Fig. 3

Qualitative and quantitative analysis of photoreceptor markers in mid-stage and mature organoids. Retinal organoids were analysed at D150 (A, B, E, F, I, J) and D225 (C, D, G, H, K, L) of differentiation. Representative images of the expression of the nuclear cone–rod homeobox protein (CRX; in green) and the calcium binding protein recoverin (RCVRN; in red) in Protocol 2 (A, C) and Protocol 3 (B, D) organoids. Representative images of the expression of the rod transcription factor NR2E3 (in green) and the cone-specific red–green opsins (RG opsin; in red) in Protocol 2 (E, G) and Protocol 3 (F, H) organoids. Representative images of rod-specific rhodopsin (in green) and cone-specific arrestin (in red) expression in Protocol 2 (I, K) and Protocol 3 (J, L) organoids. Scale bars = 20 μm. Quantification analysis of the relative areas of NR2E3 (M), RG opsin (N), arrestin (O) and rhodopsin (P) fluorescence within the ONL normalised to the area of Hoechst fluorescence in the ONL (see Additional file 1: Fig. S3). Quantification was performed on 6–13 images per organoid and 3 organoids were analysed per condition. Data are represented as mean ± SEM; *p < 0.05, n = 3; Mann and Whitney test. Quantification of rods (green bars) and cones (red bars), as determined by areas of normalised rhodopsin and arrestin fluorescence, respectively, and expressed as a percentage of the total photoreceptors at D150 (Q) and D225 (R) in Protocol 2 and Protocol 3 organoids. S Representative western blot (upper panel) of PDE6B expression in Protocol 2 and Protocol 3 organoids collected at D150, D180 and D225 of differentiation. β-tubulin was used as a loading control. Quantification (lower panel) of PDE6B expression normalised to β-tubulin expression of two independent experiments. For each time point, PDE6B levels in Protocol 3 organoids are expressed relative to those in Protocol 2 organoids. Data are represented as mean ± SEM; *p < 0.05; Mann and Whitney test