From day 18, for both hMGEOs and hCOs, neural differentiation media with vitamin A was used and replenished every 4 days. The media components were the same with the neural differentiation media used for hCOs between day 10 to day 18 described above, except that B27 supplement with vitamin A was used. In additiona, 20 ng/ml BDNF, 200 μM cAMP, and 200 μM ascorbic acid were supplemented to the media. For the majority of the experiments in the current study, HES-3 NKX2-1GFP/w cells were used unless otherwise stated in the results.
