Neural differentiation of hESCs via the dual SMAD-inhibition protocol (Chambers et al., 2009) using Noggin + SB431542 (NSB) robustly induced FOXG1+/PAX6+ precursors. Replacement of Noggin with the ALK2/3 inhibitor LDN-193189 (LSB) induced PAX6 expression equally well but showed a trend towards lower percentages of FOXG1+ cells (Figure 1B,C). Adding recombinant DKK1 or the tankyrase inhibitor XAV939 (Huang et al., 2009), inhibitors of canonical Wnt signaling, enhanced FOXG1 expression in LSB-treated cultures (Figure 1B–D). Importantly, the effect of XAV939 on FOXG1 induction was consistent (Figure 1E) across multiple independent hESC (HES-3 and WA-09) and human iPSC lines (C72 line (Papapetrou et al., 2009), SeV6 (Kriks et al., 2011)). Therefore, the use of three small molecules (XAV939, LDN-193189, and SB431542; termed XLSB) enables rapid and robust induction of forebrain fates across human ESCs and iPSC lines.
