Nuclear factor, erythroid 2 like 2 (Nrf2), is an oxidative stress induced transcription factor that regulates cytoprotective gene expression. Thus, Nrf2 is essential for cellular redox homeostasis. Loss or dysregulation of Nrf2 expression has been implicated in the pathogenesis of degenerative diseases, including diseases of the cornea. One of the most common diseases of the cornea in which Nrf2 is implicated is Fuchs’ endothelial cornea dystrophy (FECD). FECD is the leading indication for corneal transplantation; and is associated with a loss of corneal endothelial cell (CEC) function. In this review, we propose that Nrf2 is an essential regulator of CEC function. Furthermore, we demonstrate that deficiency of Nrf2 function is a hallmark of FECD. In addition, we advocate that pharmacological targeting of Nrf2 as a possible therapy for FECD.

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (p < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Fuchs Endothelial Corneal Dystrophy (FECD) is a corneal endothelial disease that causes microenvironment alterations and endothelial cell loss, which leads to vision impairment. It has a high global prevalence, especially in elderly populations. FECD is also one of the leading indications of corneal transplantation globally. Currently, there is no clearly defined canonical pathway for this disease, and it has been proposed that the combinatorial effects of genetic mutations and exogenous factors cause FECD. Clinical studies and observations have provided valuable knowledge and understanding of FECD, while preclinical studies are essential for gaining insights into disease progression and mechanisms for the development and testing of regenerative medicine therapies. In this review, we first introduce the proposed genetic and molecular pathologies of FECD. Notably, we discuss the impact of abnormal extracellular matrix deposition (guttata), endothelial-to-mesenchymal transition, cell senescence, and oxidative stress on the pathology and etiology of FECD. We review and summarize the in vitro cell models, ex vivo tissues, and in vivo animal models used to study FECD. The benefits and challenges of each model are also discussed.

The corneal endothelium is essential for maintaining corneal transparency through active fluid transport and barrier function. Corneal cell loss from disease, ageing or surgical trauma underlies a significant proportion of corneal blindness worldwide, with Fuchs’ endothelial corneal dystrophy (FECD) and pseudophakic bullous keratopathy (PBK) representing the dominant clinical indications for corneal transplantation. While Descemet’s membrane endothelial keratoplasty (DMEK) has substantially improved surgical outcomes, the procedure remains constrained by global donor tissue shortage. Regenerative medicine offers a compelling alternative by exploiting the latent proliferative and reparative potential of corneal endothelial progenitor populations. This review synthesises current knowledge on the foundational biology of corneal endothelial progenitor populations and the optimisation of expansion platforms to emerging preclinical and clinical evidence for both cell-based and pharmacological regenerative strategies. We also consider the outstanding translational challenges of potency standardisation, GMP-compliant manufacturing and regulatory navigation, as well as the longer-term potential of biomaterial-cell platforms and personalised iPSC-based medicine. The cumulative evidence positions progenitor-based approaches as viable and increasingly well-characterised alternatives to conventional donor transplantation, although their routine clinical use awaits the optimisation of manufacturing and regulatory platforms.

Objective: This was a longitudinal study of TCF4 CTG18.1 trinucleotide repeat lengths in 17 patients (27 eyes) diagnosed with Fuchs’ endothelial corneal dystrophy (FECD), and it aimed to correlate the repeat expansion status with disease severity and progression. Design: This was a prospective cohort study looking at FECD clinical progression and TCF4 CTG18.1 repeat length expansion status over time. Methods: A total of 27 eyes from 17 patients diagnosed with FECD were recruited. Only eyes with FECD disease severity of at least Grade 4 on the modified Krachmer clinical grading scale were included; eyes that had previously undergone any form of ocular surgery prior to the first genotyping or during the duration of follow-up were excluded. CTG trinucleotide repeat genotyping was performed on peripheral blood leukocytes at two time points over an average follow-up of 10 years. Over the follow-up period, the FECD progression of each subject was examined using pachymetry, Scheimpflug imaging (Pentacam), and endothelial cell density (ECD) readings, during the baseline visit, yearly thereafter, at the time of repeat CTG18.1 genotyping, and at their latest visit. Main Outcome Measures: The clinical progression of FECD patients was assessed using central corneal thickness (CCT), ECD, and any keratoplasty performed. CTG repeat length was assessed twice over the entire follow-up period. Results: The non-expanded alleles were shown to be stable over the period of follow-up and did not develop any expanded repeats. Repeat expansion did not influence the risk of attaining Threshold Disease, although more patients in the L ≥ 40 group (CTG18.1 repeat sequence of more than or equal to 40 repeats) underwent keratoplasty. Conclusions: Through this study, we found that the CTG18.1 allele lengths of <40 repeats in peripheral blood leukocytes showed minimal change over a 10-year period, and none became an expanded repeat. Hence, a single CTG expansion assessment, performed at any point in a patient’s lifetime, is likely a good representation of genetic risk. Clinicians may use this information to better advise patients on the risk of clinical progression and the best therapeutic strategy.