{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:31:13Z","timestamp":1785403873523,"version":"3.56.0"},"reference-count":31,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Lundbeck Foundation","award":["R322-2019-2611"],"award-info":[{"award-number":["R322-2019-2611"]}]},{"award":["R322-2019-2611"],"award-info":[{"award-number":["R322-2019-2611"]}],"id":[{"id":"https:\/\/ror.org\/03hz8wd80","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Carlsberg Foundation"},{"id":[{"id":"https:\/\/ror.org\/01kpjmx04","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001818","name":"Research to Prevent Blindness","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100001818","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/04drjs621","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"extramural grants","doi-asserted-by":"publisher","award":["1R21EY030215-01"],"award-info":[{"award-number":["1R21EY030215-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"extramural grants","doi-asserted-by":"publisher","award":["1R01EY029736-01A1"],"award-info":[{"award-number":["1R01EY029736-01A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007277","name":"Icahn School of Medicine at Mount Sinai","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100007277","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/04a9tmd77","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>Background: Corneal organoids derived from pluripotent stem cells have emerged as powerful tools for studying corneal development, disease modeling, and regenerative medicine applications. While previous protocols have successfully generated corneal tissue structures, there remains a need for three-dimensional models that recapitulate the complex cellular architecture and diversity of native human cornea. Methods: We developed a modified spontaneous three-dimensional corneal organoid model using human embryonic stem cells (hESCs) through an adapted Self-formed Ectoderm Autonomous Multi-zone (SEAM) protocol. hESCs were cultured as spheroids in ultra-low-binding plates under normoxic conditions and differentiated over 7\u20138 weeks. Organoids were characterized using immunofluorescence staining for corneal-specific markers and single-cell RNA sequencing to assess cellular composition and gene expression patterns. Results: Approximately 20% of organoids developed transparent regions characteristic of corneal tissue by day 30 of differentiation. Immunofluorescence analysis revealed spatially organized expression of corneal markers, including ZO-1 and E-cadherin in the outermost epithelial layers, P63\u03b1-positive putative limbal stem cells at the epithelial\u2013stromal interface, vimentin-positive stromal cells in the interior, and laminin-1 deposition that suggests Bowman\u2019s membrane formation. The organoids expressed cornea-specific keratins (K3, K12, and K15) and the master regulator PAX6 in appropriate cellular compartments. Single-cell RNA sequencing identified 18 distinct cell clusters, including three corneal epithelium subclusters with differential expression of MUC16, KRT12, and \u0394Np63\u03b1, two stromal populations with distinct inflammatory profiles, and a corneal endothelium cluster. Transcriptomic analysis confirmed expression of key corneal genes, including AQP3, CDH1, multiple keratins, mucins, and extracellular matrix components (HAS2, CD34, CD44, COL8A1, and KERA). Conclusions: This three-dimensional spheroid-based putative corneal organoid model successfully recapitulates the multilayered architecture and cellular diversity of human cornea, including stratified epithelium, putative limbal stem cells, stroma, and endothelium in spatially appropriate arrangements. The model demonstrates molecular signatures consistent with native corneal tissue and provides a valuable platform for studying corneal development, disease mechanisms, and potential therapeutic applications. Future optimization to improve organoid formation efficiency and functional maturation will enhance the utility of this system for both basic research and translational medicine.<\/jats:p>","DOI":"10.3390\/bioengineering13050518","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T09:46:11Z","timestamp":1777455971000},"page":"518","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Putative  Self-Organizing  Human Corneal Organoids Recapitulate Human Corneal Architecture and Cellular Diversity"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8095-9937","authenticated-orcid":false,"given":"Timothy A.","family":"Blenkinsop","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7224-9417","authenticated-orcid":false,"given":"Anne Z.","family":"Eriksen","sequence":"additional","affiliation":[{"name":"Department of Health Technology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6647","DOI":"10.1016\/j.biomaterials.2010.05.034","article-title":"Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells","volume":"31","author":"Ma","year":"2010","journal-title":"Biomaterials"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/s41392-022-01024-9","article-title":"Human organoids in basic research and clinical applications","volume":"7","author":"Tang","year":"2022","journal-title":"Signal Transduct. 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