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. 2021 Apr 21;12:248. doi: 10.1186/s13287-021-02316-7

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Evidence of in vitro differentiation. a Phase contrast microscopy shows key morphological changes: Human embryonic stem cell (hESC: MEL-1 line) morphology prior to differentiation, formation of differentiating cell monolayers (day 2), and anterior neuroectoderm bodies (ANEBs) (day 5). Plated ANEBs produce expanding sheets of hESC-RPE cell monolayers in the outgrowth by day 14. Scale = 100 μm. b Gene expression over the 14-day differentiation period was measured by qPCR in relation to GAPDH and normalized to maximum expression for each gene over the time period. Normalized expression profiles indicate loss of hESCs (OCT4), upregulation of neuroectoderm [PAX6(+5a), PAX6(−5a), and LHX2], eyefield formation (RAX), and differentiation of RPE cells (MITF and PMEL17). Contaminating neural retinal cell formation was monitored through expression of VSX2. Series A, B, C reflect independent time course experiments and data points reflect the average of technical triplicates. c Flow cytometry indicates expression of TRA-1-81, MITF, and PMEL17 by hESCs at day 0, and differentiation hESC-RPE (ANEBs) at day 14. Data reflect mean + S.D. (n = 3). For each marker, significant differences are detected in the staining of hESCs versus day 14 ANEBs (p < 0.0001)