Cells of the retina, photoreceptors especially, are highly metabolically active with substantial energy requirements. Accordingly, the effective delivery of oxygen and nutrients to the retina via the retinal vasculature is key to tissue function. Indeed, a disturbed retinal blood supply, especially if prolonged, can result in a loss of vision. Vascular endothelial cells, which form the inner lining of blood vessels, are pivotal in ensuring a healthy blood circulation in many tissues, including the light-sensitive retina at the back of the eye. However, the mechanism by which vascular endothelial cells maintain a functional retina (and aid the healing of a damaged one) is not understood. Here, through single-cell RNA sequencing, colony-forming assays and lineage tracing analysis, we provide data that point to a new regulatory system by which vascular endothelial cells are supplied to the retina by a reservoir of stem/progenitor cells in the optic nerve. This study finds stem-like endothelial cells in the optic nerve that supply and repair retinal blood vessels after injury, revealing a hierarchical repair system that may enable new treatments for blinding vascular eye diseases.

PURPOSE:To explore the role of the Hippo signaling pathway in the regeneration of corneal endothelial cells (CECs) and to evaluate the pro-regenerative potential of pharmacological inhibition of this pathway across species. METHODS:Corneal endothelial wound healing models were established in rabbits and mice in vivo. Hippo pathway activity was assessed by immunofluorescence for YAP localization and Western blot for phosphorylated MST1/2, LATS1/2, and YAP. Primary rabbit, monkey, and human CECs, and injury models in mouse, rabbit and monkey were used to evaluate the effect of YAP activation/inhibition on CEC regeneration. Cell proliferation was assessed by EdU labeling and Ki67 immunofluorescence staining. Recovery of corneal opacity and edema was evaluated by slit-lamp examination and anterior segment optical coherence tomography. CEC healing was observed by alizarin red staining. Long-term durability was assessed 12 months after drug withdrawal in monkeys. RESULTS:The Hippo pathway was downregulated and YAP was activated during CEC wound healing in rabbit and mouse. Knockdown of Yap1 and pharmacological inhibition of YAP suppressed CEC wound healing in rodents in both in vitro and in vivo models. XMU-MP-1, a small molecular inhibitor of Hippo signaling pathway, could promote the corneal endothelial regeneration in rodents and non-human primate in vivo models. Monkey CECs treated with XMU-MP-1 for a short term could maintain structural integrity and function for at least 12 months. CONCLUSIONS:The Hippo pathway regulates corneal endothelial regeneration among various species as a conserved signal, which may serves as a novel target for non-invasive treatment of corneal endothelium decompensation.

The complexity of cell fate decisions that underpin early eye development can be effectively modelled by leveraging the unique properties of human induced pluripotent stem cells (hiPSCs). In this study, we have utilised transcriptomic data generated from hiPSCs as they begin to self-organise and differentiate into two-dimensional eye-like organoids in vitro, and employ advanced single-cell analytical tools to dissect the cellular communication networks that direct this dynamic process. We have identified key signalling mediators and transcriptional effectors that guide the transition from pluripotency through to ocular differentiation, and our analyses reveal the conservation of developmentally defined signalling pathways. Members of the Activin, FGF, BMP, WNT, and retinoic acid families of ligands and receptors displayed communication probabilities consistent with their ocular-specific developmental roles in vivo, and this was accompanied by conserved tissue-specific activity of transcriptional regulators. These findings not only highlight the utility of hiPSCs for studying the cellular interactions and molecular pathways that drive early developmental decisions, but also advance our understanding of eye development in an accessible stem cell-based system.

This first-in-human, randomized, pivotal trial evaluated the safety and efficacy of CK2-085, a transparent, self-assembling peptide gel, for improving intraoperative visibility in glaucoma surgery. Sixty-eight glaucoma patients were randomized 1:1 to surgery with or without CK2-085. Subjective interoperative visibility evaluation (IVE), surgical feasibility evaluation (SFE) and Diathermy Evaluation (DE) scores were used to assess the surgery itself. Postoperatively, the surgical outcome was assessed by measurements of intraocular pressure (IOP), best-corrected visual acuity and corneal endothelial cell density, along with slit lamp microscopic and fundus examinations and an evaluation of adverse events. Intraoperative visibility when using CK2-085, scored at 51.5%, was significantly higher than that in the control, non-CK2-085 group (17.6%, p = 0.004). However, the surgical feasibility evaluation showed the CK2-085 group failed to establish non-inferiority against non-CK2-085 groups, which may have been affected by the surgeon’s learning curve. Analysis confirmed that the difference in diathermy use did not confound these findings. IOP one month after the operation was not significantly different between the CK2gel and control groups (p = 0.54). Occasional adverse events were temporary and occurred equally in both groups. CK2-085 is a safe and effective material for use in the maintenance of intraoperative visibility during glaucoma surgery.

