Brainphys

For generation of NSC, the commercially available PSC Neural Induction Medium (ThermoFisher, Waltham, MA, USA, A1647801) was used, following manufacturer´s instructions. NSCs were routinely maintained in NEM medium (ThermoFisher, Waltham, MA, USA). For neuronal differentiation, 42,000 cells/cm² were seeded in GFR Matrigel-coated plates in NEM medium and maintained for 48 h. After this, growth medium was replaced with differentiation medium consisting of DMEMF12 (Thermo Fisher; Waltham, MA, USA, 11330-057) supplemented with 1x N2 (Thermo Fisher Scientific, Waltham, MA, USA, 1750200), 1x B27 (Thermo Fisher Scientific, Waltham, MA, USA, 17504044), 1x NEAA (Thermo Fisher Scientific, Waltham, MA, USA, 11140035) and 100 μM β-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA, 21985023). Culture medium was changed every other day for 6 weeks. For Patch Clamp and Calcium Imaging experiments, BrainPhys (Stemcell Technologies, Grenoble, France, 05792) was used instead of DMEMF12.

Neurogenic differentiation in monolayer culture was induced when cell confluency reached 60%, and 3D spheroids were induced to differentiate 24 h after seeding. To induce differentiation preinduction media consisting of DMEM (1 g/L glucose), 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, ThermoFisher Scientific, Waltham, MA, USA), 20 ng/mL FGF, and 20 ng/mL EGF (PeproTech, London, UK) was applied for 24 h. After pre-induction step differentiation media consisting of BrainPhys™, 1% NeuroCult™ (STEMCELL Technologies, Vancouver, BC, Canada), 100 U/mL penicillin, and 100 mg/mL streptomycin, 50 ng/mL BDNF, 100 ng/mL NGF, (PeproTech, London, UK) 5 mM KCl, 2 μM RA (Sigma-Aldrich, St. Louis, MO, USA). Differentiation was carried out for 5 days.

We utilized commercial neurons from Fujifilm Cellular Dynamics International (CDI), which are a more homogeneous culture of neurons and produce more than 80% pure midbrain DA neurons The detailed nomenclatures/catalog numbers for the cells used in this paper are iCell DopaNeurons, 01279, catalog number: C1028; and MyCelliDopaNeurons, C1279, catalog number: C1113, genotype: SNCA (A53T alfa synuclein mutation). The cells were defrosted according to the protocol on the User’s Guide from CDI and counted. The medium used was Brainphys (Stem Cell Technologies), with the addition of supplements, according to the manufacturer’s instructions: iCell Neural Supplement B (catalog number: M1029) and iCell Nervous System Supplement (catalog number: M1031), N2 supplement, Laminin, and antibiotics (penicillin/streptomycin). For patch clamp electrophysiology experiments, we used Poly-L-ornithine- and Laminin-coated, 24-well plates with coverslips in each well, and we plated 4–6 × 10⁵ cells per well. Recordings started after a week in culture. For the RNA sequencing experiments, we plated 1 × 10⁶ cells per well on six-well plates. For immunostaining, we plated about 1 × 10⁵ cells per well of 8 chamber ibidi slides.

Assay-ready 384-well microBrain 3D cultures of human neurons and astrocytes were obtained from StemoniX, Inc. (BSARX-AA-0384). The source iPSCs were from a male individual without any known deleterious mutations and a normal healthy phenotype. As described previously [16 (link)], the microBrain 3D platform was generated, differentiated, and matured for 8 weeks prior to shipment. Over the time of differentiation and maturity, the spheroids spontaneously develop synchronized calcium activity. Upon receipt, the 3D co-differentiated population of cortical neurons and astrocytes [16 (link)] was maintained in and replenished every two days for one week prior to assay with BrainPhys (StemCell Technologies, 05790) media supplemented with 1x SM1 (StemCell Technologies, 05711), 20ng/mL of BDNF (StemCell Technologies, 78005), 20ng/mL of GDNF (StemCell Technologies, 78058), and 1x pen/strep (Corning, 30-002-CI).