**2. Materials and methods**

#### **2.1. Stem cell culture and neuronal differentiation**

hESCs (H1 cell line, WiCell Research Institute Inc., Madison, WI) were cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) that were mitotically inactivated using mitomycin C (Sigma-Aldrich, St. Louis, MO) in hESC media containing DMEM/ F12 supplemented with, 1% nonessential amino acids, 20% Knockout serum (Gibco), 1 mM L-glutamine, 4 ng/mL human recombinant basic fibroblast growth factor (bFGF, Invitrogen), 1% penicillin–streptomycin (Invitrogen) and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). The media was changed daily and the cells were mechanically passaged every 5–6 days. For the studies described in this chapter, hESCs at passage 55–70 were used. To generate neurons, the hESCs were taken through a four-step differentiation protocol. The hESCs were dissociated using the protease Dispase (1.5 unit/mL, Invitrogen). The hESCs were then cultured in ultra-low attachment six-well plates (Corning Inc., Corning, NY) in a normoxic incubator (20% O2 /5% CO2 , 37°C). The media was changed once per day and spherical embryoid bodies (EBs) were present 24 h after dispase digestion. Five days after digestion, EBs were moved to neural induction media containing DMEM/F12 supplemented with 1% N2 (Invitrogen), 1% nonessential amino acids, 1 mg/ mL heparin (Sigma) and 5 ng/mL bFGF. On day 9, the EBs were plated to matrigel-coated 35-mm dishes. Rosette-like structures were present within 5 days of the EBs plating down. The rosettes were manually separated from the surrounding cells using a serological pipette approximately 2 days after the morphology was clearly visible. The rosette cells were then transferred to matrigel-coated culture dishes and cultured in neural expansion media containing DMEM/F12 supplemented with 2% B27 without vitamin A, 1% N2 (Invitrogen), 1% nonessential amino acids, 20 ng/mL bFGF and 1 mg/mL heparin. The neural stem cells (NSCs) were passaged enzymatically every 5 days. NSCs were cultured in 60-mm matrigel-coated dishes (500,000 cells/dish) in neuron differentiation media containing neurobasal media (Gibco) supplemented with 2% B27, 100 ng/mL ascorbic acid (Sigma-Aldrich), 0.1 μM cyclic adenosine monophosphate, 10 ng/mL brain-derived neurotrophic factor, 10 ng/mL insulin-like growth factor 1 (PeproTech Inc., Rocky Hill, NJ) and 10 ng/mL glial cell-derived neurotrophic factor. The media was changed every other day. Following 2 weeks of culture, the cells displayed clear neuronal morphology and were used for the studies.
