Fig. 3.

Specific addition of all-trans retinoic acid (ATRA), triiodothyronine (T3), and taurine (TAU) modulates the generation and maturation of cone and rod photoreceptors in retinal organoids. A Diagram of the experimental design, showing the addition of stage-specific factors during retinal organoid differentiation in 3 phases as follows: (i) noggin, DKK1, and bFGF from day 0 to 30; (ii) ATRA (10 µM) from days 30 to 90; and (iii) medium (control), ATRA, T3 and TAU individually and in combination with each other, from days 90 to 120. Purple suns indicate retinal organoids with developed inner and outer segments (IS/OS) of photoreceptors, and red circles indicate retinal organoids without developed IS/OS. B Bright field and hematoxylin and eosin staining of retinal organoids treated with medium (control), ATRA, T3, and TAU; and a combination. Middle panels show magnification of IS/OS of photoreceptors. Arrows indicate the developing IS/OS. Scale bars: 50 µm. C Quantitative PCR of gene expression in retinal organoids at day 120. Values are normalized to GAPDH. Data presented as mean ± SD (n = 3). Statistical significance was calculated by two-way ANOVA followed by Tukey’s multiple comparison tests (p < 0.05) comparing with ATRA (*); comparing control, T3 and taurine groups (#). D Representative immunostaining images of retinal organoids treated with medium (control) or T3, stained with photoreceptor-specific markers recoverin (RECOV), blue-opsin (B-opsin), red/green-opsin (RG-opsin), and rhodopsin (RHO). Nuclei are stained with DAPI. Scale bars: 25 µm. E Quantification of number of positive cells expressing blue-opsin (Blue cones), red/green-opsin (red/green cones) or rhodopsin (Rods) in retinal organoid paraffin sections (N = 3 different experiments, n = 5–7 retinal organoids). Two-way ANOVA calculated statistical significance (p < 0.05) comparing the non-ATRA group with ATRA data (*); comparing TAU, T3, and TAU + T3 with Control (#); and comparing ATRA with the ATRA + supplement groups (†)