Multimodal objective assessment of a porcine limbal stem cell deficiency model for corneal therapy research

Reliable preclinical models that recapitulate human limbal stem cell deficiency (LSCD) and provide objective outcome measures are essential for advancing corneal cell therapies. We induced LSCD in four porcine eyes by total corneal epithelial debridement, tracked healing with serial anterior-segment optical coherence tomography (OCT) (preinjury, 0, 5–11, 17, 23, 28 days), and quantified histological changes via computer-assisted digital pathology. OCT confirmed complete epithelial removal and a reproducible stromal edema peak on days 7–9, followed by incomplete regression by day 28. Histology revealed epithelial hyperplasia, keratinization, inflammatory infiltration and neovascularization, whereas cytokeratin-3 staining revealed patchy loss of the corneal phenotype. The automated analysis revealed significant increases in epithelial roughness (p = 0.049), thickness heterogeneity (p = 0.0001), and stromal cellularity (p = 0.0179) in experimental eyes compared with control eyes. These multimodal, bias-free metrics clearly distinguish healthy corneas from LSCD corneas and provide quantifiable end points for preclinical testing of limbal epithelial, iPSC-derived or mesenchymal stromal cell advanced therapy medicinal products. All the data and analysis codes are provided in the Supplementary Information.