Embryonic E13 rats were obtained from anesthetized, timedpregnant Sprague-Dawley rats. Tissues from the forebrain and hindbrain were dissected, digested with 0.05% trypsin and EDTA acid, dissociated by gentle trituration in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal calf serum, and filtered through a sterile 60-mesh membrane. After centrifugation, cells were suspended in serum-free DMEM/F12 (1: 1) supplemented with 100 units penicillin, streptomycin (100g/ml), N2 supplement (1: 100ml medium; Gibco, Baltimore, MD), fibroblast growth factor–2 (20ng/ml; Sigma, St. Louis, MO), and epidermal growth factor (20ng/ml; Sigma). Cells were plated in six-well plates at a density of 50,000/ml in a total 5ml of the above culture medium and grown at 37 C in a humidified incubator with 5% CO2. The free-floating spheres were centrifuged and dissociated to a single-cell suspension through a Pasteur pipette with a flame-polished tip. These single cells were then resuspended into new six-well plates at a concentration of 50,000 cells/ml. Cultures were incubated for 12 days with half of the medium exchanged every 3 days for fresh medium containing fibroblast growth factor–2 and epidermal growth factor. Bromodeoxyuridine (BrdU, 1M; Sigma), which is incorporated into dividing cells, was added to the cultures on days 11 and 12. Some cultures were dissociated to a single-cell suspension on day 12 and plated at a density of 10,000 cells in DMEM supplemented with 10% fetal calf serum onto 13mm glass coverslips coated with poly-L-lysine for in vitro differentiation study. The remaining cultures were used for VHL gene transduction and transplantation. Glial cell line–derived neurotrophic factor (GDNF, 20ng/ml; Sigma) was added to some cultures 48 hours before immunocytochemistry for the in vitro differentiation study or the transplantation to assess its effect on differentiation of NPCs. von Hippel–Lindau Gene Transduction

Adenovirus vector encoding human VHL (VHL54-213 amino acids) was generated with the use of a cosmid vector pAxCAwt, as described previously. 19 The vector for green fluorescent protein (GFP) used as a control was obtained from the Riken Gene Bank. For adenovirus infection, NPCs were seeded on 6cm dishes (1 106 cells/cm2) 1 day before infection. The cells then were incubated for 1 hour with 5l