Fig. 5

NESC-spheres spontaneously differentiate into cortical neurons. a Differentiated neurons co-cultured on mouse astrocytes uniformly express a human-specific marker HN (human nucleus), suggesting that these neurons are from human NESC-spheres but not from mouse. b Quantification of NESCs early passage (P8) and late passage (P30) differentiated total neurons. HN⁺ neurons indicate human-derived neurons. c, d Differentiated BRN2⁺ and FOXP2⁺ cortical neurons from NESC-spheres. e, f Differentiated CTIP2⁺ and CUX1⁺ cortical neurons from NESC-spheres. g Quantification of deeper layers FOXP2⁺ neurons and upper layers BRN2⁺ neurons in early passage (P8) and late passage (P30) differentiation of NESCs. h Differentiated REELIN⁺ cortical neurons from NESC-spheres. i, j Differentiated mature neurons at pdD 89 expressed synapsin I protein and the post-synapse marker PSD-95 with a punctate pattern. k At day 34 after post-differentiation (pdD34), differentiated neurons from NESC-spheres express a mature neuron marker NeuN. l, m vGLUT1⁺ glutamatergic and GABA⁺ GABAergic neurons from NESC-spheres. n Quantification of NESCs early passage (P8) and late passage (P30) differentiated GABAergic neurons. Data are represented as mean ± SD (n = 3 independent experiments). *p < 0.05 by Student’s t test. Scale bars, 100 μm. Blue: DAPI, nuclear stain