Neuroepithelial Differentiation from hESCs

Neuroepithelium differentiation to primarily central nervous system epithelia requires the single-cell passaging of hESCs and the culturing of these cells to nearly 100% confluence on Matrigel-coated plates prior to beginning neural induction [19 (link), 20 (link)]. Upon reaching 100% confluence, the MEF-CM medium is replaced with N2/B27 Neural Induction medium supplemented with the small molecules SB431542 (Tocris Biosciences) and LDN193189 (Sigma). N2/B27 Neural Induction medium consists of DMEM/F12 (Gibco) as the base, N2 supplement (Invitrogen), B27 supplement minus vitamin A (Invitrogen), L-glutamine (2 mM), penicillin/streptomycin, beta-mercaptoethanol (Sigma), and MEM nonessential amino acids (Sigma). The supplemented small molecules inhibit BMP and TGF-β signalling which is paramount to successful neural induction. The medium is changed daily with a daily addition of the small molecules to final concentrations of 10 μM and 200 nM, respectively (Supplemental Figure S1). The focus for this study is the day 5 intermediate stage and the day 10 neuroepithelium. Further culturing of these cells takes place in the N2/B27 Neural Induction media without factors.