To address whether in vitro timing of SHH exposure affects the regional identity of the resulting hPSC-derived NKX2.1::GFP+ cells we compared the identity of sorted cells at day 18 of differentiation (Figure 2F). In agreement with our previous results on floor plate induction (Fasano et al., 2010), we observed that early SHH exposure (2-18) suppressed FOXG1 induction (Figure 2G, left panel) despite robust induction of NKX2.1::GFP (see Figure 1H). In contrast both 6-18 and 10-18 treatment groups showed expression of FOXG1, but differed in OLIG2 expression (Figure 2G, middle and right panels). We hypothesize that differential expression of FOXG1 and OLIG2 reflect the distinct NKX2.1+ precursor domains observed during early human development (Figure 2A–E) and mimic the Olig2+ domains during mouse forebrain development (Flames et al., 2007). More than 90% of the NKX2.1::GFP+ cells in the 6-18 and 10-18 protocol co-expressed FOXG1 and nearly 80% of the GFP+ cells in the 10-18 protocol co-expressed OLIG2 (Figure 2H).
