{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:22:53Z","timestamp":1786508573399,"version":"build-2736575974"},"reference-count":38,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2024,8,9]],"date-time":"2024-08-09T00:00:00Z","timestamp":1723161600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Santen Pharmaceutical Co. Ltd"},{"name":"Santen Pharmaceutical Co. Ltd"},{"name":"Santen Pharmaceutical Co. Ltd"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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-<jats:italic>trans<\/jats:italic> 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0308743","type":"journal-article","created":{"date-parts":[[2024,8,9]],"date-time":"2024-08-09T17:29:14Z","timestamp":1723224554000},"page":"e0308743","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":11,"title":["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"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7760-6885","authenticated-orcid":true,"given":"Tokiyoshi","family":"Matsushita","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akishi","family":"Onishi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takahiro","family":"Matsuyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomohiro","family":"Masuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoko","family":"Ogino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaaki","family":"Kageyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masayo","family":"Takahashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fumiaki","family":"Uchiumi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,8,9]]},"reference":[{"issue":"1","key":"pone.0308743.ref001","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1089\/jop.2018.0140","article-title":"New drug discovery paradigms for retinal diseases: A focus on retinal organoids","volume":"36","author":"DM Aasen","year":"2020","journal-title":"J Ocul Pharmacol Ther"},{"key":"pone.0308743.ref002","doi-asserted-by":"crossref","first-page":"668857","DOI":"10.3389\/fnins.2021.668857","article-title":"Functional 3-dimensional retinal organoids","volume":"15","author":"M Fathi","year":"2021","journal-title":"Front Neurosci"},{"issue":"2","key":"pone.0308743.ref003","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1093\/stcltm\/szab010","article-title":"Human retinal organoids provide a suitable tool for toxicological investigations: A comprehensive validation using drugs and compounds affecting the retina","volume":"11","author":"B Dorgau","year":"2022","journal-title":"Stem Cells Transl Med"},{"issue":"11","key":"pone.0308743.ref004","doi-asserted-by":"crossref","first-page":"2409","DOI":"10.1016\/j.stemcr.2022.09.004","article-title":"Human retinal organoids harboring IMPG2 mutations exhibit a photoreceptor outer segment phenotype that models advanced retinitis pigmentosa","volume":"17","author":"SJ Mayerl","year":"2022","journal-title":"Stem Cell Reports"},{"key":"pone.0308743.ref005","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1016\/j.omtn.2018.07.010","article-title":"Splice-modulating oligonucleotide QR-110 restores CEP290 mRNA and function in human c.2991+1655A>G LCA10 models","volume":"12","author":"K Dulla","year":"2018","journal-title":"Mol Ther Nucleic Acids"},{"issue":"6","key":"pone.0308743.ref006","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1016\/j.stem.2012.05.009","article-title":"Self-formation of optic cups and storable stratified neural retina from human ESCs","volume":"10","author":"T Nakano","year":"2012","journal-title":"Cell Stem Cell"},{"issue":"23","key":"pone.0308743.ref007","doi-asserted-by":"crossref","first-page":"8518","DOI":"10.1073\/pnas.1324212111","article-title":"From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium","volume":"111","author":"S Reichman","year":"2014","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0308743.ref008","doi-asserted-by":"crossref","first-page":"4047","DOI":"10.1038\/ncomms5047","article-title":"Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs","volume":"5","author":"X Zhong","year":"2014","journal-title":"Nat Commun"},{"issue":"8","key":"pone.0308743.ref009","doi-asserted-by":"crossref","first-page":"2416","DOI":"10.1002\/stem.2023","article-title":"IGF-1 signaling plays an important role in the formation of three-dimensional laminated neural retina and other ocular structures from human embryonic stem cells","volume":"33","author":"CB Mellough","year":"2015","journal-title":"Stem