Isolating and Differentiating Adult Neural Stem Cells

Adult NSCs were isolated from 8 to 12 weeks old β1^flx/flx; Rosa:YFP and β1^+/+; Rosa:YFP mice by dissecting the hippocampus as described (Bonaguidi et al., 2008 (link)). Cells were dissociated and grown as neurospheres in serum-free neurosphere medium (NM) containing DMEM:F12 (Invitrogen) with N2, B27, penicillin/streptomycin/glutamine, and heparin (Sigma). NM was supplemented with 20 ng/mL epidermal growth factor (EGF, Millipore) and 10 ng/mL basic fibroblast growth factor (Millipore). Cells were incubated at 37°C in humidified 5% CO₂. Neurospheres were passaged every 3–5 days. After the third passage, cultures were infected with adenovirus expressing Cre (Vector Biolabs) and either LV-caILK or control lentivirus (LV-mCherry). Seven days postinfection, cells were dissociated and plated for differentiation at 2 × 10⁴ cells/cm² on to glass coverslips coated with poly-d-lysine (Sigma) and laminin (Roche) in 24-well plates. Cells were allowed to differentiate for 7 days in NM supplemented with 0.2 ng/mL EGF. Differentiated cells were then fixed in 4% paraformaldehyde for 15 min at room temperature (RT) and immunostained.