Poly ornithine

Primary mouse astrocyte cell cultures were obtained from the brain cortex of 1-day-old neonatal C3H/He (H-2^k) mice as previously described [39 (link)]. In a set of experiments, 1-day-old neonatal C57BL/6 (H-2^b) mice were also used. Briefly, mice were decapitated and meninges removed. Cortices were isolated, minced and a single cell suspension obtained by mechanical dissociation. The cells from each brain were seeded into a 25 cm² culture flask precoated with poly-ornithine (0.1 mg/mL; Sigma, USA) in DMEM/F-12 medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and maintained at 37°C kept in 95% humid atmosphere with 5% of CO₂. Microglial and other non-adherent cells were removed by soft shaking the culture flasks. Astrocytes from seven to ten days of culture were detached with trypsin 0.125% EDTA and plated at a density of 10⁵ cells per 13 mm diameter poly-ornithine-coated coverslip in 24-well plates (NUNC, Denmark). Cells were kept 24-hours in culture for to allow adhesion of astrocytes to coverslips. Details of each experiment are described in the figure legends.

Control NPCs were derived from human iPSCs (WT83 c9 and c6), as previously described^{4 (link)}. All iPSC lines were negative for mycoplasma contamination. Briefly, high-passage iPSC/hESC colonies on feeder-free plates were grown to 80% confluency. The medium was changed to N2 (DMEM/F12 medium containing 1 × N2 supplement [Invitrogen], 1 μM dorsomorphin [Tocris], and 1 μM SB431542 [Stemgent]) for 48 h. Colonies were then detached from the plate and cultured in suspension as embryoid bodies for 5 days at 90 rpm in N2 medium. Embryoid bodies were plated on Matrigel (Corning) in NGF medium (DMEM/F12 medium supplemented with 0.5 × N2, 0.5 × B27 supplement [Gibco], 20 ng/ml fibroblast growth factor, and 1% penicillin/streptomycin) for 1 week. Rosettes were manually picked, dissociated, and added to plates double-coated with polyornithine (10 μg/mL, Sigma-Aldrich) and laminin (2.5 μg/mL, Gibco) in NGF medium. To generate neurospheres, NPCs were scraped from the plates and continuously shaken for 2–5 days at 90 rpm in NGF medium. Neurospheres were then treated with NG medium supplemented with 10 μM ROCK inhibitor (Y-27632, Calbiochem) for 48 h. Neurospheres were maintained in NG medium for 4 weeks to allow neuronal maturation.

HEK 293 were seeded at a density of 3 × 10⁶ cells/plate in 10 cm dishes. MNSC and GSC neurospheres were trypsinized to obtain a single cell suspension and seeded at the same cell density on polyornithine (Sigma-Aldrich) coated 15 cm dishes. The following day, media was changed to DMEM supplemented with 5% FBS and 2 μM ATRA. Cells were incubated at 37 °C at designated times. If cells were incubated for longer than 48 h, the media was removed and replaced with fresh media.

Neural stem cells were plated on poly-ornithine (Sigma-Aldrich) and laminin (Life Technologies) coated culture plates in complete StemPro neural stem cell SFM (Life Technologies) at 2.5 × 10⁴cells per cm². After 24 h, the medium was replaced with neural differentiation medium (neurobasal A medium containing 2% B27 and 0.5 mM L-glutamine; Life Technologies) for neuron differentiation. 3 days after differentiation, 10 μM fluorodeoxyuridine (Sigma-Aldrich) and 10 μM uridine (Sigma-Aldrich) were added to remove mitotic cells. After 2 days differentiation cells were infected with expressing lentivirus at an m.o.i. of 5, then were treated with 500 nM Aβ for 4 days after 7 days differentiation.

PCs were isolated from E17-E19 mice embryos following a method previously described (Tabata et al., 2000 (link)) with slight modifications. Briefly, the cerebella were dissected in ice-cold HBSS supplemented with 20 µg/ml gentamicin (both from Invitrogen), then incubated with 10 U/ml papain (Sigma Millipore) and 2.5 U/ml DNase I (Roche Diagnostic) and 4 mm MgCl₂ (Sigma Millipore) at 33°C for 20 min. The cerebella were titrated in HBSS with 2.5 U/ml DNase I and 4 mm MgCl₂ and were filtered with 200 µm Nylon mesh (Millipore). After washing twice in HBSS, the cells were plated on precleaned, poly-ornithine (500 µg/ml, Sigma Millipore) coated 1.5H glass-bottomed slide (Ibidi) at the density of 1.2 × 10⁶ cells/cm². For tracking experiments, the cells were transfected before plating with L7-mCherry or -GFP (a gift from J Hammer III) (see Wagner et al., 2011 (link)) using Nucleofector 4D (Lonza Walkersville) according to manufacturer's protocol. The culture medium contained PNBM neural basal medium (Lonza Walkersville), GS21 neural supplement (1:50, Globalstem), 5 µg/ml gentamicin, and 2 mm Glutamax (Invitrogen); half-volume of the medium was changed once a week and the day before the experiment.