Human iPSC-derived motor neuron differentiation and maintenance

Motor neurons (MN) were generated using a modified version of previously established protocols^50,51. iPSC colonies were dissociated into single cells using Accutase. Cells were then seeded into ultralow attachment dishes in mTESR1 medium supplemented with ROCK inhibitor, Y-27632 (10 µM; catalog #1254 Tocris) and basic fibroblast growth factor (FGF-2; 20 ng/mL; catalog #233-FB-010 Tocris) for the first 24 h to allow for embryoid body (EB) formation. The following day, ROCK inhibitor was removed, and fresh mTESR1 was added to the cultures. Forty-eight hours after EB aggregation, cells were switched to MN Differentiation Medium: Advanced DMEM/F-12 (catalog #12634010 ThermoFisher) and NB Medium (50:50 v/v), 1× N-2 supplement, 1× B27 + Insulin, 1× GlutaMAX (catalog #A1286001 ThermoFisher), 1× pen/strep, and 0.1 mM 2-mercaptoethanol (catalog #31350010 ThermoFisher). To specify neural patterning, dual SMAD inhibition was used with small molecules SB431542 (10 µM) and LDN193189 (100 nM) from day 0 to day 6 of differentiation. From day 0 to day 4 the glycogen synthase kinase 3 inhibitor, CHIR99021 (3 µM; catalog #4423 Tocris) was added to increase the population of Olig2 positive MN progenitors. Beginning on day 2, MN specification was induced with 1 µM All-trans Retinoic Acid (at-RA; catalog #0695 Tocris) and 1 µM Smoothened Agonist (SAG; catalog #4366 Tocris) until day 16. The y-secretase inhibitor, (2S)-N-[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT, catalog #2634 Tocris) was used at 10 µM in conjunction with neurotrophic factors BDNF and GDNF (10 ng/mL each) starting on day 8 until day 16 of differentiation, with 50% medium changes every 2 days. Spheroids were then dissociated with a solution containing 0.25% Trypsin-EDTA and DNAse (25 µg/mL; catalog #18047019 ThermoFisher), and plated onto poly-l-ornithine (25 µg/mL; catalog #P2533-10MG Sigma-Aldrich), Fibronectin (10 µg/mL; catalog #11051407001 Sigma-Aldrich), and Laminin (10 µg/mL)-coated plates in medium supplemented with BDNF and GDNF (10 ng/mL each). See Supplementary Table 5 for detailed protocol steps and refer to Supplementary Fig. 2 for differentiation protocol outline and representative images of MN cultures.