Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

Differentiation of neural retina in organoid cultures from human pluripotent stem cells (PSCs) is highly variable and requires an extended period of as many as 200 days. To provide objective assessment of different protocols and culture conditions, we performed transcriptome analysis and molecular staging using RNA-seq data from developing and adult human retina. Using a modified protocol with 9-cis retinal (9CRAL) instead of widely-used all-trans retinoic acid (ATRA) in the culture media, we show comparable differentiation of human embryonic and induced PSCs into a polarized, laminated architecture with all retinal cell types in retinal organoids. Transcriptome comparisons of multiple developing organoids to that of human fetal and adult retina have permitted molecular staging of the differentiation state and uncovered expedited rod photoreceptor maturation by 9CRAL. A direct comparison with the ATRA-organoid cultures revealed higher expression of rhodopsin and other rod genes in 9CRAL-organoids. Our studies thus provide a framework for transcriptome-based staging of retinal organoids to facilitate advances in human disease modeling and evaluation of therapies. Overall design: Five cell lines, one ESC line (H9) and four iPSC lines (PEN8E, PEN8E_2, NEI377 and ESP) were cultured across two different institutions and four researchers. Samples were harvested at various time points during differentiation. Differentiation of H9, PENE8E, PENE8E_2, and NEI377-derived organoids followed similar protocols. ESP-derived organoids were differentiated via previously published protocols. Starting at day (D)63, H9 and PEN8E media was supplemented with 9-cis retinal (9CRAL), whereas PEN8E_2, NEI377 and ESP were supplemented with all-trans retinoic acid (ATRA). In addition, H9 experiments were performed a second time and direct comparisons performed at D90 and D120 with either ATRA or 9CRAL.