Differentiation of human postmitotic neurons derived from hESCs. (A) Differentiation of H9 hESCs into neurons. hESCs (line H9) were dissociated from feeder cells and cultured in aggregates to develop into EBs, which were transferred to flasks containing neural induction medium. Rosette structures were formed and, 12 days after neural induction, were isolated from the surrounding cells to develop into neurospheres. One month later, terminal neuronal differentiation was induced in the presence of BDNF and GDNF. (B) Human neural progenitors at the rosette stage were immunostained with anti-nestin antibody (green) and anti-BF1 antibody (red). Bars: 50 mm. (C) Terminally differentiated human neural cells were immunostained with anti-MAP2 antibody (red) and anti-GFAP antibody (green). Bars: 50 μm. (D) Human neurons derived from hESCs were immunostained with anti-MAP2 antibody (green) and anti-PSD-95 antibody (red). The merged image shows an enlarged image of the boxed area. Bars: 20 μm.