Cells"},{"key":"pone.0308743.ref010","doi-asserted-by":"crossref","first-page":"6286","DOI":"10.1038\/ncomms7286","article-title":"Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue","volume":"6","author":"A Kuwahara","year":"2015","journal-title":"Nat Commun"},{"issue":"13","key":"pone.0308743.ref011","doi-asserted-by":"crossref","first-page":"7081","DOI":"10.3390\/ijms22137081","article-title":"ten Asbroek ALMA, Bergen AA. The role of small molecules and their effect on the molecular mechanisms of early retinal organoid development","volume":"22","author":"EL Wagstaff","year":"2021","journal-title":"Int J Mol Sci"},{"issue":"1","key":"pone.0308743.ref012","article-title":"Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines","volume":"146","author":"EE Capowski","year":"2019","journal-title":"Development"},{"issue":"39","key":"pone.0308743.ref013","doi-asserted-by":"crossref","first-page":"16698","DOI":"10.1073\/pnas.0905245106","article-title":"Modeling early retinal development with human embryonic and induced pluripotent stem cells","volume":"106","author":"JS Meyer","year":"2009","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"pone.0308743.ref014","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1002\/stem.3082","article-title":"Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived retinal organoids","volume":"38","author":"D Zerti","year":"2020","journal-title":"Stem Cells"},{"issue":"1","key":"pone.0308743.ref015","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1186\/s13287-020-01883-5","article-title":"COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids","volume":"11","author":"D Pan","year":"2020","journal-title":"Stem Cell Res Ther"},{"key":"pone.0308743.ref016","first-page":"663","article-title":"Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal","volume":"25","author":"KD Kaya","year":"2019","journal-title":"Mol Vis"},{"key":"pone.0308743.ref017","doi-asserted-by":"crossref","first-page":"3594","DOI":"10.1038\/srep03594","article-title":"A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells","volume":"4","author":"M Nakagawa","year":"2014","journal-title":"Sci Rep"},{"issue":"1","key":"pone.0308743.ref018","doi-asserted-by":"crossref","first-page":"21421","DOI":"10.1038\/s41598-021-00703-x","article-title":"Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion","volume":"11","author":"M Nishida","year":"2021","journal-title":"Sci Rep"},{"issue":"19\u201320","key":"pone.0308743.ref019","doi-asserted-by":"crossref","first-page":"6505","DOI":"10.1007\/s00018-021-03917-4","article-title":"A look into retinal organoids: methods, analytical techniques, and applications","volume":"78","author":"TAV Afanasyeva","year":"2021","journal-title":"Cell Mol Life Sci"},{"issue":"1","key":"pone.0308743.ref020","doi-asserted-by":"crossref","first-page":"18936","DOI":"10.1038\/s41598-019-55130-w","article-title":"Preconditioning the initial state of feeder-free human pluripotent stem cells promotes self-formation of three-dimensional retinal tissue","volume":"9","author":"A Kuwahara","year":"2019","journal-title":"Sci Rep"},{"issue":"3","key":"pone.0308743.ref021","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/nbt.1529","article-title":"Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling","volume":"27","author":"SM Chambers","year":"2009","journal-title":"Nat Biotechnol"},{"issue":"8","key":"pone.0308743.ref022","doi-asserted-by":"crossref","first-page":"1206","DOI":"10.1002\/stem.674","article-title":"Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment","volume":"29","author":"JS Meyer","year":"2011","journal-title":"Stem Cells"},{"issue":"1","key":"pone.0308743.ref023","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.stemcr.2022.12.002","article-title":"Comprehensive characterization of fetal and mature retinal cell identity to assess the fidelity of retinal organoids","volume":"18","author":"H. J. Kim","year":"2023","journal-title":"Stem Cell Reports"},{"issue":"3","key":"pone.0308743.ref024","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1080\/02713683.2019.1697453","article-title":"Understanding the complexity of retina and pluripotent stem cell derived retinal organoids with single cell RNA sequencing: current progress, remaining challenges and future prospective","volume":"45","author":"D Zerti","year":"2020","journal-title":"Curr Eye Res"},{"issue":"7594","key":"pone.0308743.ref025","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1038\/nature17000","article-title":"Co-ordinated ocular development from