In vitro hESC neural differentiation yields cortical neurons.

<p>(<b>A</b>) Schematic representation of the strategy for differentiating hESCs into cortical neurons, displaying culture and treatments conditions. (<b>B</b>) RT-PCR of H9 cells shows differential expressions of pluripotent markers (<i>Pou5F1</i>, <i>Nanog</i>), dorsal progenitors (<i>Pax6, Emx2, Otx2, Foxg1</i>), cortical neurons (<i>Fezf2, Nfib, Tbr1</i>), and ventral marker (<i>Ascl1</i>) at neural differentiation day-6, 12 and 32. (<b>C–E</b>) Immunofluorescence staining of differentiated H9 cells shows expression of neural progenitor markers NESTIN, PAX6, TBR2 and immature neuronal marker TUJ1. (<b>F–H</b>) Immunohistochemistry shows that differentiated H9 cells express cortical neuron marker BCL11B (F), NFIB (G) and TBR1 (H). Scale bars in C, D, 50 µm; E, 25 µm; F–H, 100 µm. (<b>I</b>) Immunofluorescence staining of differentiated <i>Fezf2</i>-YFP-HUES 9 reporter line on day 40 with GFP antibody. Scale bar, 200 µm. (<b>J</b>) Differentiated <i>Fezf2</i>-YFP reporter line expressing GFP and the human specific nuclear antigen. Scale bar, 25 µm. Abbreviations: DAPI, 4′,6′-diamidino-2-phynylindole; YFP, yellow fluorescent protein; GFs, growth factors;