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Fig. 1 Generation of retinal organoids from human iPSCs. (A) Differentiation protocol to generate retinal organoids. Feeder-free human iPSCs were preconditioned 1 day before the differentiation. Serum-free floating culture of embryoid body–like (EB-like) aggregates with the quick aggregation (SFEBq) method (see Methods) was employed. SAG, smoothened agonist; RA, retinoic acid; RPE, retinal pigment epithelium. (B) Bright-field view of differentiated retinal organoids on day 100 and day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. Scale bars: 200 μm. (C) Representative immunofluorescence images of retinal organoids on day 100 and day 180 stained for the panphotoreceptor marker recoverin (RCVRN), rod cell marker arrestin 1 (ARR1), cone cell marker G protein subunit α transducin 2 (GNAT2), mitochondrial marker translocase of outer mitochondrial membrane 20 (TOM20), cilia marker ADP ribosylation factor–like GTPase 13B (ARL13B), and outer segment marker peripherin 2 (PRPH2). Scale bars: 50 μm. (D) Scanning electron microscopy images of photoreceptor cells in retinal organoids on day 180. Middle and right panels are higher-magnification images of the dotted boxes in the left panels. BB, basal body; CC, connecting cilium; DS, disc stack; IS, inner segment; OS, outer segment. Scale bars: 1 μm.