Poly l ornithine po

ReNcell-VM (Millipore®) is a human neural progenitor cell line derived from the ventral mesencephalon region of the fetal brain and immortalized by retroviral transduction with the v-myc oncogene. ReNcell-VM were first expanded in T-flasks (Falcon®, Corning) previously coated with poly-L-ornithine (PO) (20 μg mL⁻¹, Sigma-Aldrich) for 30 min and laminin (LN) (10 μg mL⁻¹ in PBS, Sigma-Aldrich) for 4 h at 37°C and 5% CO₂. After seeding, cells were expanded in N2 medium, consisting of DMEM/F12 with glucose (1.6 g L⁻¹, Sigma-Aldrich), N2-supplement (1%, Thermo Fisher), penicillin/streptomycin (1%, Thermo Fisher) and insulin (20 μg mL⁻¹, Sigma-Aldrich), and supplemented with EGF (20 ng mL⁻¹, Peprotech), FGF-2 (20 ng mL⁻¹, Peprotech), and B27 supplement (20 μl mL⁻¹, Thermo Fisher).

The dissociated cells were plated at a density of 10,000 cells in an uncoated 96-well plate, containing neural culture medium supplemented with hLIF and bFGF. The cells were then cultured in an atmosphere containing 4% O₂ and 5% CO₂ for 5 d, and the resulting neurosphere formation was analyzed. For neural differentiation, the neurospheres were dissociated into single cells and plated at a density of 10,000 cells per well on coverslips for 24-well plates coated with poly-L-ornithine (PO) (Sigma-Aldrich) and fibronectin (Sigma-Aldrich). For the induction of neuronal differentiation, the cells were further cultured for 3 d in the neural culture medium (without hLIF or bFGF) supplemented with 2% (vol/vol) B27 (Life Technologies), 10 ng/ml brain-derived neurotrophic factor (R&D Systems), 10 ng/ml glial-derived neurotrophic factor (R&D Systems), 200 μM ascorbic acid (Sigma-Aldrich) and 1 mM dibutyryl-cAMP (Sigma-Aldrich).

Neural crest induction was performed using monolayer culture protocols as previously described [30 (link)]. In brief, hiPSCs were dissociated into single cells with Accutase and then replated on Matrigel-coated dishes at a density of 20,000 cells/cm². At the beginning of the induction (day 1), cells were incubated with E6 medium for 24 h and then cultured in NCN2 medium for 6 days (7 days in total). p75^highHNK1⁺ NCSCs were isolated by fluorescence-activated cell sorting (FACS) using a BD Influx Cell Sorter (BD-Pharmingen), cultured and expanded in neural crest culture medium (NCCM).
In the migration assay, day 7 differentiated cells were disaggregated and suspended in neural crest induction medium (NIM) to form spheres in ultralow-attachment culture dishes for 1 day. Then, the spheres were attached to dishes coated with poly-L-ornithine (PO; Sigma-Aldrich) and laminin (LN; Millipore, Temecula, CA, USA) and cultured in NIM for 24–48 h.