Fig. 3

a Under dual restriction and dual inhibition, when FGF2 were supplemented to the culture medium during dual inhibition, SOX1, FGF5, LMX1A and MASH1 were all significantly down-regulated. b Under dual restriction and dual inhibition, when PD173074 were added to the culture medium on day 1, there was significant up-regulation of SOX1, FGF5, LMX1A and MASH1 expression. c Under dual restriction and dual inhibition, a significantly higher percentage of SOX1⁺ cells was observed for cells treated with PD173074 on day 1 only and harvested on day 5, as compared to the absence of PD173074 treatment. d Morphologically, cells formed networked structures on day 5 when switched to differentiation medium from restriction medium. e After 5 days of culture, Nestin⁺ (Red) and Tuj1⁺ (Green) were found co-localized within the colonies formed. f When sub-cultured, columnar neural rosettes were observed on day 10. g When sub-cultured, columnar neural rosettes were SOX2⁺ (Red) and Tuj1⁺ (Green) on day 10. Cell nuclei were stained with DAPI (Blue). Scale Bars: D, 100 μm. E, F & G, 200 μm. n = 3 for each group