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

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 reproducible platform for long-term experimental studies, facilitating mechanistic investigations and the development and evaluation of novel therapeutic approaches.

Primary limbal epithelial cells were isolated from biopsies of two patients with distinct PAX6 variants and from a healthy donor. Immortalization was performed by InSCREENex GmbH. The resulting cell lines were characterized using microscopy, BrdU assay, qPCR, flow cytometry, immunocytochemistry, and mRNA sequencing.

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 differences, except for reduced ΔNp63α protein levels in one aniridia line. mRNA sequencing detected more than 14,000 transcripts, including subsets uniquely expressed in control and aniridia-derived lines.

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.