Papain and dnase

DMEM/F-12, antibiotics and antimycotics were obtained from Gibco Laboratories (Grand Island, NY, USA), papain and DNase from Worthington Biochemical Corporation (Lakewood, NJ, USA) and FBS from Lonza (Basel, Switzerland). Calcium Green-1 AM was purchased from Molecular Probes (Eugene, OR, USA), tetrodotoxin from EMD Chemicals (San Diego, CA, USA), and ω-conotoxin, ω-agatoxin and SNX-482 from Alomone Labs (Jerusalem, Israel). DMPP, poly-d-lysine, fibronectin, collagen and nifedipine were obtained from Millipore Sigma (St. Louis, MO, USA). All other chemicals were reagent grade and purchased from standard commercial sources.

iPS cell lines were obtained from public biobanks (GM25256-Corriell Institute; NDS00262, NDS00209-NINDS) and maintained in mTeSR1 media (StemCell Technologies) on Matrigel (Corning). iPS cells were fed daily and split every 4–7 days using ReLeSR (StemCell Technologies) according to the manufacturer’s instructions. Differentiation of iPS cells into motor neurons was carried out as previously described^{41 (link)}. In brief, iPS cells were dissociated and placed in ultra-low adhesion flasks (Corning) to form 3D spheroids in media containing DMEMF12/Neurobasal (Thermo Fisher), N2 Supplement (Thermo Fisher), and B-27 Supplement-Xeno free (Thermo Fisher). Small molecules were added to induce neuronal progenitor patterning of the spheroids, (LDN193189, SB-431542, Chir99021), followed by motor neuron induction (with retinoic acid, Smo agonist and DAPT). After 14 days, neuronal spheroids were dissociated with Papain and DNAse (Worthington Biochemical) and plated on poly-d-lysine/laminin coated plates in Neurobasal Medium (Thermo Fisher) containing neurotrophic factors (BDNF, GDNF and CNTF; R&D Systems). For viral transductions, neuronal cultures were incubated for 18 h with media containing lentivirus particles for shScramble, or shTDP-43. Infection efficiency of over 90% was assessed by RFP expression. Neuronal cultures were analysed for RNA and protein 7 days post transduction.