Isolation and Culture of Human Astrocytes

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.