Glial cell derived neurotrophic factor gdnf

Neural stem cells (NSCs) were generated and differentiated into neural cultures as described previously^{72 (link)}. For patterning, NSCs were plated on polyornithin-/laminin-coated culture flasks at 1E4 cells/cm² in DMEM/F-12 with GlutaMax (#31331093) and neurobasal medium (#21103049) supplemented with 1X B27 (ThermoFisher #12587010), 1X N2 (ThermoFisher #17502048), 50 µM 2-mercaptoethanol (ThermoFisher #31350010) and 100 ng/ml FGF-8 (Peprotech), 200 ng/ml sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma) and cultured for one week. For differentiation, the resultant progenitors were plated at 5E4 cells/cm² in basal medium supplemented with 20 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF; Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), and 100 μM ascorbic acid 2-phosphate.

NCSCs were cultured on polyornithine‐laminin‐fibronectin‐coated culture dishes in DMEM/F12 medium supplemented with 1× N2, 10 ng/mL brain‐derived neurotrophic factor (BDNF) (PeproTech), 10 ng/mL glial cell‐derived neurotrophic factor (GDNF) (PeproTech), 10 ng/mL nerve growth factor (NGF) (PeproTech), 200 μmol/L ascorbic acid and 0.1 mmol/L dibutyryl cyclic AMP (dbcAMP) (all from Sigma‐Aldrich) for 10‐14 days. The medium was changed every other day and assayed by immunocytochemistry.

The generation of neuronal precursor cells (NPCs) from the patient and isogenic control iPSCs as well as the differentiation from NPCs to dopaminergic neurons, was performed according to ref. ^{52 (link)}. NPCs were cultured in N2/B27 medium, consisting of DMEM-Ham’s F12 medium and neurobasal medium (1:1), 0.5% N2 (Gibco), 1% B27 (Gibco), 1% P/S, and 1% GlutaMAX supplemented with 3 µm CHIR99021 (Axon Medchem), 0.5 µM puromycin (Merck Millipore), and 150 µM ascorbic acid (AA, Sigma–Aldrich). NPCs were split into Matrigel-coated wells every 5–6 days at a ratio of 1:10. To start differentiation, 1.25 × 10⁶ cells were split into a six-well plate, and the following day, the medium was changed to differentiation medium [N2/B27 medium supplemented with 100 ng/ml FGF8 (Peprotech), 1 µM phorbol 12-myristate 13-acetate (PMA) and 200 µM AA]. On day 8, the medium was changed to maturation medium [N2/B27 medium supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) (Peprotech), 10 ng/ml glial cell-derived neurotrophic factor (GDNF) (Peprotech), 1 ng/ml TGF-β3 (Peprotech), 0.5 mM dibutyryl cAMP (dbcAMP) (Applichem), and 200 µM AA] supplemented with 1 µM PMA for 2 days. From day 10 onward, the cells were cultured in a maturation medium without PMA. Maturation was reached after 14 days in the maturation medium.