(A) hPSCs were directed to generate retinal organoids in a step wise manner as previously described31,32,55,57. (B,C). By 40 days, ganglion cells were readily identified by the expression of POU4F2 and PAX6 within inner layers of organoids, and remained distinct from retinal progenitors (LHX2). Early organoids expressed ELAVL3/4 and POU4F2 in a ring-like manner within basal layers of organoids, (C, 200 µm, C’-C” 50 µm), while mitotic marker PH3 was localized towards the apical surface of organoids. (D–F). By 70 days, OTX2 positive photoreceptors were seen toward the apical surface of organoids (D), and became increasingly abundant by Day 80 (E). By 110 days, multiple layers of photoreceptors were visible on the apical surface, and further differentiation yielded NR2E3 positive rod photoreceptors (F). (G) Bulk RNAseq analysis demonstrate that organoids mimic fetal retina gene expression patterns at comparative stages. Supercluster 1 and 2 genes marking retinal progenitor, cell cycle, and ganglion cells genes decrease in expression while supercluster 4 genes indicative of later-born retinal cell types increase expression at later timepoints. (H) DNAm (years) age plotted against the day since the differentiation was initiated (in days) (p < 0.0073).