferative conditions. For the analysis of NPC migration, neurospheres grew attached to polyornithine/laminin (Invitrogen, Carlsbad, California, United States) coated coverslips for 48 h (Fig. 2A ). Spheres generated from six SPG11-NPC lines (SPG11-1, SPG11-2 and SPG11-3) were compared to controls.
For the neurosphere assay (3 (link)), NPCs were kept in suspension in 96 well ultra-low attachment plates (Corning, Amsterdam, the Netherlands) for 72 h at a density of 5 × 104 cells/cm2 under proliferative conditions. For the analysis of NPC migration, neurospheres grew attached to polyornithine/laminin (Invitrogen, Carlsbad, California, United States) coated coverslips for 48 h (
