Feline Lindhout

2 50 AUTHOR CONTRIBUTIONS FWL, RK and SP designed the research, conducted experiments, and wrote the article together with LJH. RS conducted and analyzed the mass spectrometry experiments supervised by MA, LJH performed and interpreted the electrophysiology data supervised by CJW, RK and BLS provided and interpreted the RNA sequencing analysis. HDM edited the article. CCH designed the experimental plan, supervised the research and wrote the article. DECLARATION OF INTERESTS Casper Hoogenraad is an employee of Genentech, Inc., a member of the Roche group. The authors declare that they have no additional conflict of interest. MATERIALAND METHODS hiPSC-derived neuronal cell culture Human iPSC-derived cortical neuronal progenitor cells (NPCs; ax0016, Axol Bioscience) from a female newborn donor were obtained, expanded and differentiated following Axol Bioscience protocols (Human iPSC-derived Neural Stem Cells, Protocol version 5.0). For expansion of hiPSC-derived NPCs, cells were thawed and quenched with Neuronal Expansion-XF Medium (ax0030, Axol Bioscience), centrifuged (200 g, 5 min), resuspended in Neuronal Plating-XF medium (ax0033, Axol Bioscience), and plated (~500k per well) on six- wells plates pre-coated with freshly-thawed SureBond (ON, 37°C; ax0041, Axol Bioscience) in PBS at 37°C with 5% CO2. Medium was replaced on the next day by Neuronal Expansion- XF Medium supplemented with EGF (20 ng/ml; AF-100-15, Peprotech) and FGF (20 ng/ml; 100-18B, Peprotech). Medium was refreshed every two days, and cells were passaged when cultures reached a 70-80% confluency. For passaging, cells were washed once with PBS, incubated with Unlock (5 min, 37°C; ax0044, Axol Bioscience), quenched with Neuronal Expansion-XF Medium, and centrifuged (200 g, 5 min). Cell pellets were resuspended in Neuronal Plating-XF medium and plated in a 1:3 ratio on six-wells plates pre-coated with freshly-thawed SureBond (see above). After three passaging rounds, cells were frozen in KnockOut Serum Replacement (10828028, Life Technologies) with 10% DMSO and stored in liquid nitrogen. For neuronal differentiation of hiPSC-derived NPCs, cells were thawed and quenched with Neuronal Expansion-XF Medium, centrifuged (200 g, 5 min), resuspended in Neuronal Plating-XF medium, and plated on 12 mm (~100k per well) or 18 mm (~200k per well) pre-coated glass coverslips in respectively a 24-wells or 12-wells plate at 37°C with 5% CO2. Coating of coverslips was performed directly before plating: coverslips were first incubated with ReadySet (45 min, 37°C; ax0041+, Axol Bioscience), washed four times with sterilized water, and incubated with freshly-thawed 1x SureBond (1h, 37°C; ax0041+, Axol Bioscience) in PBS. After 24 hours (day 1), the medium was fully replaced by Neuronal Maintenance-XF Medium (ax0032, Axol Bioscience), and after another 24 hours (day 2) by Neuronal Differentiation-XF Medium (ax0034, Axol Bioscience). Three days later (day 5), half of the medium was replaced by Differentiation-XF Medium. Next day (day 6), half of the medium was replaced by Neuronal Maintenance-XF Medium, again one day later (day 7),