Fuchs endothelial corneal dystrophy (FECD) is characterized by the progressive degeneration of the corneal endothelium (CE). The purpose of this article is to review the diagnostic tools available to image and assess the CE in FECD. Slit-lamp biomicroscopy with specular reflection and retroillumination are important techniques to assess the CE. Objective diagnostic tests, such as retroillumination photographic analysis, specular microscopy, in vivo confocal microscopy (IVCM), and anterior segment optical coherence tomography, are valuable tools to evaluate the CE in FECD. Specular microscopy can be performed rapidly without touching the eye but requires a clear cornea with a smooth CE. In contrast, IVCM can image all layers of the cornea, even in advanced FECD. However, IVCM is contact-based and more technically challenging. It is important to select the appropriate objective diagnostic test to image and assess the CE in managing patients with FECD.

To treat unilateral limbal stem cell (LSC) deficiency, we developed cultivated autologous limbal epithelial cells (CALEC) using an innovative xenobiotic-free, serum-free, antibiotic-free, two-step manufacturing process for LSC isolation and expansion onto human amniotic membrane with rigorous quality control in a good manufacturing practices facility. Limbal biopsies were used to generate CALEC constructs, and final grafts were evaluated by noninvasive scanning microscopy and tested for viability and sterility. Cultivated cells maintained epithelial cell phenotype with colony-forming and proliferative capacities. Analysis of LSC biomarkers showed preservation of “stemness.” After preclinical development, a phase 1 clinical trial enrolled five patients with unilateral LSC deficiency. Four of these patients received CALEC transplants, establishing preliminary feasibility. Clinical case histories are reported, with no primary safety events. On the basis of these results, a second recruitment phase of the trial was opened to provide longer term safety and efficacy data on more patients.

To investigate if corneal endothelial cells (CECs) in Fuchs endothelial corneal dystrophy (FECD) have altered cellular migration compared with normal controls. Comparative analysis. Descemet’s membrane and CECs derived from patients with FECD undergoing endothelial keratoplasty or normal cadaveric donors. Ex vivo specimens were used for live cell imaging and generation of immortalized cell lines. Live imaging was performed on FECD and normal CECs and on ex vivo specimens transfected with green fluorescent protein. Migration speeds were determined as a function of cellular density using automated cell tracking. Ex vivo specimens were classified as either FECD or normal low cell density (nonconfluent) or high cell density (confluent). Scratch assay was performed on CECs seeded at high confluence to determine migration speed. Genetic analysis from blood samples or CECs was performed to detect a CTG repeat expansion in the TCF4 gene. Mean cell migration speed. Fuchs endothelial corneal dystrophy CECs in low cell density areas displayed increased mean speed (0.391 ± 0.005 μm/minute vs. 0.364 ± 0.005 μm/minute; P < 0.001) and mean maximum speed (0.961 ± 0.010 μm/minute vs. 0.787 ± 0.011 μm/minute; P < 0.001) compared with normal CECs, and increased mean maximum speed (0.778 ± 0.014 μm/minute vs. 0.680 ± 0.011 μm/minute; P < 0.001) in high cell density areas ex vivo. Similarly, FECD CECs displayed increased mean speed compared with normal CECs (1.958 ± 0.020 μm/minute vs. 2.227 ± 0.021 μm/minute vs. 1.567 ± 0.019 μm/minute; P < 0.001) under nonconfluent conditions in vitro. Moreover, FECD CECs also displayed increased mean speed compared with normal CECs under high confluent conditions as detected by scratch assay (37.2 ± 1.1% vs. 44.3 ± 4.1% vs. 70.7 ± 5.2%; P < 0.001). Morphologic analysis showed that FECD CECs displayed an increased fibroblastic phenotype as detected by filamentous-actin labeling. Fuchs endothelial corneal dystrophy CECs demonstrated increased migration speed compared with normal CECs. Further investigation into the mechanisms of heightened cell migration in FECD is needed and may provide insight into its pathogenesis, as well as having implications on descemetorhexis without endothelial keratoplasty.

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, withTUBB4A(tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.