Differentiation and FACS purification of A2B5+ cells from U-iPSCs. (A) Scheme of our differentiation protocol. (B–C) Before sorting, about 80% of differentiated cells have A2B5 immunoreactivity; after A2B5+ sorting, 95–99% of cells are A2B5+. (D–E) On the contrary, the percentage of TRA1-81+ iPSCs is decreased to 0. (F–H) Before sorting, human iPSCs differentiate along neural lineage express A2B5, and NSC markers SOX1 and PAX6. However, a small percentage of cells express pluripotent marker TRA1-81 (I), indicating contamination of undifferentiated cells. (J–L) After A2B5+ sorting, cells maintain A2B5, SOX1 and NESTIN expression. (M) Most importantly, the undifferentiated TRA1-81+ cells have been depleted. Bar, F, I, J–M, 50 μm; G, H, 20 μm.