Multimodal Imaging of the Corneal Endothelial Transition Zone Reveals Progenitor Cell Population

The assessment of donor corneas is currently based solely on central endothelial cell (EC) density, which potentially overlooks the transition zone (TZ) regenerative potential. Therefore, the present study characterizes TZ using multimodal imaging techniques to understand its regenerative potential and refine the assessment of donor tissue. Ex vivo donor corneas (n = 41) were examined using phase-contrast microscopy for EC counting and reflectance confocal microscopy (HRTII/RCM) for non-invasive visualization of the TZ. A subset of eight of these corneas underwent ultrastructural analysis using field-emission scanning electron microscopy (SEM) and immunostaining analysis using confocal microscopy. We observed a significant decrease in central EC density (<i>p</i> < 0.001) with increasing storage duration and donor age, while TZ width and TZ surface cell count remained stable. HRTII/RCM and SEM revealed distinct morphological differences (small, polygonal cells, irregular arrangement) in the TZ compared to the peripheral endothelium (PE). Immunostaining revealed elevated expression of progenitor markers (Nestin, ABCG2, SOX2, Lgr5, Vimentin) and reduced expression of endothelial markers (ZO1 and Na/K-ATPase) in the TZ compared to the PE, indicating the presence of a stem cell-like population. These findings suggest that TZ may contribute to endothelial cell regeneration, and HRTII/RCM could serve as a novel tool for TZ evaluation in low EC count donor corneas.