Figure 5.

9CRAL-expedited photoreceptor development. A: Representative images of immunostained sections of H9 retinal organoids supplemented with either all-trans retinoic acid (ATRA; left) or 9-cis retinal (9CRAL; right). D90 (top) 10 μm sections were immunolabeled for pan-photoreceptor marker recoverin (green) and retinal progenitor cell marker CHX10 (red). D120 sections (bottom) were immunolabeled for rod photoreceptor marker rhodopsin (green), cone photoreceptor marker OPN1SW (red), and L/M cone photoreceptor marker OPN1L/MW (magenta). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Arrowheads indicate relevant staining of a specific marker. Scale bar, 10 μm. B: Immunoblot showing rhodopsin expression in H9 ATRA and 9CRAL organoids at D120 (individual replicates are shown). The asterisk denotes a second band which is likely to be dimerization of rhodopsin. γ-tubulin is included as the protein loading control for protein amounts. C: Transmission electron microscopy of longitudinal sections at D130, showing inner segments and cilia. Hollow, solid, and v-shaped arrowheads indicate the relevant structure of the photoreceptor cilium, mitochondria, and inner segments, respectively. Magnifications (scale bars) in the right panel are the same as shown in the left.