Selection of NPCs (Step 2)
The EBs maintained for a total of 4 days (2 days in hanging drops and 2 days in suspension) were plated onto gelatin-coated culture dishes in ES medium. After 24 h of culture to allow cell attachment and surface spreading, ES medium was replaced with serum-free ITSFn medium containing DMEM/F12 (1:1) supplemented with insulin (10 |xg/ml), sodium selenite (6.7 pg/ml), transferrin (5.5 |xg/ml), and fibronectin (5 |xg/ml; Invitrogen). The resulting EB outgrowths were maintained in serum-free ITSFn medium for 7 days, with medium replenishment every 2 days. For immu-nocytochemistry (ICC), NPCs were plated onto polyornithine- and lami-nin-coated glass coverslips (polyornithine, 15 mg/ml; laminin, 1 mg/ml; Sigma). ddvap bed wetting
Expansion of NPCs (Step 3)
The EB outgrowths were incubated with 1-2 ml of collagenase IV (1 mg/ml, 5-10 min) followed by the addition of DMEM/F12 (1:1) plus 10% FBS. Dispersed cell clumps were collected by centrifugation (1000 X g, 10 min) and replated onto polyornithine- and laminin-coated six-well plates or glass coverslips in N2 medium (DMEM/F12 [1:1] supplemented with laminin [1 |xg/ml; Invitrogen], FGF-2 [10 ng/ml; R&D Systems, Minneapolis, MN], and N2 supplement [1%; Invitrogen]). The medium was changed every 2 days, whereas FGF-2 was added daily. Cells plated onto coverslips were fixed in 2% paraformaldehyde for 10 min and held in 30% ethylene glycol and 20% glycerol in 0.05 M sodium phosphate buffer at -200C for ICC analysis.
Differentiation of NPCs into Neural Lineages (Step 4)
The NPCs, either attached on substrates or developing as suspended spherical structures, were cultured in N3 medium that was changed every 2 days for 7-12 days until cells were fixed for ICC assays. The N3 medium was the same as the N2 medium, but it did not contain FGF-2.