Neural basal

Established pancreatic cancer tumorspheres [10] (link) were maintained in sphere media as described previously [19] (link), [20] (link) with modifications [50% NeuralBasal (Invitrogen, Carlsbad, CA), 1% N2 Supplement (Invitrogen, Carlsbad, CA), 2% B27 supplement (Invitrogen, Carlsbad, CA), 1% Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA), 10 ng/mL BMP4 (Sigma), 10 ng/mL LIF (Sigma), 20 ng/mL human bFGF-2 (Invitrogen, Carlsbad, CA), all in 1∶1 DMEM/F12 (Invitrogen, Carlsbad, CA)]. Tumorspheres were passaged every 6 days. For passaging, tumorspheres were dissociated with 0.05% trypsin for 2–5 min and then immediately washed twice with 40 mL PBS. Cells were then passed through a 40 μm nylon mesh cell strainer, counted and plated in fresh sphere medium in Costar ultra low-attachment 6 well plates (Corning, Lowell, MA).

R1 mESCs (a kind gift from Dr. Shaorong Gao [59 (link)]) were grown on gelatin-coated dish with ESC medium, which consisted of DMEM (Hyclone), 15% (v/v) fetal bovine serum (FBS; Thermo Fisher), 0.1 mM β-mercaptoethanol (Sigma), 2 mM L-glutamine (Thermo Fisher), 0.1 mM nonessential amino acids (Thermo Fisher), 1% (v/v) nucleoside mix (Sigma), and 1000 U/mL recombinant LIF (Millipore). MEFs, mRuby3-NONO-KI HeLa cells [60 (link)], and H2B-GFP stably expressed HeLa Cells were cultured in DMEM (Hyclone) with 15% (v/v) fetal bovine serum (FBS; Thermo Fisher). NPCs were grown in neural basal (Gibco) supplemented with 1× NEAA (Gibco), 0.5 × GlutaMAX (Gibco), 1 × B27 (Gibco), 1 × N2 (Gibco), 0.075% BSA (Sigma), 1 × PS (Hyclone), 10 ng/mL bFGF (PeproTech), 10 ng/mL mEGF (PeproTech).
To induce NPC differentiation [61 (link)], monolayer culture for neural differentiation was performed. Briefly, mESCs were dissociated and plated at a density of 1 × 10⁴ cells/cm² in N2B27 medium (DMEM/F12, Neurobasal, N2 (Thermo Fisher 17502-048), B27 (Gibco 17504044)) supplemented with 1 mM l-glutamine and 0.1 mM β-mercaptoethanol.
The MEFs isolated from E13.5 to 14.5 days of pregnancy could be maintained in culture consistently for more than 10 passages, and the third or fourth passage was used for experiments.
The cell lines have been authenticated and are available upon request.

The hippocampus of six newborn rats was put into Ca²⁺/Mg²⁺-free Hank’s balanced salt solution (HBSS) and cut into 1-mm³ pieces. The tissue was placed in PBS containing 0.25% trypsin for 15 min at 37 °C, and then digestion was stopped with a medium containing serum. After centrifugation, the cells were resuspended and blown into a single-cell suspension at a density of 1 × 10⁶ cells/ml. Cells were then seeded on poly-l-lysine-coated (1 mg/ml; Sigma-Aldrich, MO, USA) glass coverslips on a 6-well culture plate, and incubated in neural-basal (Gibco, Grand Island, NY, USA) containing 2% B27 supplement (Gibco) at 37 °C in 5% CO₂ and 95% air with saturated humidity. The culture medium was changed every other day.