human iPS cells and recovery of corneal function","volume":"531","author":"R Hayashi","year":"2016","journal-title":"Nature"},{"key":"pone.0308743.ref026","doi-asserted-by":"crossref","first-page":"732382","DOI":"10.3389\/fcell.2021.732382","article-title":"Generation and staging of human retinal organoids based on self-formed ectodermal autonomous multi-zone system","volume":"9","author":"J Li","year":"2021","journal-title":"Front Cell Dev Biol"},{"issue":"23","key":"pone.0308743.ref027","doi-asserted-by":"crossref","first-page":"6332","DOI":"10.1093\/hmg\/ddu351","article-title":"Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation","volume":"23","author":"EE Capowski","year":"2014","journal-title":"Hum Mol Genet"},{"issue":"1","key":"pone.0308743.ref028","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.ydbio.2017.09.030","article-title":"Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis","volume":"432","author":"LA Remez","year":"2017","journal-title":"Dev Biol"},{"issue":"4","key":"pone.0308743.ref029","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1016\/S0092-8674(00)80439-0","article-title":"Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation","volume":"91","author":"T Furukawa","year":"1997","journal-title":"Cell"},{"key":"pone.0308743.ref030","doi-asserted-by":"crossref","first-page":"174","DOI":"10.3389\/fcell.2020.00174","article-title":"Crx is posttranscriptionally regulated by light stimulation in postnatal rat retina","volume":"8","author":"Y Wu","year":"2020","journal-title":"Front. Cell Dev. Biol"},{"issue":"1","key":"pone.0308743.ref031","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1186\/s13287-022-03146-x","article-title":"Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid","volume":"13","author":"C Sanjurjo-Soriano","year":"2022","journal-title":"Stem Cell Res Ther"},{"issue":"8","key":"pone.0308743.ref032","doi-asserted-by":"crossref","first-page":"2091","DOI":"10.1242\/dev.120.8.2091","article-title":"Retinoic acid promotes differentiation of photoreceptors in vitro","volume":"120","author":"MW Kelley","year":"1994","journal-title":"Development"},{"issue":"23","key":"pone.0308743.ref033","doi-asserted-by":"crossref","first-page":"13298","DOI":"10.1073\/pnas.93.23.13298","article-title":"Retinoic acid alters photoreceptor development in vivo","volume":"93","author":"GA Hyatt","year":"1996","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"2","key":"pone.0308743.ref034","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1242\/dev.124.2.363","article-title":"Expression of Sonic hedgehog and its putative role as a precursor cell mitogen in the developing mouse retina","volume":"124","author":"AM Jensen","year":"1997","journal-title":"Development"},{"issue":"16","key":"pone.0308743.ref035","doi-asserted-by":"crossref","first-page":"6277","DOI":"10.1523\/JNEUROSCI.17-16-06277.1997","article-title":"Sonic hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro","volume":"17","author":"E. M. Levine","year":"1997","journal-title":"J Neurosci"},{"issue":"1","key":"pone.0308743.ref036","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1006\/mcne.1999.0806","article-title":"Activin A promotes progenitor differentiation into photoreceptors in rodent retina","volume":"15","author":"AA Davis","year":"2000","journal-title":"Mol Cell Neurosci"},{"issue":"1","key":"pone.0308743.ref037","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1007\/s13238-013-0011-2","article-title":"Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors","volume":"5","author":"K Zhang","year":"2014","journal-title":"Protein Cell"},{"issue":"3","key":"pone.0308743.ref038","doi-asserted-by":"crossref","first-page":"838","DOI":"10.1016\/j.stemcr.2017.06.021","article-title":"Activin signals through SMAD2\/3 to increase photoreceptor precursor yield during embryonic stem cell differentiation","volume":"9","author":"AQ Lu","year":"2017","journal-title":"Stem Cell Reports"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0308743","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,9]],"date-time":"2024-08-09T17:29:37Z","timestamp":1723224577000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0308743"}},"subtitle":[],"editor":[{"given":"Li-Ping","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,8,9]]},"references-count":38,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2024,8,9]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0308743","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,9]]}}}