Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 89 interesting to direct future research in addressing the long-term functional consequences of an altered axonal microtubule network due to centrosome dysfunction. ACKNOWLEDGEMENTS We thank Dr. Didier Trono for kindly providing the lentiviral vector. This work was supported by the Netherlands Organization for Scientific Research (NWO-ALW-VICI, 865.10.010, CCH), the Netherlands Organization for Health Research and Development (ZonMW-TOP, 912.16.058, CCH), the European Research Council (ERC) (ERC-consolidator, 617050, CCH), and the research program of the Foundation for Fundamental Research on Matter (FOM, #16NEPH05, CJW). AUTHOR CONTRIBUTIONS FWL initiated the study, designed and performed experiments, and wrote the article together with SP, RK and LJH. SP designed and performed experiments for the growth cone analysis, RK provided the microtubule live-imaging data, RS conducted the mass spectrometry experiments supervised by MA, LJH performed and interpreted the electrophysiology experiments supervised by CJW, and NS provided the STED microscopy data supported by HDM. HDM edited the article. CCH designed the experimental plan, supervised the research and edited 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 Human iPSC-derived neuron culture Human iPSC-derived neuronal stem cells (ax0016, Axol Bioscience) were purchased, expanded and subjected to neuronal differentiation and maintenance as previously prescribed (Lindhout et al. 2020). In brief, for expansion of human iPSC-derived NSCs, cells were plated on SureBond-coated plastic wells of a six-wells plate, and kept in Neuronal Plating-XF medium (ax0033, Axol Bioscience) at 37°C with 5% CO2. On the next day, medium was replaced by Neuronal Expansion-XF Medium supplemented with EGF (20 ng/ml; AF-100-15, Peprotech) and FGF (20 ng/ml; 100-18B, Peprotech), which was refreshed every two days. For cell passaging, cells were washed once with PBS, dissociated with Unlock (ax0044, Axol Bioscience), and plated and maintained on pre- coated wells as described above. For neuronal differentiation, human iPSC-derived NSCs were plated on ReadySet/SureBond-coated glass coverslips and kept in Neuronal Plating- XF medium (ax0033, Axol Bioscience) at 37°C with 5% CO2. Cells were plated at a density of ~40k cells per well for control and ~60k cells per well or Centrinone-B-treated conditions, to compensate for the decreased proliferating rates caused by dysfunctional centrosomes. Neuronal induction was induced two days after plating by gradually replacing the medium