Fig. 1

Protocols and results of stem cell differentiation in vitro. A A schematic illustrating cell cultures of WA09 and LT2e human ES cell lines in vitro. B Immunocytochemistry results at day 0 (a,d,g,j, and m), day 7 (b, e, h, k, and n), and day 14 (c, f, I, l, and o) of the WA09 cell line. SIX1 and OCT4 (a–c), EYA1 (d–f), PAX6 and OCT4 (g–i), DLX5 and OCT4 (j–l), and PAX2 and PAX8 staining (m–o) are shown. Pluripotent marker; OCT4 is expressed strongly on day 0, slightly on day 7, and not expressed on day 14. Pre-placodal ectoderm markers; SIX1, EYA1, and PAX6, and Dlx5 starts expressing at day 7 and continue to increase their expression as seen on day 14. Otic markers; PAX2 and PAX8 are slightly expressed on day 14. Scale bar represents 50 μm. C Representative semi-qPCR results showing the levels of each gene expression. D Quantification of the expression levels of each gene. OCT4 expression decreases, while PPE markers and OP markers increase with time of differentiation. Biological sample numbers were 3 and each sample was replicated twice. *P < 0.05; **P < 0.01. E Schematic shows our protocol to differentiate human ES cells in vitro. ES cell, embryonic stem cell; PPE, preplacodal ectoderm; OP, otic progenitor