Patient-Derived Immortalized Limbal Epithelial Cells as In Vitro Models of Congenital Aniridia

Abstract

Purpose: To establish and comprehensively characterize immortalized limbal epithelial cell lines derived from patients with PAX6 haploinsufficiency-associated congenital aniridia, as well as from a healthy donor. These well-defined cell models provide a reliable and repro ducible platform for long-term experimental studies, facilitating mechanistic investigations and the development and evaluation of novel therapeutic approaches. Methods: Primary limbal epithelial cells were isolated from biopsies of two patients with distinct PAX6 vari ants and from a healthy donor. Immortalization was performed by InSCREENex GmbH. The resulting cell lines were characterized using microscopy, BrdU assay, qPCR, flow cy tometry, immunocytochemistry, and mRNA sequencing. Results: Immortalized aniridia and control cell lines displayed typical polygonal epithelial morphology and comparable proliferation rates. Total PAX6 mRNA and protein levels were similar among groups; however, nuclear PAX6 immunosignals were significantly reduced in aniridia-derived lines. Expression of ABCG2, TP63, FOSL2, ALDH1A1, and FABP5 showed no significant differ ences, except for reduced ∆Np63α protein levels in one aniridia line. mRNA sequencing detected more than 14,000 transcripts, including subsets uniquely expressed in control and aniridia-derived lines. Conclusions: Immortalized aniridia limbal epithelial cell lines preserve key epithelial characteristics and overall transcriptomic similarity to controls while exhibiting disease-relevant molecular alterations. These cell lines represent models of PAX6-associated ocular surface disease.