Purpose:To study the structural arrangement of crystallin proteins in the human lens during development. Methods:Fetal human lenses were acquired from the UK Human Developmental Biology Resource and examined at four developmental stages; postconception weeks (pcw) 8 to 9 (n = 5), 12 to 13 (n = 3), 16 to 17 (n = 6), and 20 to 21 (n = 3). Small-angle X-ray scattering patterns were obtained as raster scans across the entirety of each lens using a 0.1 nm-wavelength, synchrotron X-ray beam measuring 200 × 150 µm at the specimen. Analysis of each small-angle X-ray scattering pattern provided a measure of the average nearest neighbor spacing and the extent of spatial order in the crystallin protein array. Results:Crystallins in the lens center became compacted as development progressed, with the average spacing measuring 19.9 nm at 8 to 9 pcw, 19.6 nm at 12 to 13 pcw, 18.7 nm at 16 to 17 pcw, and 17.7 nm at 20 to 21 pcw. The spatial order of the crystallin proteins in the lens center also decreased with time as indicated by a parameter called the coherence distance, which measured 26.9 nm at 8 to 9 pcw, 24.7 nm at 12 to 13 pcw, 24.6 nm at 16 to 17 pcw, and 24.9 nm at 20 to 21 pcw. Spacing and spatial order were consistently higher at the lens periphery, compared with the center, at all developmental stages studied. Conclusions:Spatiotemporal modifications in the array of crystallin proteins occur as the human lens develops. These are perhaps reflective of a shift in the relative proportions of crystallin subtypes present and have potential implications for the lens's developing refractive index.

During eye development, surface ectoderm cells that express PAX6 differentiate into corneal, limbal and conjunctival epithelia. However, several aspects of this differentiation process -- such as the developmental origin of the limbal epithelium and the mechanisms that underlie PAX6-mediated lineage specification -- are not properly understood. To explore these issues, we used single-cell RNA sequencing to study ocular surface epithelial cells derived from human induced pluripotent stem cells. Our analysis reveals that the corneal and conjunctival epithelial cell lineages originate from the surface ectoderm, and that the conjunctival lineage contributes to limbal epithelial cell populations. We also show that primordial conjunctival epithelial cells express limbal epithelial markers before lineage bifurcation. Finally, the activity and expression of PAX6 are highest in corneal epithelial cells, followed by those of the limbal epithelium and conjunctival epithelium, suggesting that lineage-specific differentiation is regulated by levels of PAX6 activity. These findings provide deeper understanding of the early-stage human ocular surface development.

Early detection of keratoconus is essential for maximizing the potential of cross-linking treatments designed to halt keratoconus progression, minimizing the risks of iatrogenic ectasia as well as reducing the need for corneal transplantation. This review focuses on the progress that has been made in the early detection of keratoconus using biomechanical and topographical properties derived from three different technologies, namely the ocular response analyser (ORA), corneal visualization Scheimpflug tonometer (Corvis ST) and optical coherence tomography (OCT). A PubMed search was performed using the keywords of ‘early keratoconus’, ‘subclinical keratoconus’, ‘forme fruste keratoconus’, ‘very asymmetric ectasia with normal topography/tomography’ and ‘ocular response analyser’ and/or ‘Corvis ST’/‘corneal visualized Scheimpflug tomographer/tomography’ and/or ‘optical coherence tomography/tomographer’. The integration of biomechanical parameters and corneal morphological data from the topography/tomography or OCT, or the assessment of bilateral asymmetry, has demonstrated improvement in the accuracy of diagnosing early-stage keratoconus. As measurement principles differ depending on the technique used for keratoconus assessment, comprehensive metrics may be needed to reflect subtle anterior or posterior corneal changes and help identify eyes with very early ectasia. Although clinical experts have always, and will most likely, continue to play a pivotal role in decision-making for early keratoconus diagnosis, future developments in technology and AI may lead to enhanced early detection in the future.

