N2 components

Human fetal astrocytes (HFA), isolated at approximately 20 weeks of gestation, were purchased from Lonza (Lonza Biologics, Portsmouth, NH). U138MG cells were obtained from ATCC (ATCC HTB-16, Manassas VA) and cultured as described in ([12] (link)). Human Progenitor-Derived Astrocytes (PDA) were generated from neural progenitor cells, as previously described ([13] (link)). Briefly, progenitor cells were provided by Dr. Eugene Major (National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD (NIH) and seeded on poly-D-lysine- coated T-75 tissue culture flasks at 2×10⁶ cells/flask. Cells were maintained in progenitor medium consisting of neurobasal media (Life Technologies Invitrogen, Carlsbad, CA) supplemented with 0.5% bovine albumin (Sigma, St. Louis, MO), neurosurvival factor (Lonza), N2 components (Life Technologies Invitrogen), 25 ng/ml fibroblast growth factor, 20 ng/ml epidermal growth factor (R&D Systems, Minneapolis MN), 50 μg/ml gentamicin (Lonza) and 2 mM L-glutamine (Life Technologies Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM (Life Technologies Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma), 2 mM L-glutamine, and 50 μg/ml gentamicin. L-Wnt3a (ATCC CRL-2647) were cultured in complete DMEM with 0.4 mg/ml G-418. Media was supplemented every three days.

Progenitor-derived astrocyte (PDAs) were generated from neural progenitor cells (kindly provided by Dr. Eugene Major, NINDS, NIH, MD) as previously described [14 (link)]. Briefly, PDAs were maintained in progenitor medium consisting of Neurobasal media (Invitrogen) supplemented with 0.5 % bovine albumin (Sigma-Aldrich), neurosurvival factor (NSF; Lonza), N2 components (Invitrogen), 25 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml EGF (R&D Systems), 50 μg/ml gentamicin (Lonza), and 2 mM L-glutamine (Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicin. Cultures were >90 % positive for glial fibrillary acidic protein (GFAP) after 30 days of differentiation. Fetal-derived Normal Human Astrocytes (NHAs, Lonza) were maintained in Astrocyte Growth Media (AGM) BulletKit (Lonza) supplemented with 0.3 % heat-inactivated fetal bovine serum, 1 ml/ml ascorbic acid, 1 ml/ml rhEGF, 1 ml/ml AG-1000 (30 mg/ml gentamicin and 15 μg/ml amphotericin), 2.5 ml/ml insulin, and 10 ml/ml L-glutamine. Adherent primary cells were removed by treatment with 1 mM EDTA for 5 min with gentle scraping or pipetting multiple times.