Fig. 2

Differentiation of CTX0E16 cells generates glutamatergic cortical neurons. a-d Representative images of differentiated (DD 28) CTX0E16 neurons co-stained for 4′,6-diamidino-2-phenylindole (DAPI), MAP2 and either the deep cortical layer marker Ctip2 (a), the upper cortical layer marker Cux1 (b), GABAergic interneuron marker calretinin (c) or calbindin (d). Yellow arrows indicate neurons expressing these markers. e Quantification revealed that 41.6 % of MAP2-positive neurons expressed Ctip2, whereas less than 10 % of MAP2-labelled neurons were positive for Cux1, calretinin or calbindin; n = approximately 1,500 cells per condition from three independent experiments carried out in triplicate; error bars represent SD. (f, g) Images of DD28 CTX0E16 neurons co-stained for DAPI, MAP2 and the glutamatergic and excitatory markers VGlut1 (f) or CaMKIIα (g). h, i DD 28 CTX0E16 neurons were also co-stained for DAPI, MAP2 and the GABAergic markers GAD65/67 (h) or VGAT (i). j Quantification of F-I revealed that over 65 % of MAP2-psoitive neurons also expressed VGlut1 or CaMKIIα, whereas less than 45 % of MAP2-neurons were also positive for either GAD65/67 or VGAT; n = approximately 1,500 cells per condition from three independent experiments carried out in triplicate; error bars represent SD. Scale bar, 20 μm