EpiLCs induction was modified from47 (link). Briefly, ES cells were gently dissociated using trypsin/EDTA, and a single-cell suspension was plated onto Geltrex (GIBCO_#A1413202)-coated plates at a density of 5000 cells/cm2 in ESC growth medium. One day after plating, EpiLC induction started by adding the N2B27 medium supplemented with 20 ng/ml ActivinA (GIBCO_#PHC9564) and 12 ng/ml bFGF (GIBCO_#PHG0026). The cells were splitted 1:3 in small clumps using 1 mg/ml Collagenase IV (GIBCO_#17104019). Medium was changed daily. EpiLCs were collected for DNA, RNA and protein analyses after 14 days of induction.
N2B27 medium is composed by 50% advanced DMEM/F12 (GIBCO_#12634028) and 50% Neurobasal medium (GIBCO_#21103049), supplemented with 0.5% N2 Supplement (GIBCO_#17502048), 1% B27 Supplement (GIBCO_#17504044), 0.033% BSA solution (SIGMA_#A9647), 50 µM β-mercaptoethanol (Sigma_#M3148), 2 mM Glutamax (GIBCO_#35050038), 25 U/ml penicillin and 25 μg/ml streptomycin (Invitrogen_#15070063).
N2B27 medium is composed by 50% advanced DMEM/F12 (GIBCO_#12634028) and 50% Neurobasal medium (GIBCO_#21103049), supplemented with 0.5% N2 Supplement (GIBCO_#17502048), 1% B27 Supplement (GIBCO_#17504044), 0.033% BSA solution (SIGMA_#A9647), 50 µM β-mercaptoethanol (Sigma_#M3148), 2 mM Glutamax (GIBCO_#35050038), 25 U/ml penicillin and 25 μg/ml streptomycin (Invitrogen_#15070063).
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