Integrated transcriptomic and metabolomic analyses reveal distinct energy metabolic signatures and functional properties of RPE cells under two culture conditions.

Retinal pigment epithelium (RPE) degeneration is a major cause of vision loss in multiple retinal diseases, including age-related macular degeneration, Bietti crystalline dystrophy and Stargardt disease. 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–related genes and higher transepithelial resistance. In contrast, KSR-RPE cells exhibited comparable tricarboxylic acid cycle activity but higher expression of oxidative phosphorylation–related 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.