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bioRxivDevelopmental BiologyPreprint

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

Koray Dogan Kaya, Holly Y. CheniD, Matthew J. BrooksiD, Ryan A. Kelley, Hiroko Shimada, Kunio NagashimaiD, Natalia de ValiD, Charles T. Drinnan, Linn M. Gieser, Kamil Kruczek, Slaven ErcegiD, Tiansen LiiD, Dunja LukoviciD, Yogita K. AdlakhaiD, Emily WelbyiD, Anand SwaroopiD

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Abstract

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.\n\nSummary 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.

Abstract summary

Claude Opus 5.5 · xhigh effort · 18 Oct 2026 · from the abstract · CC BY-NC-ND 4.0

Comparing retinal organoid transcriptomes against human fetal and adult retina places each culture on a developmental scale; replacing all-trans retinoic acid with 9-cis retinal sped rod differentiation.

Retinal organoids grown from different pluripotent stem cell lines mature at different rates, so cultures treated as equivalent may not be, which weakens comparisons between labs and the reading of disease models. Benchmarking organoid expression profiles against human fetal and adult retina offers a maturity readout that does not rely on days in culture alone, and applying it surfaced a change of retinoid that the authors report speeds rod photoreceptor differentiation.

Key results

  1. SuggestedVariation in how fast organoids develop is visible in their expression profiles, so a culture's differentiation state can be staged by matching it to human fetal and adult retina transcriptomes.

    Reported from transcriptome analyses of organoids from human embryonic and induced pluripotent stem cell lines; the abstract gives no correlation values, stage assignments or number of organoids. · Abstract

  2. SuggestedSupplying 9-cis retinal in place of all-trans retinoic acid accelerated rod photoreceptor maturation in organoid culture.

    Rhodopsin expression was higher and mitochondrial morphology more mature at day 120 under 9-cis retinal; no effect sizes, replicate counts or statistics are reported in the abstract. · Abstract

  3. ClaimedThe authors propose transcriptome-based staging as an objective way to compare organoid maturity across cell lines and across studies, intended to support disease modelling and in vitro testing of therapies.

    Stated as the purpose of the work; no cross-laboratory or cross-platform validation of the staging approach is described in the abstract. · Abstract

Methods, data and reproducibility

Transcriptomes were generated from retinal organoids at successive stages of development, derived from both human embryonic and induced pluripotent stem cell lines, and compared with human fetal and adult retina expression data to assign a maturity stage. Organoid cultures were also grown with 9-cis retinal rather than all-trans retinoic acid, and assessed for rhodopsin expression and mitochondrial morphology.

  • Transcriptomes of developing retinal organoids from embryonic and induced pluripotent stem cell lines · human
  • Reference fetal and adult retina transcriptome data used for molecular staging · human
Limitations (2)
  • The abstract reports no quantities: how many organoids, lines or timepoints were profiled, how stages were scored, and how large the 9-cis retinal effect was all remain unstated. · noted in this summary
  • The retinoid comparison is reported only through rod markers at a single timepoint, so whether other retinal cell types, later stages or organoid yield are affected cannot be judged from the text. · noted in this summary

Read this if you culture human retinal organoids and need an objective maturity scale across lines, or are weighing 9-cis retinal against all-trans retinoic acid for rod differentiation.

Citations
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