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. 2019 Mar 14;10:1205. doi: 10.1038/s41467-019-08961-0

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© The Author(s) 2019

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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Fig. 1 — Identification and C-Kit/SSEA4 expression in hEROs. a Induction of the 24-day neural retina structure (black arrowhead). b Induction of the 30-day neural retina. c Magnification of the neural retina (black arrowheads) shown in b. d Quantitative analysis showing that the number of generated neural retinas increased with induction time (18-day: 11.1 ± 1.2%; 24-day: 63.3 ± 1.9%; 30-day: 86.7 ± 1.9%; n = 3 independent experiments/group; each experiment containing 96 hEROs.). e–g Expression of the RPC markers RAX, PAX6, and CHX10 and the proliferation marker Ki67 in hEROs. h Identification of the apical side of hEROs on day 30 via ZO-1 staining. i–k Distribution of C-Kit and SSEA4 antigens in 30-day, 45-day, and 60-day hEROs. l Flow cytometry analysis of C-Kit and SSEA4 expression in hEROs at different induction times (30-day, 45-day, and 60-day). m Statistical analysis of C-Kit and SSEA4 expression in hEROs (C-Kit: 30-day: 32.60 ± 10.58%; 45-day: 8.53 ± 1.36%; 60-day: 1.58 ± 0.45%; SSEA-4: 30-day: 24.6 ± 4.16%; 45-day: 9.35 ± 2.23%; 60-day: 1.17 ± 0.88%, n = 3 independent experiments/group; each experiment containing 192 hEROs). n Percentage of C-Kit⁺/SSEA4⁻ cells determined by flow cytometry analysis (30-day: 17.80 ± 4.06%; 45-day: 7.99 ± 2.96%; 60-day: 0.96 ± 0.42%; n = 3 independent experiments/group; each experiment containing 192 hEROs). o–q Co-expression of C-Kit and RPC markers in 30-day hEROs. r Quantitative analysis of RPC marker expression in C-Kit⁺/SSEA4⁻ cells in 30-day hEROs (RAX: 92.86 ± 1.88%; CHX10: 75.79 ± 7.46%; PAX6: 32.74 ± 1.58%; n = 3 independent experiments/group; each experiment containing 3 hEROs). Data are presented as mean ± SD. Scale bars, 200 μm (a, c, i–k), 1 mm (b), 50 μm (e–h, o–q). hEROs hESC-derived retinal organoids