4.2. Differentiation of iPSC Lines into Striatal Medium Spiny Neurons (MSNs)

The striatal differentiation of iPSCs into medium spiny neurons (MSNs) was adapted from previous protocols published by Stanslowsky et al. [37] and Capetian et al. [38]. Briefly, feeder-free iPSC colonies were detached and suspended in mTeSR (Stemcell Technologies, Vancouver, BC, Canada) supplemented with 1 µM dorsomorphin (Tocris, Bio-Techne, Minneapolis, MN, USA), 10 µM SB-431542 (Tocris, Bio-Techne, Minneapolis, MN, USA), 1 µM IWP2 (Merck, Darmstadt, Germany), and 10 µM Rho kinase (ROCK) inhibitor Y27632 (Stemcell Technologies, Vancouver, BC, Canada) to form free-floating embryoid bodies (EBs) at day 0. On day 2, medium was replaced with 1:1 mTeSR/N2 medium (Knockout-DMEM/F-12, with 1:100 N2 supplement, Thermo Fisher Scientific, Waltham, MA, USA and 1% penicillin, streptomycin, L-glutamine, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with the above-mentioned concentrations of dorsomorphin, SB-431542, and IWP2. On day 4, N2 medium was supplemented with dorsomorphin, SB-431542, IWP2, and 0.2 µM purmorphamine (PMA; Enzo Life Sciences, Lörrach, Germany). On day 6 and 8, medium was replaced with N2 medium supplemented with 1 µM IWP2 and 0.2 µM PMA. On day 10, only N2 medium without supplements was used. On day 12, EBs were plated and kept in N2B27 maturation medium (1:1 DMEM/F-12 and Neurobasal medium containing 1:200 N2, 1:100 B27 without vitamin A, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin, streptomycin, L-glutamine) with 20 ng/mL brain-derived neurotrophic factor (BDNF; PeproTech, Cranbury, NJ, USA), 10 ng/mL glial cell line-derived neurotrophic factor (GDNF; PeproTech, Cranbury, NJ, USA) and 50 µM dibutyryl-cAMP (dbcAMP; Sigma-Aldrich, St. Louis, MO, USA). Once the outgrowing cells reached full confluency, they were replated on laminin/poly-DL-ornithine hydrobromide (Thermo Fisher Scientific, Waltham, MA, USA) coated dishes for terminal differentiation. Maturation medium was changed every other day. Fully differentiated neuronal cells were characterized after 70 days ± 7 days. A total of two to three independent MSN differentiations of each iPSC line (two control and two SGCE lines) were analyzed.