Rcb1127

R1 mESCs (Nagy’s lab, MSH, Toronto, Canada) were cultured and differentiated on PA6 stromal feeders (RCB1127, Riken BRC Cell Bank, Japan)43 (link). Specifically, mESCs were plated at low density (100 cells/cm²) on a confluent layer of PA6 cells and were grown in Serum Replacement Medium with Noggin (300 ng/ml; R&D Systems) as previously described44 (link). At day 5, 200 ng/ml Shh (R&D Systems) and 25 ng/mL Fgf8b (R&D Systems) were added to the medium. At day 8, the medium was switched to N2 medium containing Shh, Fgf8b, and Fgf2 (10 ng/ml, R&D Systems). At day 10 of differentiation the cells were pulsed with EdU (10 μM, Life Technologies). From day 11 of differentiation, Shh, Fgf8b and Fgf2 were removed from the N2 medium and replaced by BDNF (20 ng/ml, R&D Systems), GDNF (20 ng/ml, R&D Systems), and ascorbic acid (0.2 mM). Between day 8–15, cells were treated daily with either dopamine (10 μM), haloperidol (1 μM), quinpirole (10 μM), sulpiride (10 μM) or media only. At day 15, cells were fixed in 4% PFA and processed for immunocytochemistry as described below.

All experiments were carried out in accordance with our institutional research guidelines. Human iPS cells were cultured according to a previously described method and previously well confirmed by the provider [8 (link)]. Briefly, the adult human dermal fibroblast-derived human iPS cell line (201B7) and PA6 cell line (as feeder cells, RCB1127) were purchased from the RIKEN Bioresource Center (Ibaraki, Japan). Human iPS cells were grown on a radiation-treated mouse embryonic fibroblast (MEF) feeder layer in standard iPS culture medium, containing DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 20% knockout serum replacement (Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 100 U/mL penicillin/streptomycin (Invitrogen), and 1% non-essential amino acids (Invitrogen). Cells were passaged every week, and the medium was changed every two days [14 (link)].