Host-microbiome interplay during development governs the homeostasis of various bodily surfaces, however, postnatal colonization of the microbiome and its impact on the homeostasis of ocular surface is still unclear. Here, the changes of the conjunctival microbiome in C57BL/6 J mice were tracked in 1-week-old neonates through to 8-week-old adult mice. This disclosed that changes in the conjunctival microbiome correlate with age, especially at the 2-week and 3-week time points, which, respectively, are accompanied by eyelid-opening and weaning. Antigen presenting cells were also recruited to the conjunctival epithelium after eyelid-opening, whilst an inhibition of microbial colonization at 2-to-3 weeks of age led to a disruption of mucosal homeostasis and aggravated the development of allergic eye disease. This study improves our understanding of the development of the conjunctival microbiome in mice, and provides an indication that early microbial colonization is required for the establishment of mucosal ocular surface homeostasis, the perturbation of which leads to increased susceptibility to allergic eye disease. Colonization of conjunctival microbiota during early postnatal life in mice promotes ocular surface homeostasis, whereas microbiota disruption by topical antibiotic delays ocular mucosal development and aggravates allergic eye disease.

Corneal endothelial cells (CECs) have limited regeneration capacity in primates while display a potent restorative capability in some species such as rodents after wounding. The mechanism which determines the regenerative capability of CECs remains poorly understood. Here, we identified that the Hippo pathway was inhibited and the downstream effector YAP was activated during CEC wound healing in rabbits. Knockdown of Yap1 and pharmacological inhibition of YAP suppressed CECs wound healing in rodents in both in vitro and in vivo models. XMU-MP-1, a specific small molecular inhibitor for Hippo pathway, could promote the corneal endothelial regeneration in rodents and corneal endothelial proliferation in cultured primate CECs, as well as in vivo non-human primate CEC wounding model. These findings suggest that Hippo pathway serves as a conserved signal to regulate corneal endothelial regeneration among various species, providing a novel target for non-invasive treatment of corneal endothelium decompensation. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, https://ror.org/01h0zpd94, 82471047, 81970773, 82401223 China Postdoctoral Science Foundation, https://ror.org/0426zh255, 2024M751351, 2023M741610, BX20240154 Natural Science Foundation of Hunan Province, China, 2025JJ90130, 2024JJ6403

Although cell-encapsulating hydrogels are of tremendous interest in regenerative medicine, few of them have been used in clinics and rarely used natural extracellular matrices as polymer precursors. One successful example is to use riboflavin (RF)/ultraviolet A (UVA) to cross-link corneal collagen, which has been used in clinics to halt disease progression in patients with corneal ectatic diseases. However, high-energy UVA and its action on RF cause tissue damage, particularly irreversible endothelium loss, and thus standard RF/UVA protocol may have limitations in treating patients at the advanced stage with thin cornea. Photo-initiators lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) has high efficiency under visible 405 nm light, and is widely applied in 3D bioprinting to cross-link synthesized monomers. Here, we introduced a new strategy to cross-link cornea using LAP and visible light (VL). The LAP/VL protocol could effectively increase corneal stiffness with equivalent efficiency to the RF/UVA protocol in both porcine and rabbit cornea. As LAP and VL were used, the loss of corneal endothelial and stromal cells was minimized, and epithelial wound healing and stromal cell repopulation were accelerated. In summary, we propose that the LAP/VL protocol is an effective and safe alternate for cornea CXL with advantages for relatively thin cornea. Our study also expands the application scope of LAP, indicating it is a suitable photocatalyst for in situ natural extracellular matrices CXL.

