We further adapted our protocol to utilize NPC monolayers during differentiation. To this end, we plated EBs treated with anticaudalizing factors onto laminin/polyornithine-coated tissue culture surfaces at differentiation day 20 to allow rosette formation. The rosettes were manually dissociated after 1 week to isolate the NPC population (Figure 2A). Using immunostaining and quantitative PCR (qPCR), we found that the NPCs obtained using this method showed similar levels of SOX2 but higher levels of the hippocampal NPC markers PAX6, FOXG1, and EMX2 compared to NPCs derived without the anticaudalizing cocktail treatment (Figures 2B and 2C).
