We next wanted to test our ability to induce ventral fates using XLSB in the presence of SHH activators (Figure 1A). The transcription factor NKX2.1 is a marker of ventral prosencephalic progenitor populations (Sussel et al., 1999; Xu et al., 2004). Using a previously established NKX2.1::GFP knock-in reporter hESC line (Goulburn et al., 2011), we observed GFP induction by day 10 following activation of the SHH pathway by recombinant SHH (R&D Systems, C-25II) and the smoothened activator purmorphamine (Figure 1F). We found maximal induction of NKX2.1::GFP at day 18 upon treatment with purmorphamine at 1 μM and with SHH at 5nM (Figure 1G), a condition referred to as “SHH” for the remainder of the manuscript. Our past studies on the derivation of hESC derived floor plate cells demonstrated that early treatment with SHH (day 1 of differentiation) is critical for the efficient induction of FOXA2 (Fasano et al., 2010). In contrast, we observed here that cells exposed to SHH at late differentiation stages (day 10 of the XLSB protocol) remain competent for inducing NKX2.1::GFP as measured by FACS (Figure 1H). Therefore, XLSB+SHH treatment represents a strategy to generate NKX2.1+ progenitors at high efficiencies.