Glaucoma is a series of irreversible and progressive optic nerve degenerations, often accompanied by astrocyte remodeling as the disease progresses, a process that is insufficiently understood. Here, we investigated the morphology of retinal and optic nerve head (ONH) astrocytes under mechanical stress, and explored whether a specific phase is present that precedes astrocyte remodeling. A mouse model of transient ocular hypertension (OHT) and an in vitro cell stretch model were established to mimic the pathological conditions of increased intraocular pressure and mechanical stress on cultured cells. Glial fibrillary acidic protein (GFAP), S100B, and actin staining were used to characterize astrocyte morphology and cytoskeleton, with qPCR used to measure mRNA expression. We also silenced S100B expression and conduct RNA sequencing on ONH astrocytes. Astrocytes displayed weaker GFAP intensity (p < 0.0001) in the early-stage OHT mouse model, prior to the onset of hypertrophy, which was accompanied by an increase in GFAP mRNA expression (p < 0.0001) and a decrease in S100B mRNA expression (p < 0.001). In vitro-stretched astrocytes tended to contract and had fewer cellular processes and more elongated cell bodies. Downregulation of S100B expression occurred in in both the in vivo (p = 0.0001) and in vitro (p = 0.0023) models. S100B-silenced ONH astrocytes were similarly characterized by a slender morphology. In the RNA-seq analysis, genes downregulated by more than fivefold were predominantly enriched in terms related to nutrient metabolism, motor proteins and morphogenesis. Meanwhile, genes upregulated by more than fivefold were primarily associated with terms related to histone modification and visual perception. As an early response to mechanical stress, S100B expression is downregulated in astrocytes, which assume a slender morphology, reminiscent of cell "weakening." Silencing intracellular S100B expression induced similar morphology changes and altered the transcriptome. Stress-induced changes were reversible, with evidence of enhanced late-stage reactivation that is likely related to S100B.

PURPOSE:To investigate the presence and characteristics of mitochondria-associated membranes (MAMs) in Fuchs endothelial corneal dystrophy (FECD) and to assess the relationship between endoplasmic reticulum (ER) stress and MAM formation in corneal endothelial cells, given the established roles of mitochondrial dysfunction and ER stress in FECD pathogenesis. STUDY DESIGN:Experimental laboratory investigation. METHODS:Corneal endothelial tissues from FECD patients and controls were examined by use of transmission electron microscopy to evaluate the ultrastructural features of mitochondria-ER contacts. An established FECD cell model was used for immunofluorescence colocalization analysis and protein expression profiling. Experimental models of protein misfolding (MG132) and direct ER stress induction (tunicamycin) were implemented to explore the relationship between ER stress and MAM formation. RESULTS:The FECD specimens exhibited extensive mitochondria-ER contacts with evident tethering complexes and distances reduced to <20 nm when compared with normal corneal endothelium. Quantitative analysis showed significantly increased mitochondria-ER colocalization in iFECD cells (P <0.01). The FECD cell model showed significant upregulation of MAM-associated proteins, including GRP75, Mfn1, Mfn2, Sigma1 receptor, VDAC, and IP3R. MG132 and tunicamycin treatments both increased MAM formation while activating all UPR pathways. CONCLUSIONS:This study provides the first evidence of enhanced MAM formation in FECD and identifies ER stress as a key driver of this structural change. While these findings suggest a potential role for MAMs in linking ER stress and mitochondrial dysfunction in FECD pathogenesis, further investigation is needed to clarify whether such changes are protective adaptations or whether they contribute to disease progression.

Background The loss of corneal epithelial stem cells from the limbus at the edge of the cornea has severe consequences for vision, with the pathological manifestations of a limbal stem-cell deficiency (LSCD) difficult to treat. Here, to the best of our knowledge, we report the world's first use of corneal epithelial cell sheets derived from human induced pluripotent stem cells (iPSCs) to treat LSCD. Methods This non-randomised, single-arm, clinical study involved four eyes of four patients with LSCD at the Department of Ophthalmology, Osaka University Hospital. They comprised a woman aged 44 years with idiopathic LSCD (patient 1), a man aged 66 years with ocular mucous membrane pemphigoid (patient 2), a man aged 72 years with idiopathic LSCD (patient 3), and a woman aged 39 years with toxic epidermal necrosis (patient 4). Allogeneic human iPSC-derived corneal epithelial cell sheets (iCEPSs) were transplanted onto affected eyes. This was done sequentially in two sets of HLA-mismatched surgeries, with patients 1 and 2 receiving low-dose cyclosporin and patients 3 and 4 not. The primary outcome measure was safety, ascertained by adverse events. These were monitored continuously throughout the 52-week follow-up period, and during an additional 1-year safety monitoring period. Secondary outcomes, reflective of efficacy, were also recorded. This study is registered with UMIN, UMIN000036539 and is complete. Findings Patients were enrolled between June 17, 2019 and Nov 16, 2020. We had 26 adverse events during the 52-week follow-up period (consisting of 18 mild and one moderate event in treated eyes, and seven mild non-ocular events), with nine recorded in the additional 1-year safety monitoring period. No serious adverse events, such as tumourigenesis or clinical rejection, occurred during the whole 2-year observational period. At 52 weeks, secondary measures of efficacy showed that the disease stage had improved, corrected distance visual acuity was enhanced, and corneal opacification had diminished in all treated eyes. Corneal epithelial defects, subjective symptoms, quality-of-life questionnaire scores and corneal neovascularisation mostly improved or were unchanged. Overall, the beneficial efficacy outcomes achieved for patients 1 and 2 were better than those achieved for patients 3 and 4. Interpretation iCEPS transplantation for LSCD was found to be safe throughout the study period. A larger clinical trial is planned to further investigate the efficacy of the procedure. Funding The Japan Agency for Medical Research and Development, the Ministry of Education, Culture, Sports, Science, and Technology—Japan, and the UK Biotechnology and Biological Sciences Research Council.

