Differentiation of Stem Cells to Induced Neurons

TRE3G-NGN2 was integrated into the adeno-associated virus integration site (AAVS) of the hESCs and iPSCs as previously described^{50 (link)}. To start differentiation to iNeurons from stem cells (day 0), cells were plated at 2 × 10⁵ cells ml⁻¹ onto Matrigel-coated plates into ND1 medium (DMEM/F12, 1X N2 (Thermo Fisher Scientific), human BDNF (10 ng ml⁻¹; PeproTech), human neurotrophin-3 (NT3, 10 ng ml⁻¹; PeproTech), 1X NEAA, human laminin (0.2 μg ml⁻¹) and doxycycline (2 mg ml⁻¹) also containing Y27632 (ROCK inhibitor, 10 mM). The medium was replaced with ND1 without Y27632 the next day. The following day, the medium was replaced with ND2 (neurobasal medium, 1X B27, 1X GlutaMAX, BDNF (10 ng ml⁻¹), NT3 (10 ng ml⁻¹) and doxycycline at 2 mg ml⁻¹. On days 4 and 6, 50% of the medium was changed with fresh ND2. On any day in the day 4–7 range, cells were replated at 4 × 10⁵ cells well⁻¹ in ND2 medium with Y27632. The medium was replaced the next day with fresh ND2 (without Y27632). Every other day, 50% of the medium was changed with ND2. At day 9 and onwards, doxycycline was removed from the ND2 mixture. iNeurons were fed every other day with 50% medium change until the experimental day (day 12 of differentiation, unless otherwise noted).