Exploring single-cell RNA sequencing as a decision-making tool in the clinical management of Fuchs' endothelial corneal dystrophy.

Single-cell RNA sequencing (scRNA-seq) has enabled the identification of novel gene signatures and cell heterogeneity in numerous tissues and diseases. Here we review the use of this technology for Fuchs' Endothelial Corneal Dystrophy (FECD). FECD is the most common indication for corneal endothelial transplantation worldwide. FECD is challenging to manage because it is genetically heterogenous, can be autosomal dominant or sporadic, and progress at different rates. Single-cell RNA sequencing has enabled the discovery of several FECD subtypes, each with associated gene signatures, and cell heterogeneity. Current FECD treatments are mainly surgical, with various Rho kinase (ROCK) inhibitors used to promote endothelial cell metabolism and proliferation following surgery. A range of emerging therapies for FECD including cell therapies, gene therapies, tissue engineered scaffolds, and pharmaceuticals are in preclinical and clinical trials. Unlike conventional disease management methods based on clinical presentations and family history, targeting FECD using scRNA-seq based precision-medicine has the potential to pinpoint the disease subtypes, mechanisms, stages, severities, and help clinicians in making the best decision for surgeries and the applications of therapeutics. In this review, we first discuss the feasibility and potential of using scRNA-seq in clinical diagnostics for FECD, highlight advances from the latest clinical treatments and emerging therapies for FECD, integrate scRNA-seq results and clinical notes from our FECD patients and discuss the potential of applying alternative therapies to manage these cases clinically.

Single nuclei transcriptomics of the in situ human limbal stem cell niche.

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

Geographical Inequality on Cataract Surgery Uptake in 200,000 Australians: Findings from the "45 and Up Study".

Using a geographical information system (GIS), we investigated the spatiotemporal evolution of a cataract surgery service and its association with socioeconomic factors and private insurance, based on 10-year real-world medical claim data in an Australian population. The data collected cover a decade (2007-2016) from the "45 and Up Study". A total of 234,201 participants within the cataract surgery service were grouped into 88 Statistical Area Level 3 (SA3s) according to their residential postcodes in New South Wales Australia. We analyzed the spatiotemporal variations and geographical distribution inequality in cataract surgery incidence and its respect to socioeconomic status (SES) and private health insurance coverage by Spearman correlation analysis and Moran's I test. Then these variations were intuitive displayed by six-quartile maps and a local indicator of spatial association (LISA) maps based on GIS. The average cumulative age-gender-standardized of the incidence of cataract surgery (ICS) was 8.85% (95% CI, 5.33-15.6). Spatial variation was significant (univariate Moran's I = 0.45, P = 0.001) with incidence gradually decreasing from the coastal regions to the north-western inland regions, suggesting inequality in the cataract surgery service across the state of New South Wales. Notably, clustering of the low incidence areas had gradually disappeared over the decade, suggesting that the cataract surgery service has improved over time. Low scores on the "index of socioeconomic disadvantages" (IRSD) and high private health insurance coverage were significantly associated with a higher incidence of cataract surgery (bivariate Moran's I = -0.13 and 0.23, P < 0.01; Spearman correlation r = 0.25 and -0.25, P = 0.02), which is displayed on the map visually and obviously. Spatiotemporal variations in the incidence of cataract surgery are significant, but the low incidence area had gradually disappeared over time. High socioeconomic status and private insurance contribute to a higher incidence of cataract surgery in Australia.

Corneal supply and the use of technology to reduce its demand: A review.

Recovery and access to end-of-life corneal tissue for corneal transplantation, training and research is globally maldistributed. The reasons for the maldistribution are complex and multifaceted, and not well defined or understood. Currently there are few solutions available to effectively address these issues. This review provides an overview of the system, key issues impacting recovery and allocation and emphasises how end-user ophthalmologists and researchers, with support from administrators and the wider sector, can assist in increasing access long-term through sustaining eye banks nationally and globally. We posit that prevention measures and improved surgical techniques, together with the development of novel therapies will play a significant role in reducing demand and enhance the equitable allocation of corneas.

Biodegradable and biocompatible poly(ethylene glycol)-based hydrogel films for the regeneration of corneal endothelium.

Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-µm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties.

Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa.

PURPOSE: Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa. METHODS: Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry. RESULTS: MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa. CONCLUSIONS: MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

Ultrathin chitosan-poly(ethylene glycol) hydrogel films for corneal tissue engineering.

Due to the high demand for donor corneas and their low supply, autologous corneal endothelial cell (CEC) culture and transplantation for treatment of corneal endothelial dysfunction would be highly desirable. Many studies have shown the possibility of culturing CECs in vitro, but lack potential robust substrates for transplantation into the cornea. In this study, we investigate the properties of novel ultrathin chitosan-poly(ethylene glycol) (PEG) hydrogel films (CPHFs) for corneal tissue engineering applications. Cross-linking of chitosan films with diepoxy-PEG and cystamine was employed to prepare ~50 µm (hydrated) hydrogel films. Through variation of the PEG content (1.5-5.9 wt.%) it was possible to tailor the CPHFs to have tensile strains and ultimate stresses identical to or greater than those of human corneal tissue while retaining similar tensile moduli. Light transmission measurements in the visible spectrum (400-700 nm) revealed that the films were >95% optically transparent, above that of the human cornea (maximum ~90%), whilst in vitro degradation studies with lysozyme revealed that the CPHFs maintained the biodegradable characteristics of chitosan. Cell culture studies demonstrated the ability of the CPHFs to support the attachment and proliferation of sheep CECs. Ex vivo surgical trials on ovine eyes demonstrated that the CPHFs displayed excellent characteristics for physical manipulation and implantation purposes. The ultrathin CPHFs display desirable mechanical, optical and degradation properties whilst allowing attachment and proliferation of ovine CECs, and as such are attractive candidates for the regeneration and transplantation of CECs, as well as other corneal tissue engineering applications.

Unique residues on the H2A.Z containing nucleosome surface are important for Xenopus laevis development.

Critical to vertebrate development is a complex program of events that establishes specialized tissues and organs from a single fertilized cell. Transitions in chromatin architecture, through alterations in its composition and modification markings, characterize early development. A variant of the H2A core histone, H2A.Z, is essential for development of both Drosophila and mice. We recently showed that H2A.Z is required for proper chromosome segregation. Whether H2A.Z has additional specific functions during early development remains unknown. Here we demonstrate that depletion of H2A.Z by RNA interference perturbs Xenopus laevis development at gastrulation leading to embryos with malformed, shortened trunks. Consistent with this result, whole embryo in situ hybridization indicates that endogenous expression of H2A.Z is highly enriched in the notochord. H2A.Z modifies the surface of a canonical nucleosome by creating an extended acidic patch and a metal ion-binding site stabilized by two histidine residues. To examine the significance of these specific surface regions in vivo, we investigated the consequences of overexpressing H2A.Z and mutant proteins during X. laevis development. Overexpression of H2A.Z slowed development following gastrulation. Altering the extended acidic patch of H2A.Z reversed this effect. Remarkably, modification of a single stabilizing histidine residue located on the exposed surface of an H2A.Z containing nucleosome was sufficient to disrupt normal trunk formation mimicking the effect observed by RNA interference. Taken together, these results argue that key determinants located on the surface of an H2A.Z nucleosome play an important specific role during embryonic patterning and provide a link between a chromatin structural modification and normal vertebrate development.