Figure 1.

Transient Shh inhibition by GANT61 and Olig2 overexpression sequentially promoted NG2⁺ OPC differentiation from hiPSCs. A Strategy for generating OPCs derived from Olig2/hiPSCs treated with GANT61 during neural induction and sequentially inducing Olig2 overexpression by doxycycline treatment. Adherent hiPSC colonies were differentiated into NPCs (GANT61-NPCs) by adding GANT61 to NIM1 and NIM2 from d-7 to d0. The cultured GANT61- NPCs were dissociated into single cells in six-well plates precoated with poly-L-ornithine/laminin and then subjected to glial induction with GIM and DM containing Y-27632. B The mRNA expression levels of oligodendroglial lineage genes (NKX2.2, CSPG4, ST8SIA1, and PDGFRa) involved in OL development at d4 after Olig2 induction (** p < 0.01, *** p < 0.001, by a two-tailed Student's t test). C Representative flow cytometry analyses for the expression of NG2 and PDGFRα in the control and Olig2 induction cultures at d4 after Olig2 induction. D The corresponding quantification of NG2⁺ and PDGFRα⁺ cells at d4 in control and Olig2-induced cultures (ns p > 0.05, ** p < 0.01, by a two-tailed Student's t test). Representative flow cytometry analyses (E) and quantification (F) of the coexpression of NG2 and PDGFRα in the control and Olig2-induced cultures at d7 of differentiation after Olig2 induction (*** p < 0.001, by a two-tailed Student's t test). The graphs represent the individual data points and the mean ± SEM of three independent experiments. Immunofluorescence images are representative of n = 3 biological replicates.