Cellular senescence promotes meibomian gland dysfunction in a chronic graft-versus-host disease mouse model.

PURPOSE: Aging is a well-established risk factor for meibomian gland dysfunction (MGD). We previously reported an accelerated cellular senescence phenomenon in the lacrimal glands of a murine model of chronic graft-versus-host disease (cGVHD). Herein, we aimed to elucidate the relationship between cellular senescence and MGD in cGVHD mice, utilizing the senolytic agent ABT-263. METHODS: A cGVHD mouse model was established through allogeneic bone marrow transplantation (BMT) from B10.D2 to BALB/c mice. Subsequently, cGVHD mice were treated with either ABT-263 or vehicle. The eyelids of recipients were analyzed at 4-week intervals post-BMT in both groups. RESULTS: Meibomian gland (MG) area was significantly smaller in cGVHD mice than in syngeneic control mice. ABT-263-treated mice retained a significantly larger MG area than their vehicle-treated counterparts. Pathological and immunohistochemical examinations revealed significant reductions in eyelid tissue inflammation and pathological fibrosis in the ABT-263 group compared to that in the vehicle-treated group. Additionally, expression of DNA damage markers, senescent cell markers, and senescence-associated secretory phenotype (SASP) factors was elevated in the eyelids of cGVHD mice compared with that in syngeneic mice. The expression of these cellular senescence-associated molecules was considerably suppressed in ABT-263-treated eyelids compared to that in vehicle-treated ones. CONCLUSIONS: Cellular senescence, along with expression of SASP factors, exhibited increased activity in the eyelids, particularly in the MGs of cGVHD mice. ABT-263 mitigated the severity of MGD. These findings highlight the potential of targeting cellular senescence as an effective approach for MGD treatment in cGVHD.

The Cornea: An Ideal Tissue for Regenerative Medicine.

Regenerative medicine is a highly anticipated field with hopes to provide cures for previously uncurable diseases such as spinal cord injuries and retinal blindness. Most regenerative medical products use either autologous or allogeneic stem cells, which may or may not be genetically modified. The introduction of induced-pluripotent stem cells (iPSCs) has fueled research in the field, and several iPSC-derived cells/tissues are currently undergoing clinical trials. The cornea is one of the pioneering areas of regenerative medicine, and already four cell therapy products are approved for clinical use in Japan. There is one other government-approved cell therapy product approved in Europe, but none are approved in the USA at present. The cornea is transparent and avascular, making it unique as a target for stem cell therapy. This review discusses the unique properties of the cornea and ongoing research in the field.

Eyes open on stem cells.

Recently, the murine cornea has reemerged as a robust stem cell (SC) model, allowing individual SC tracing in living animals. The cornea has pioneered seminal discoveries in SC biology and regenerative medicine, from the first corneal transplantation in 1905 to the identification of limbal SCs and their transplantation to successfully restore vision in the early 1990s. Recent experiments have exposed unexpected properties attributed to SCs and progenitors and revealed flexibility in the differentiation program and a key role for the SC niche. Here, we discuss the limbal SC model and its broader relevance to other tissues, disease, and therapy.

A Comprehensive Assessment of Tear-Film-Oriented Diagnosis (TFOD) in a Dacryoadenectomy Dry Eye Model.

Tear film instability is a major cause of dry eye disease. In order to treat patients with short tear film breakup time (TBUT)-type dry eye, the development of tear film stabilizing agents is essential. However, the lack of an appropriate animal model of tear film instability has made drug development difficult. Although rabbit dry eye models have been reported in the past, there are only a few reports that focus on tear film instability. Herein, we assessed the tear film stability of a rabbit dry eye model induced by dacryoadenectomy. A clinical evaluation of the ocular surface, interferometry, and histological assessments of the cornea and conjunctiva were performed. Following the removal of the lacrimal glands, TBUT was shortened significantly, with dimple and random breakup patterns prominently observed. Furthermore, the blink rate in this model increased after dacryoadenectomy, suggesting that this model partially captured the phenotypes of human short TBUT-type dry eye and may be useful as an animal model for investigating potential drug candidates.

Non-apoptotic regulated cell death in Fuchs endothelial corneal dystrophy.

