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

Purpose: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. Methods: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. Results: 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 demonstrate that the addition of 9-cis retinal, instead of the 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. Conclusion: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.