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. 2018 Nov 8;8:16550. doi: 10.1038/s41598-018-34845-2

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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SEAM formation and the insolation of human iPSC-derived CE lineage cells. (a) Schematic representation of the procedure used for the generation of a SEAM from human iPSCs and the subsequent isolation of CE lineage cells. CECs appeared in zone 3 of the SEAM at 3 to 4 weeks after the initiation of differentiation. Non-epithelial cellular zones 1 and 2 were removed by pipetting at week 7. At weeks 10 to 15, iPSC-derived CE lineage cells were isolated by cell sorting using anti-SSEA-4, anti-ITGB4, and anti-TRA-1-60 antibodies. Bars, 200 μm. (b) Representative immunostaining images showing how the expression of corneal epithelial markers p63 (green) and K12 (red) in non-CEC colony (no. 1) emerged in the iPSC-derived CEC sheets. (c,d) Representative immunostaining images of epithelial cell markers, pan-cytokeratin or K14 (green) and CD200 (red), in non-CEC colonies (nos 2, 3) in the iPSC-derived CEC sheets. Nuclei, blue. Scale bars, 50 μm.