Differentiation of hiPSCs into Dopaminergic Neurons

Neuronal differentiation of human iPSCs was induced as previously described (Arioka et al., 2018 (link)). Briefly, iPSCs were pretreated with 3 µM SB431542 (Cayman, Ann Arbor, MI, United States), 3 µM CHIR99021 (Cayman), and 3 µM dorsomorphin (Sigma, Burlington, MA, United States) for 1 week (day 0–7) and dissociated into single cells by incubation with TripLE Select (Gibco) for 5 min. The cells were cultured in neurosphere medium containing MHM [DMEM/F12, supplemented with 1× N2 (Gibco), 0.6% glucose (Sigma), 5 mM HEPES (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin, 1× B27 (Gibco), 20 ng/mL bFGF, 10 ng/mL hLIF (Nacalai, Kyoto, Japan), 10 µM Y-27632 (Cayman), 3 µM CHIR99021, 2 μM SB431542, 100 ng/mL FGF8b (Peprotech, Cranbury, NJ, United States), and 1 µM purmorphamine (Cayman) for 1 week (day 7–14)]. On day 14, neurospheres were passaged by dissociation into single cells in the same manner as primary neurosphere formation. For terminal differentiation into dopaminergic neurons, secondary neurospheres were dissociated 1 week after passage (day 21) and plated onto poly-L-ornithine/laminin/fibronectin-coated dishes at the density of 3 × 10⁴ cells/cm² in dopaminergic neuron medium [MHM supplemented with B27, 10 µM DAPT (Cayman), 20 ng/mL BDNF (Peprotech), 20 ng/mL GDNF (R&D), 0.2 mM ascorbic acid (Sigma), 1 ng/mL TGF-β3 (R&D), and 0.5 mM dbcAMP (Sigma)].