Imr90 4

The hiPSC line IMR90-4 (WiCell Reasearch Institute, Madison, WI, USA) was cultured on growth factor reduced Matrigel-coated (Corning, Corning, NY, USA) 6-well plates in mTeSR plus medium (Stemcell Technologies, Vancouver, BC, Canada) at 37 °C, 5% CO₂, and 95% humidity. At 60% confluency, cells were passaged with Accutase (PAN Biotech, Aidenbach, Germany) and seeded for differentiation at a density of 7500 cells/cm². Differentiation was performed with minor modifications according to a protocol published by Lippmann et al. and Haferkamp et al. [20 (link),50 (link)]. In short, two days after seeding, medium was switched to unconditioned medium composed of DMEM/F12 supplemented with 20% knockout serum replacement, 0.1 mM mercaptoethanol, 1 mM L-Glutamine, and 1% MEM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, daily medium changes with UM were performed for five consecutive days. On day 6, medium was switched to serum-free EC medium (ECM +/+) (human endothelial serum-free medium supplemented with 0.5% B27 Supplement (Thermo Fisher Scientific, USA), 10 µM retinoic acid (Sigma-Aldrich, St. Louis, MO, USA), and 20 ng/mL human basic FGF (Thermo Fisher Scientific, USA)). On day 8, cells were harvested using Accutase and seeded onto collagen IV/fibronectin (40% collagen (1 mg/mL) and 20% fibronectin (0.5 mg/mL) (Sigma-Aldrich, USA) in distilled water and incubated overnight at 37 °C) coated polyester tissue culture (TC) inserts in serum-free ECM +/+ (0.33 cm², pore size 0.4 µm, transparent) (Sarstedt) at a density of 1 × 10⁶ cells/cm². The next day, medium was switched to human endothelial serum-free medium supplemented with 0.5% B27 Supplement without human basic FGF and retinoic acid (ECM −/−).

Human iPSCs (IMR90-4, WiCell) were cultured on Matrigel-coated culture dishes or 6-well plates with mTeSR1 plus media (100-0276, STEMCELL Technologies). Cells were passaged every 3 to 4 d using the Gentle Cell Dissociation Reagent (100-0485, STEMCELL Technologies) to dissociate iPSC colonies into small cell clumps. Organoids derived from spontaneous aggregation (SA) and single-cell aggregation in a 96-well ultra-low attached plate (96W) were produced as previously described [15 (link)]. UCO generation was based on the SA organoid method with modifications. When the iPSCs reached a confluence of 70% to 80%, the cells were detached from the culture dishes as clumped colonies using a low concentration of dispase (Invitrogen: 17105-041; 0.7 mg/ml) for 30 min in a 37 °C incubator. Suspended iPSC clumps were resuspended in mTeSR1 plus media with 10 μM Y-27632 and gently pipetted using a 5-ml serological pipette. Small pieces of iPSC clumps were spread into the 400, 600, and 1,000 μm of microwells (StemFIT 3D, MICROFIT) and cultured for 1 d to obtain EBs homogeneously. Subsequently, EBs were collected and cultured in 100-mm ultra-low attachment (ULA) dishes (4615, Corning) with mTeSR1 plus media for an additional day. Neural induction was started by replacing the neural induction media [Dulbecco’s modified Eagle’s medium/F12 (12634-010, Gibco) containing Glutamax (35050-061, Gibco), 20% KnockOut Serum replacement (10828-010, Gibco), 1% Eagle’s minimum essential medium–nonessential amino acid solution (MEM-MEAA; 11140-050, Gibco), 1% penicillin/streptomycin (15140-122, Gibco), and 0.1 mM β-mercaptoethanol] with dual SMAD inhibitors (10 μM dorsomorphin; P5499, Sigma and 10 μM SB431542; S4317, Sigma) for 3 d. On day 4, the EBs were transported to 6-well ULA plates, and 2.5 μM IWP-2 (HY13912, MedChem Express) was added to the neural induction media. From days 10 to 14, the media were changed daily to neural progenitor cell (NPC) expansion media [Neurobasal A medium (10888-022, Gibco) containing B27 supplement minus VitA (12587-010, Gibco), Glutamax, penicillin/streptomycin] supplemented with fibroblast growth factor 2 (FGF2) (3718-FB-01M, R&D Systems) and epidermal growth factor (EGF) (236-EG-01M, R&D Systems). On day 15, NPC expansion media were changed every other day until day 24. On day 25, media were changed to neuronal differentiation media, which were basally the same as the NPC expansion media, and growth factors were changed to brain-derived neurotrophic factor (BDNF; 248-BDB-01M, R&D Systems and NT-3;267-N3-025, R&D Systems). On day 43, the organoids were cultured by a basal medium by excluding the growth factors from the neuronal differentiation media, and the media were replenished every 3 d.

hiPSC line (IMR90-4, WiCell) derived from a healthy donor and fully characterized was cultured with mTeSR™ Plus Medium (STEMCELL Technologies) on growth factor–reduced matrigel-coated 6-well plates (Corning) and used for all experiments in this study. The hiPSCs were routinely passaged using Versene (Gibco, Thermo Fisher Scientific) at a ratio of 1:4–1:6 in mTeSR™ Plus Medium supplemented with 10 µM of ROCK inhibitor Y-27632 (Focus Biomolecules) and maintained at 37 °C in a humidified atmosphere containing 5% CO₂.