Introduction: Fuchs endothelial corneal dystrophy (FECD) is the leading cause of corneal blindness in developed countries. Corneal endothelial cells in FECD are susceptive to oxidative stress, leading to mitochondrial dysfunction and cell death. Oxidative stress causes many forms of cell death including parthanatos, which is characterized by translocation of apoptosis-inducing factor (AIF) to the nucleus with upregulation of poly (ADP-ribose) polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). Although cell death is an important aspect of FECD, previous reports have often analyzed immortalized cell lines, making the evaluation of cell death difficult. Therefore, we established a new in vitro FECD model to evaluate the pathophysiology of FECD. Methods: Corneal endothelial cells were derived from disease-specific induced pluripotent stem cells (iPSCs). Hydrogen peroxide (H2O2) was used as a source for oxidative stress to mimic the pathophysiology of FECD. We investigated the responses to oxidative stress and the involvement of parthanatos in FECD-corneal endothelial cells. Results: Cell death ratio and oxidative stress level were upregulated in FECD with H2O2 treatment compared with non-FECD control, indicating the vulnerability of oxidative stress in FECD. We also found that intracellular PAR, as well as PARP-1 and AIF in the nucleus were upregulated in FECD. Furthermore, PARP inhibition, but not pan-caspase inhibition, rescued cell death, DNA double-strand breaks, mitochondrial membrane potential depolarization and energy depletion, suggesting that cell death was mainly due to parthanatos. Conclusions: We report that parthanatos may be involved in the pathophysiology of FECD and targeting this cell death pathway may be a potential therapeutic approach for FECD.

Advances in corneal regenerative medicine with iPS cells.

The cornea is a pioneering area of regenerative medicine, and Japanese researchers have led the world in this field. In Japan, 3 different epithelial sheet regenerative medicine products have been approved for corneal epithelial stem cell deficiency, and the first-in-human studies of cultured corneal endothelial cell suspension transplants, induced pluripotent stem cell (iPS cell)-derived corneal epithelial sheet transplants, and iPS cell-derived corneal endothelial substitute cell transplants were all conducted and reported globally for the first time by Japanese researchers. In the field of corneal epithelial regenerative medicine, Pellegrini et al.  (Lancet 349:990-3, 1997) performed the first in-human transplant of autologous cultured corneal epithelial sheets. More than 20 years later, autologous cultivated corneal epithelium and autologous cultivated oral mucosal epithelium products were launched in Japan. In addition, clinical studies of iPS cell-derived corneal epithelial cell sheet transplant have begun, which may solve the issues with conventional autologous epithelial sheets. In corneal endothelium regenerative medicine, a clinical study of transplant of allogenic cultured corneal endothelial cell suspension for bullous keratopathy was reported, in which corneal endothelial cells derived from donor corneas were grown in culture and then injected into the anterior chamber with a ROCK inhibitor (Kinoshita et al. in N Engl J Med 378:995-1003, 2018). Our research group is also developing iPS-cell-derived corneal endothelium-like cells, termed corneal endothelial cell substitute from iPS cells (CECSi cells), and we are conducting a clinical study to treat bullous keratopathy with these cells (Hatou et al. in Stem Cell Res 55:102497, 2021). This review describes the progress and challenges of corneal epithelial and endothelial regenerative medicine and the promising future of corneal regenerative medicine with iPS cells.

Graft Size and Double Scroll Formation Rate in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: This study aimed to evaluate the usefulness of intentional double scroll formation of donor Descemet membrane (DM) inside a glass tube inserter (the Fogla technique) in DM endothelial keratoplasty (DMEK) for controlled insertion and unfolding of grafts. METHODS: Eleven consecutive patients who underwent DMEK were included in this study. We sought to specify graft characteristics in which double scroll configuration was successfully formed using the Fogla technique. We compared donor age, graft size, surgical time, unfolding time, and visual outcomes between patients with and without double scroll configuration. The ability to form double scroll formation of DM grafts of various diameters and unfolding time of DM grafts was evaluated using total seven eye-bank eyes in ex vivo experiments. RESULTS: A double scroll configuration inside a glass tube was successfully obtained in six DMEK grafts (54.5%). When comparing clinical features between those with and without double scroll configuration, only graft size was significantly larger in those with double scroll configuration (7.9 ± 0.2 mm) than in those without (7.4 ± 0.4, P = 0.03). There were no significant differences in other features and clinical outcomes, although unfolding-time was shorter in eyes with double scroll configuration (4.6 ± 2.0 min) compared to those without (8.6 ± 8.1, P = 0.21). Ex vivo experiments showed that unfolding time was significantly shorter in double scroll configuration (2.71 ± 0.49 min) than in single scroll (5.02 ± 0.79, P = 0.01). CONCLUSIONS: A double scroll configuration within a glass tube can be obtained more frequently in larger DMEK grafts (8.0 mm in diameter), which may result in easier and faster DMEK procedures.

