Differentiation of iPSCs into Midbrain Dopaminergic Neurons

A modified dual-SMAD inhibition protocol was used to direct cells towards floor plate-based mDA neurons^{3 (link),5 (link)}. At day 30 of differentiation, cells were replated on dishes pre-coated with polyornithine (PO; 15 µg mL⁻¹)/ laminin (1 µg mL⁻¹)/ fibronectin (2 µg mL⁻¹) in Neurobasal/B27/L-glutamine-containing medium (NB/B27; Life Technologies) supplemented with 10 µM Y-27632 (until day 32) and with BDNF (brain-derived neurotrophic factor, 20 ng mL⁻¹; R&D), ascorbic acid (AA; 0.2 mM, Sigma), GDNF (glial cell line derived neurotrophic factor, 20 ng mL⁻¹; R&D), dibutyryl cAMP (0.5 mM; Sigma), TGFβ3 (transforming growth factor type β3, 1 ng mL⁻¹; R&D), and DAPT (10 nM; Tocris). Two days after plating, cells were treated with 1 µg mL⁻¹ mitomycin C (Tocris) for 1 h to kill any remaining proliferative contaminants. iPSC-derived mDA neurons were fed every 2 to 3 days and maintained without passaging until they were assayed at day 65. To prevent neurons from lifting off, laminin and fibronectin were supplemented into the media every 7–10 days.