Isolation and culture of hindbrain cells

Hindbrain regions of HH18 embryos were dissected in sterile PBS with penicillin-streptomycin (Pen-Strep, 1:100; Gibco, USA), then placed in a tube containing human embryonic stem cell medium [hESC; DMEM/F-12 1:1 with 20% KnockOut serum replacement, GlutaMax L-alanyl-L-glutamine (2 mM), non-essential amino acids (0.1 mM; all from Gibco), β-mercaptoethanol (0.1 mM; Sigma-Aldrich), Pen-Strep (1:100) and Fungizone (1:500)]. Media was next replaced with 1 ml of TrypLE Express (Gibco) to dissociate the tissue into single cells. After a manual disassociation by pipetting up and down, TrypLE was neutralized with 10:1 hESC medium and cells were passed through a 100 μm mesh strainer to detach adherent cells. Cells were cultured in hESC media at density of 1×10^5–6 cells/ml, seeded in a 48- or 96-well Nunclon Delta Surface culture plate (Thermo Fisher Scientific) and incubated at 37°C in 5% CO₂ (Peretz et al., 2016 (link), 2018 (link)). For live imaging, cell plates were imaged every 3-6 h in IncuCyte S3 Zoom HD/2CLR time-lapse microscopy system, equipped with a 20× Plan Fluorobjective (Sartorius). Time-lapse movies were generated by capturing phase images for up to 5 days of incubation (Wang et al., 2020a (link)).