Fig. 6.

Otic placode marker expression of the induced cells in the presence or absence of RA from Day 14. (A) Schematic figure of the culture conditions used to investigate the role of RA in the differentiation of the otic placode. The cells were treated with LDN, CHIR, and FGF3/FGF10 from Day 14 to Day 22 of differentiation. Culture conditions with or without RA (1 μM) treatment were compared. (B) qPCR analysis of the otic placode markers SIX1, PAX8, PAX2, DLX5, and GATA3 on Day 22 of cultures with or without RA treatment from Day 14 to Day 22. The relative gene expression levels were normalized against those of undifferentiated hESCs. The bars and error bars represent the mean ± SEM (three independent experiments). Asterisks indicate significant differences (∗p < 0.05, two-tailed unpaired Student's t test). (C) Representative bright field images and low-magnification immunocytochemical images on Day 18 of spheres with or without RA treatment. The nuclei were stained with Hoechst 33,342. Scale bars, bright field images, 100 μm; immunostaining images, 100 μm. (D) Representative immunocytochemical images of induced cells on Day 18 in cultures treated with RA. The cells were coimmunostained for multiple otic placodal markers (ECAD/PAX8/SOX2, SIX1/PAX8/SOX2, or SIX1/FOXG1/SOX2). The nuclei were stained with Hoechst 33,342. Scale bars, 50 μm. (E) Representative immunocytochemical images of induced cells on Day 22 of culture treated with RA. The cells were coimmunostained for multiple otic placodal markers (ECAD/PAX2/SOX2, SIX1/PAX8/SOX2, or PAX8/PAX2/SOX2). The nuclei were stained with Hoechst 33,342. Scale bars, 50 μm.