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. Author manuscript; available in PMC: 2014 May 2.
Published in final edited form as: Cell Stem Cell. 2013 May 2;12(5):559–572. doi: 10.1016/j.stem.2013.04.008

Figure 1. Wnt inhibition and activation of SHH signaling yields highly efficient derivation of forebrain fates and NKX2.1 induction.

Figure 1

(A) Schematic of the differentiation protocol in the dual SMAD inhibition paradigm to generate anterior neural progenitors. NSB: Noggin+ SB431542. LSB: LDN193189+ SB31542.

B–E): When either DKK1 or XAV939, both Wnt signaling antagonists, were added to the dual SMAD inhibition protocol (DLSB or XLSB), there was a significant increase in the percentage of FOXG1+ cells (B) without loss of PAX6 expression (C): ** p < 0.01; *** p < 0.001; using ANOVA followed by Scheffe test. D) Representative immunofluorescent image for FOXG1 (red) and PAX6 (green) expression at day 10 following XLSB treatment. Single channel fluorescent images of marked region are shown in the three right panels E) Robust telencephalic specification using XLSB was also observed at comparable efficiencies in human induced pluripotent stem cells (hiPSC lines SeV6, C72; n = 4). F–H) Addition of SHH signaling to the XLSB protocol significantly enhanced the production of NKX2.1::GFP expressing progenitors. (F) 5nM SHH (Sonic C24II) and 1μm Purmorphamine, added from day 4, showed synergistic effects in inducing NKX2.1::GFP expression at day 10 (*** p < 0.001; compared to SHH). A range of concentrations of SHH and Purmorphamine are compared at day 18 in (G), and again co-treatment was greatly superior to quite high concentrations of either SHH or purmorphamine alone (*** p < 0.001; compared to no SHH using ANOVA followed by Scheffe test). (H) Delaying the timing of SHH exposure between 2 and 10 days of differentiation did not dramatically affect the efficiency of NKX2.1::GFP induction measured at day 18 (*** p < 0.001 compared to 0–18 using ANOVA followed by Scheffe test). P: purmorphamine, S: Sonic hedgehog. Data are from hESC line HES-3 (NKX2.1::GFP) in panels B,C,F,G,H from hESC line WA-09/H9 (panel D) and from hESC line WA-09/H9 and hiPSC lines SeV6 and C72 (panel E). Scale bar in (D) represents 125μm. Data in (B,C,E–H) are represented as mean ± SEM.