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Induced pluripotent stem cell (iPSC)-derived RPE cells hold promise for regenerative therapies. B27- and KSR-based media are among the most commonly used for RPE culture; however, how these culture conditions shape RPE cell identity and features remain incompletely understood. Here, we performed comprehensive transcriptomic and metabolomic profiling of iPSC-derived RPE cells cultured in B27 or KSR media to systematically compare their gene expression and metabolic features. B27- and KSR-cultured RPE cells exhibited distinct morphologies and barrier properties. Integrated multi-omics analyses revealed that KSR-cultured RPE cells displayed a relative bias toward fatty acid oxidation and oxidative phosphorylation, whereas B27-cultured RPE cells showed a relative bias toward glycolytic metabolism. The glycolytic tendency observed in B27-RPE cells was accompanied by increased expression of extracellular matrix\u2013related genes and higher transepithelial resistance. In contrast, KSR-RPE cells exhibited comparable tricarboxylic acid cycle activity but higher expression of oxidative phosphorylation\u2013related genes compared with B27-RPE cells. Together, these results demonstrate that RPE cells cultured under different conditions adopt distinct but partially overlapping metabolic and transcriptional states, which are associated with differences in RPE-related features and barrier properties. Our findings highlight the importance of metabolic balance between glycolysis and fatty acid oxidation in shaping in vitro RPE phenotypes.<\/jats:p>","DOI":"10.1038\/s41598-026-39689-9","type":"journal-article","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T03:57:25Z","timestamp":1775793445000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated transcriptomic and metabolomic analyses reveal distinct energy metabolic signatures and functional properties of RPE cells under two culture conditions"],"prefix":"10.1038","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-1442-3006","authenticated-orcid":false,"given":"Fan","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenlu","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qinxue","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tangyan","family":"Ao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,10]]},"reference":[{"key":"39689_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.preteyeres.2021.100969","volume":"85","author":"SM George","year":"2021","unstructured":"George, S. 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