Fig 4.

Expansion of human ES cell-derived FOXA2⁺ neural progenitor cells by SHH-C24II and human FGF8a for 28 days leads to the generation of FOXA2 expressing DA neurons. (A) Human ES cell-derived neural progenitor cells were grown in medium supplemented with Shh and FGF8 splice variants before neuronal differentiation. (B–E) Immunocytochemistry at DIV49 revealed many β-tubulin⁺ neurons (green) and TH⁺ neurons (blue) but very few FOXA2⁺ cells (red) differentiated from human ES cells after exposure to Fgf8b with (B) or without mouse Shh (E). In contrast, 500 ng/ml SHH-C24II with FGF8a (C) or Fgf8b (D) generated many FOXA2⁺ cells. Importantly, FOXA2⁺/β-tubulin^High/TH⁺ neurons were only observed after exposure to FGF8a (C, arrowheads). (F) Cell counts revealed a significantly greater percentage of human FOXA2⁺ cells (* p < 0.05 ANOVA) and FOXA2⁺ neurons (# p < 0.05 ANOVA) after exposure to 200 or 500 ng/ml SHH-C24II and FGF8a. A significant increase in the percentage of FOXA2⁺ DA neurons was observed after exposure to 500 ng/ml SHH-C24II and FGF8a (δ p < 0.05 ANOVA). Scale bar = 50 µm.