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. 2020 Jul;46:101868. doi: 10.1016/j.scr.2020.101868

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© 2020 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 1 — Schematic description of ocular surface lineage epithelial development and differentiation of p63-EGFP knock-in hiPSCs into early ocular surface ectoderm. (A) Schematic description of ocular surface ectodermal development in an embryonic eye and a pre-SEAM structure of differentiated human iPSCs at 10 days and a SEAM structure at 6 weeks of differentiation. The cell culture duration, mediums, cell treatments and analysis time points are shown. The cells were treated 1) from 0 to 4 days with BMP4, LDN 193189 (LDN), TGFβ, SB431542 (SB), retinoic acid (RA), JNK-IN-8 (JI8), IWP2, IWP2 + BMP4 (I + B), IWP2 + LDN (I + L), CHIR99021 (CHIR), CHIR + BMP4 (C + B), and CHIR + LDN (C + L), or 2) from 4 to 7 days with BMP4, IWP2, or I + B. CE, corneal epithelium; CDM, Corneal Differentiation Medium; DM, Differentiation Medium; EK, epidermal keratinocyte; CNS, central nervous system; NR, neuroretina; OSE, ocular surface ectoderm; SE, surface ectoderm; LE, lens; NC, neural crest; OC, optic cup; RPE, retinal pigment epithelium; NE, neural ectoderm. (B) A representative phase contrast image of a colony of undifferentiated hiPSCs at 0 day (scale bar, 200 μm). (C) Characterization of differentiated p63-EGFP knock-in hiPSCs into ocular cells in the pre-SEAM structure at 10 days of differentiation (scale bar, 100 μm). Immunofluorescence staining for ocular cells markers; p63 expression (green) was detected in the paracentral zone and PAX6 expression (red) was detected in the central and paracentral zone. Nuclei, blue. (D) Higher magnification images of highlighted zone from C. The white arrows indicate p63+/PAX6+ cells in the paracentral zone (scale bar, 100 μm). (E) p63+/Pax6+ cells in mouse embryonic eyes at E8.5 and E9.5 (left panels; upper and bottom, scale bar, 200 μm and the other pictures, scale bar, 100 μm). p63 (green) and Pax6 (red) in the embryos. Nuclei, blue. The white arrows indicate p63+/Pax6+ cells. The yellow allows indicate ventral and dorsal. OSE, ocular surface ectoderm; PLE, presumptive lens ectoderm; OV, optic vesicle.