aNPCs maintained in FGF give rise to cortical neurons by default and are responsive to patterning cues.

<p>(<b>A</b>)<b>:</b> Upon withdrawal of FGF2, aNPCs display time-dependent upregulation of dorsal telencephalic marker <i>EMX2</i> as determined by qRT-PCR (D = days <i>in vitro</i> differentiation. <i>EMX2</i> expression levels are normalised to levels detected in proliferating aNPCs ( = 1), β-actin is used as housekeeping control. <i>EMX2</i> D10 expression = 130.4±29.2, n = 4. (<b>B</b>)<b>:</b> Gene expression in human fetal brain (FB) and aNPC cultures differentiated for 6 days. All expression levels are normalized to levels detected in human tissue ( = 1). Data are represented as mean ± SEM, n = 4 for differentiated aNPCs, passage >20. For tissue, n = 1. aNPC derived neurons express REELIN by 4 weeks of differentiation as determined by qRT-PCR (<b>C</b>; aNPCs vs neurons: 0.7±0.2 vs 35.3±5.4, n = 4, <i>P</i><0.001, un-paired <i>t</i>-test) and immunofluorescence (<b>D</b>). (<b>I</b>)<b>:</b> Quantitative immunohistochemical analysis of neurons differentiated from aNPCS revealed expression of both deep-layer and upper-layer cortical neuronal markers (CTIP2, BRN2, CUX1, SATB2) (<b>E–H</b>). Data from n = 4–7 differentiation experiments from three H9 hESC- (passages >20) and three hIPSC-derived (passages 10–28) aNPC lines shown. (<b>J</b>)<b>:</b> aNPCs are responsive to patterning cues, sequentially upregulating HOXB4, OLIG2, ISL1 and HB9 expression in response to treatment with RA and SHH agonist purmorphamine during motor neuron differentiation. (<b>K</b>)<b>:</b> Quantitative immunohistochemical analysis of OLIG2 induction efficiency in early and late passage aNPCs upon treatment with RA and purmorphamine (n = 3 differentiation experiments from independent, early (<15) and late (>25) passage aNPC lines). Data are represented as mean ± s.e.m. Scale bars are 20 µm.