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. 2022 Feb 19;11(2):159–177. doi: 10.1093/stcltm/szab010

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© The Author(s) 2022. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Impact of drugs on photoreceptors of retinal organoids derived from WT3 iPSCs. (A): Immunofluorescence of Crx (green), recoverin (Recov; red), rhodopsin (Rho; green), and OPN1MW/LW at day 150 of differentiation. Expression of Crx and recoverin was found at the apical edge of organoids in all conditions, except digoxin and thioridazine exposure where a disorganization of the photoreceptor layer was observed. Rods identified by rhodopsin immunofluorescence were rare in all retinal organoids at this time point of differentiation. Expression of OPN1MW/LW for middle and long-wavelength indicated mature and premature (nuclear staining) cones in all conditions. Higher magnification images revealed subcellular compartments of middle-/long-wavelength cones (OPN1; red) and rods (Rho; green) as well as rod OS (Gαt1; red). Nuclei were counterstained with Hoechst (blue). Scale bars, 20 μm (B): Immunofluorescence quantification of recoverin, Crx, rhodopsin, OPN1MW/LW, and OPN1SW for all conditions. The exposure of digoxin and thioridazine resulted in a reduction of recoverin- and Crx-positive cells while rhodopsin expression was not affected after drug treatments at this time point of differentiation. OPN1MW/LW⁺ cells were reduced in all drug-treated conditions, except for Thioridazine whereas OPN1SW expression was affected only after sildenafil exposure. Data shown as mean ± SEM, N = 2 (independent experiments), and 5-20 images from different retinal organoids were quantified per condition. Differences were considered statistically significant at ∗P <.05, ∗∗P < .01 and ∗∗∗P <.001. (C): Single-cell RNA-Seq data at day 200 of differentiation showed a reduction in the percentage of rods (clusters 0 and 7) after digoxin and thioridazine treatment, while the percentage of cones (clusters 2 and 19) decreased after digoxin, thioridazine, and sildenafil exposure. Data are shown as an individual percentage for each cell type and condition. The mean (black line) ± SEM (dashed line) of control and ketorolac conditions are shown. Hoe, Hoechst; Recov, Recoverin; Rho, Rhodopsin.