Characterization of generated iPSCs and neuronal differentiation. (A) SCN1A sequencing of the indicated cell material. Solid arrowheads point to the c.4933C>T substitution. (B) iPSC morphology and immunostaining of pluripotency markers (Oct 4, Nanog, Tra-1-60, Tra-1-81, and SSEA4 without SSEA1). Scale bar, 500 μm. (C) iPSC-derived teratomas generated in NOD-SCID mouse testes comprised tissues from all three germ layers. Scale bar, 200 μm (neural rosettes and respiratory epithelium) and 400 μm (others). (D) Real-time PCR analysis showed suppressed expression of the four reprogramming factors in both patient and control iPSCs compared to patient fibroblasts transduced with the same four factors (D1-HDF-4F). (E) G-band karyotyping showed normal chromosome numbers (46,XX) in all tested colonies (N = 20 each). (F) Representative images of embryoid bodies (EB) and neurospheres (NS). Scale bar, 500 μm. (G) Expression of βIII-tubulin, a neuronal marker (green) and GFAP, an astrocyte marker (red) in iPSC-derived neural cells. Scale bar, 200 μm. Day numbers indicate the days of differentiation in adherent culture after neurosphere formation.