Fig. 4

After dual restriction and dual inhibition, cells were subsequently patterned with Shh and FGF8. Cells were Tuj1⁺ (a Green) and Nurr1⁺ (a Red), CaMKII⁺ (b Green) and DAT⁺ (b Red) after 10 days of patterning. Cell nuclei were stained with DAPI (Blue). c After 10 days of patterning, for cells subjected to dual lineage restriction followed by culture in N2 supplemented medium, 4.57 ± 0.9 % of the cells were Nurr1⁺, as compared to more than 98 % of Nurr1⁺ cells with continuous LDN193189 treatment or dual inhibition. d Dopamine release was found to increase continuously after patterning for up to 28 days. When 5 and 10 μM of amantadine were added to the culture medium, higher dopamine secretion levels were observed and a plateau was reached on day 25 of patterning. e Compared with neurons generated from neurosphere, neurons generated from dual restriction and dual inhibition from hESCs single cells displayed a significantly higher neurite outgrowth rate over 90 h culture. Scale Bar: A, B, 100 μm. n = 3 for each group