Rapid and efficient generation of mature retinal organoids derived from human pluripotent stem cells via optimized pharmacological modulation of Sonic hedgehog, activin A, and retinoic acid signal transduction.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Activins*/pharmacology ; Activins*/metabolism ; Organoids*/drug effects ; Organoids*/metabolism ; Organoids*/cytology ; Hedgehog Proteins*/metabolism ; Tretinoin*/pharmacology ; Retina*/metabolism ; Retina*/cytology ; Retina*/drug effects ; Signal Transduction*/drug effects ; Cell Differentiation*/drug effects; Humans ; Pluripotent Stem Cells/drug effects ; Pluripotent Stem Cells/cytology ; Pluripotent Stem Cells/metabolism

Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
Competing Interests: The study was funded by Santen Pharmaceutical Co. Ltd and Tokiyoshi Matsushita, Takahiro Matsuyama, and Masaaki Kageyama are employed by Santen Pharmaceutical Co., Ltd. Tokiyoshi Matsushita, Akishi Onishi, and Masayo Takahashi are co-inventors of a patent related to this work, filed by RIKEN and Santen Pharmaceutical Co., Ltd [WO2022138803A1]. None of these alter our adherence to PLOS ONE policies on sharing data and materials.
(Copyright: © 2024 Matsushita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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104625-48-1 (Activins)
0 (Hedgehog Proteins)
5688UTC01R (Tretinoin)
0 (activin A)
0 (SHH protein, human)

Date Created: 20240809 Date Completed: 20240809 Latest Revision: 20240815

20240815

PMC11315325

10.1371/journal.pone.0308743

39121095