Human iPSCs were differentiated using published protocols [28,29] with few modifications to increase differentiation efficiency as illustrated in S1 Fig. Briefly, hiPSCs at between 10 and 25 passages were rinsed with 1X Ca2+/Mg2+ free Dulbecco’s Phosphate-Buffered Saline (HyClone; DPBS) and then treated with Accutase (MP Biomedicals) for 5 ~ 7 minutes at 37°C to make a single cell suspension. Live cells were counted using Vi-Cell (Beckman-Coulter). At day 0 of differentiation, three million cells were added to each well of an AggreWell800 plate (Stem Cell Technologies, Inc.) following manufacturer’s protocol in NIM with 250 ng/ml noggin (R&D Systems) and 10 μM SB431542 (R&D Systems). 50 ~ 75% of the media was replaced daily for 5 days with fresh NIM supplemented with noggin and SB431532. On the fifth day of differentiation, cell clusters were collected from a well of the AggreWell800 plate and re-plated in 6-well plates coated with Matrigel™ in NIM supplemented with noggin and SB431532. Media was replaced every other day for an additional four to five days until neural rosettes composed of NPs were detected in most areas of the wells.
