Motor Neuron Differentiation and Maturation Protocol

Motor neuron differentiation was performed as described before, with some modifications^{32 (link)}. iPSC clones were treated with collagenase type IV to form small clusters and resuspended in Essential^TM 8 medium. During the first 2 days, medium was changed every day with Neuronal basic medium (DMEM/F12 plus Neurobasal medium with N2 and B27 supplement without vitamin A) supplemented with 40 μM SB431542 (Tocris Bioscience), 0.2 μM LDN-193189 (Stemgent), 3 µM CHIR99021 (Tocris Bioscience), and 5 µM Y-27632 (Merck Millipore). From day 3 on, 0.1 µM retinoic acid (Sigma) and 500 nM SAG (Merck Millipore) was added. From day 8 on, BDNF (10 ng/ml, Peprotech) and GDNF (10 ng/ml, Peprotech) were added. DAPT (20 µM, Tocris Bioscience) was added on day 9. Floating clusters were dissociated into single cells for plating on day 11 by using 0.05% trypsin (Gibco^TM). Motor neuron progenitors were subsequently plated on laminin (20 μg/ml)-coated 12-well plates at 0.5–2 × 10⁵ cells per well. From day 17 on, the cells were switched to motor neuron maturation medium supplemented with BDNF, GDNF, and CNTF (each 10 ng/ml, Peprotech) to keep long term cultures. Media were changed every other day by replacing half of the medium. For rescue experiments, motor neurons were treated overnight with either 1 µM Tubastatin A (Sigma), 1 µM ACY-738 (Acetylon Pharmaceuticals Inc., Boston, USA) or an equivalent amount of DMSO.