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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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Detection of early photoreceptors after 45 days and markers typical of the neural retina derived from IMR90.4 iPSCs. (a) A D45 RC culture with a curved (yellow arrows) pseudostratified appearance. (b,c) RCVN (+) PRs at D45 line the prospective outer retina. (d–f) Quantitative PCR analysis of the early retinal markers VSX2, RCVRN, and CRX was performed on cDNA samples pooled (n = 10) from IMR90.4 iPSCs to confirm their relative levels of expression. (g) At D160 OS-like structures were observed protruding from the surface of RCs. A higher magnification image of the POSs illustrates the fine radial architecture (inset box). (h) PAX6 immunolabeling at D160 labels neurons in the presumptive INL and GCL (arrows). (i) PSD95 immunolabeling at D120 in the outer retina where PRs are located. (j–l) CtBP2 staining in nuclei and synapses in D160 retina is highlighted by arciform structures corresponding to ribbon synapses (j, inset panel). (j) The position of CtBP2 labeling at synapses occurs at the base of photoreceptor terminals, a position verified by double labeling with PNAL, a marker for PR inner segments and synaptic terminals (k,l; arrow). An inset box in (l) corresponds to the region marked by the arrow in panels (k and l). Data from pooled samples (n = 10 per time point) was calculated using three replicates per time point and error bars are 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. INL = inner nuclear layer; OS-like = outer segment-like; PNAL = peanut lectin agglutinin; POS = photoreceptor outer segment; PR = photoreceptor, RC = retina cup; RGC = retinal ganglion cell. Scale bars (a) = 300 μm, (b) = 50 μm, (c) = 25 μm, (g) = 40 μm, (h,i,k,l) = 60 μm, (j) = 50 μm.