Fuchs’ endothelial corneal dystrophy (FECD) is a common sight-threatening condition characterised by pathological changes in the posterior cornea. Here we report observations by light, transmission and volume scanning electron microscopy on changes in the endothelium and matrix associated with the characteristic deformations of Descemet’s membrane, termed guttae. Specimens were archived full-thickness human corneal tissue, removed during graft surgery, that had been fixed, stained and embedded by conventional processing methods for examination by transmission electron microscopy more than 40-years previously. Intact archived samples can be extremely valuable where, as with FECD, new cell-based methods of therapy now avoid excision of the full cornea thickness and any tissue excised is inferior for study. Volume electron microscopy, in particular serial block face scanning electron microscopy (SBF SEM), employing backscatter electron detection from resin-embedded specimens, has become an invaluable technique for 3D imaging of biological samples. However, archived specimens are normally considered unsuitable for imaging as conventional processing methods generate low backscatter electron yield. To overcome this for SBF SEM, we subjected epoxy resin-embedded specimens to de-plastination, then applied additional contrasting agents, uranyl acetate and lead acetate, prior to re-embedding. Selected regions of interest in the new resin blocks were examined in a scanning electron microscope equipped for SBF SEM and serial image datasets acquired. Enhanced contrast enabled 3D reconstruction of endothelium and guttae in Descemet’s membrane over large tissue volumes.

Meibomian gland dysfunction (MGD) is a chronic abnormality of the Meibomian glands (MGs) that is recognized as the leading cause of evaporative dry eye worldwide. Despite its prevalence, however, the pathophysiology of MGD remains elusive, and effective disease management continues to be a challenge. In the past 50 years, different models have been developed to illustrate the pathophysiological nature of MGD and the underlying disease mechanisms. An understanding of these models is crucial if researchers are to select an appropriate model to address specific questions related to MGD and to develop new treatments. Here, we summarize the various models of MGD, discuss their applications and limitations, and provide perspectives for future studies in the field.

The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.

The generation of a self-formed, ectodermal, autonomous multi-zone (SEAM) from human induced pluripotent stem cells (hiPSCs) offers a unique perspective to study the dynamics of ocular cell differentiation over time. Here, by utilising single-cell transcriptomics, we have (i) identified, (ii) molecularly characterised and (iii) ascertained the developmental trajectories of ectodermally-derived ocular cell populations which emerge within SEAMs as they form. Our analysis reveals interdependency between tissues of the early eye and delineates the sequential formation and maturation of distinct cell types over a 12-week period. We demonstrate a progression from pluripotency through to tissue specification and differentiation which encompasses both surface ectodermal and neuroectodermal ocular lineages and the generation of iPSC-derived components of the developing cornea, conjunctiva, lens, and retina. Our findings not only advance the understanding of ocular development in a stem cell-based system of human origin, but also establish a robust methodological paradigm for exploring cellular and molecular dynamics during SEAM formation at single-cell resolution and highlight the potential of hiPSC-derived systems as powerful platforms for modelling human eye development and disease. Single-cell transcriptomic analyses delineate the differentiation of human iPSCs into ectodermal ocular lineages, highlighting defined developmental trajectories and the sequential formation and maturation of distinct ocular cell types in vitro.

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior-posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman's layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles.