Differentiation of retinal organoids using the original serum-free floating cultures of embryoid body-like aggregates with quick reaggregation (SFEBq) protocol. No significant morphological differences were observed among neural retinas (NRs) differentiated from ESCs or iPSCs, and the representative figures of NR are shown. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). (A) Morphology of NR. Immunostaining with anti-rhodopsin (Rho, red) antibody marks rod photoreceptors, whereas anti-calbindin (Calb, green) labels both horizontal and amacrine cells. S-opsin (Opnsw, red) is a marker for S-cones. Amacrine and ganglion cells are labeled by Calretinin (red) and Brn3a (red) immunostaining. Bipolar cells are indicated by Pkcα (red) and Chx10 (red). Scale bar: 100 μm. (B) Relative viability of cells in 30–60 organoids that were counted in each experiment. The survival data from D18 organoids in High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids (HIPRO) protocol was set at 100%. Gating by DAPI and forward scatter was used to evaluate cell viability. (C) Percentage of GFP+ cells (rods) in the total cell population from 30 to 60 organoids. No significant differences were observed in the percentage of GFP+ cells from ESCs or iPSCs-derived retinal organoids. The data in (B) and (C) are represented as mean ± SEM and was obtained from 4 independent experiments each from ESC and iPSC organoids (n=4). *p<0.05. To access the data, click or select the words “Appendix 1.”