Neurobasal b27 l glutamine containing medium nb b27

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.

Human embryonic stem cells (hESCs) were differentiated with a modified dual-SMAD inhibition protocol towards floor plate-based midbrain dopaminergic (mDA) neurons as described previously^{64 (link)}. Briefly, hESCs were maintained on mouse embryonic fibroblasts and passaged with Dispase (STEMCELL Technologies). For each differentiation, hESCs were harvested with Accutase (Innovative Cell Technology). At day 30 of differentiation, hESC-derived mDA neurons were replated and maintained on dishes precoated with polyornithine (PO; 15 μg mL⁻¹), Laminin (1 μg mL⁻¹), and 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 brain-derived neurotrophic factor (BDNF; 20 ng mL⁻¹; R&D), ascorbic acid (AA; 0.2 mM, Sigma), glial cell line-derived neurotrophic factor (GDNF; 20 ng mL⁻¹; R&D), transforming growth factor type β3 (TGFβ3; 1 ng mL⁻¹; R&D), dibutyryl cAMP (0.5 mM; Sigma), and DAPT (10 nM; Tocris). Two days after replating, mDA neurons were treated with 1 μg mL⁻¹ mitomycin C (Tocris) for 1 h to kill any remaining non-post mitotic contaminants. Toxicity assays were performed at 65 days of neuron differentiation. PU-AD was added to the cells for 72 h and the CellTiter-Glo Luminescent Cell Viability Assay (Promega) assay was performed according to the manufacturer’s indications.