Fig. 3.

Defect in neuronal maturation of mutant RTT-iPSCs. (A) Schematic depicting the neural differentiation procedure. See Materials and Methods for a detailed description. Representative images showing morphological changes during neural differentiation of hESCs (H1), and RTT-iPSCs (RTT4-iPS-24w, RTT4-iPS-19bi). (B and C) H1 hESCs and RTT-iPSCs were induced for neuronal differentiation and stained with neuronal stem cell marker (NESTIN), mature neuronal marker (TuJ), and astrocyte marker (GFAP) during the differentiation. H1 hESCs and WT and mutant RTT-iPSCs all show a similar neuronal commitment, represented by NESTIN staining after 11 d of neural differentiation (B). H1 hESCs and WT RTT1-iPS-13w cells are fully differentiated into neuronal differentiation for 25 d and display a robust expression of the mature neuronal marker TuJ, whereas mutant RTT1-iPS-15m cells demonstrate less neural maturation. In contrast, glial differentiation (GFAP⁺ cells) is similar in all cell lines (C). **P < 0.01. Bar graphs show the percentages of TuJ⁺ and GFAP⁺ cells. The densities of TuJ⁺ and GFAP⁺ cells vs. DAPI per square mm were analyzed using Image J analysis software. Data are mean ± SEM of one experiment performed in triplicate.