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. 2017 Apr 10;7:766. doi: 10.1038/s41598-017-00774-9

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© The Author(s) 2017

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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Localization of cone and rod opsins is restricted to outer segments late in Day 300 retina cups derived from IMR90.4 iPSCs. (a) Schematic diagram of the important stages during the transition from pluripotent stem cells into 3D retina cups bearing rod and cone outer segments. (b) A brightfield image of exuberant POS growth from the surface of RCs. The distal tips of POS are prominently labeled with antibodies against (c) rhodopsin and (d) red-green opsin. Black arrows indicate outer segment-like structures while white arrows indicate inner segment structures. (e) Quantitative analysis of select photoreceptor genes (NRL, NR2E3, Rhod, SW-, MW- and LW-opsin, arrestin and the calcium channel proteins CNGA3, and CNGB3) was carried out to show quantifiable changes in gene expression. Target genes were normalized against the geometric means of the reference housekeeping genes CREBBP, FBXL12, and SRP72. Data reflects the expression levels from pooled samples (n = 10 per time point) from three replicates per time point and error bars represented as s.e.m. The difference between the indicated time points is statistically significant by Student’s t-test where ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant. POS = photoreceptor outer segment; PR = photoreceptors; RC = retina cup. Scale bars = 50 μm.