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Retinal organoids generated from human pluripotent stem cells exhibit considerable variability in temporal dynamics of differentiation. To assess the maturity of neural retina in vitro, we performed transcriptome analyses of developing organoids from human embryonic and induced pluripotent stem cell lines. We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrated that addition of 9-cis retinal, instead of widely-used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Our studies thus provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids, which should facilitate disease modeling and evaluation of therapies in vitro.Summary StatementThree-dimensional organoids derived from human pluripotent stem cells have been extensively applied for investigating organogenesis, modeling diseases and development of therapies. However, substantial variations within organoids pose challenges for comparison among different cultures and studies. We generated transcriptomes of multiple distinct retinal organoids and compared these to human fetal and adult retina gene profiles for molecular staging of differentiation state of the cultures. Our analysis revealed the advantage of using 9-cis retinal, instead of the widely-used all-trans retinoic acid, in facilitating rod photoreceptor differentiation. Thus, a transcriptome-based comparison can provide an objective method to uncover the maturity of organoid cultures across different lines and in various study platforms.