The Anterior Eye Chamber as a Visible Medium for In Vivo Tumorigenicity Tests.

Pluripotent stem cell (PSC)-based cell therapies have increased steadily over the past few years, and assessing the risk of tumor formation is a high priority for clinical studies. Current in vivo tumorigenesis studies require several months and depend strongly on the site of grafting. In this study, we report that the anterior eye chamber is preferable to the subcutaneous space for in vivo tumorigenesis studies for several reasons. First, cells can easily be transplanted into the anterior chamber and monitored in real-time without sacrificing the animals due to the transparency of the cornea. Second, tumor formation is faster than with the conventional subcutaneous method. The median tumor formation time in the subcutaneous area was 18.50 weeks (95% CI 10.20-26.29), vs. 4.0 weeks (95% CI 3.34-.67) in the anterior chamber (P = .0089). When hiPSCs were spiked with fibroblasts, the log10TPD50 was 3.26, compared with 4.99 when hiPSCs were transplanted without fibroblasts. There was more than a 40-fold difference in the log10TPD50 values with fibroblasts. Furthermore, the log10TPD50 for HeLa cells was 1.45 and 100% of animals formed tumors at a concentration greater than 0.1%, indicating that the anterior chamber tumorigenesis assays can be applied for cancer cell lines as well. Thus, our method has the potential to become a powerful tool in all areas of tumorigenesis studies and cancer research.

Transplantation of iPSC-derived corneal endothelial substitutes in a monkey corneal edema model.

OBJECTIVE: In order to provide regenerative therapy for millions of patients suffering from corneal blindness globally, we derived corneal endothelial cell substitute (CECSi) cells from induced pluripotent stem cells (iPSCs) to treat corneal edema due to endothelial dysfunction (bullous keratopathy). METHODS AND RESULTS: We developed an efficient xeno-free protocol to produce CECSi cells from both research grade (Ff-MH09s01 and Ff-I01s04) and clinical grade (QHJI01s04) iPSCs. CECSi cells formed a hexagonal confluent monolayer with Na, K-ATPase alpha 1 subunit expression (ATP1A1), tight junctions, N-cadherin adherence junction formation, and nuclear PITX2 expression, which are all characteristics of corneal endothelial cells. CECSi cells can be cryopreserved, and thawed CECSi cell suspensions also expressed N-cadherin and ATP1A1. Residual undifferentiated iPSCs in QHJI01s04-derived CECSi cells was below 0.01%. Frozen stocks of Ff-I01s04- and QHJI01s04-derived CECSi cells were transported, thawed and transplanted into a monkey corneal edema model. CECSi-transplanted eyes significantly reduced corneal edema compared to control group. CONCLUSION: Our results show a promising approach to provide bullous keratopathy patients with an iPS-cell-based cell therapy to recover useful vision.

Observation of Chronic Graft-Versus-Host Disease Mouse Model Cornea with In Vivo Confocal Microscopy.

Graft-versus-host disease (GVHD) is a major complication after hematopoietic stem cell transplantation (HSCT), and ocular GVHD can cause severe dry eye disease that can lead to visual impairment. Epithelial damage, vascular invasion, corneal fibrosis, and corneal perforation may occur in severe cases. It is generally accepted that inflammatory cells such as dendritic cells and T cells contribute to this pathological condition. However, it is still unknown what pathological condition occurs on the ocular surface after HSCT, and when. We therefore observed the dynamics of inflammatory cells in the cornea of chronic GVHD (cGVHD) model mice from 1 to 4 weeks after bone marrow transplantation (BMT) by in vivo confocal microscopy (IVCM) and considered the relationship with the pathophysiology of ocular GVHD (tear volume, corneal epithelial damage). In the allogeneic group, neovascularization occurred in all eyes at 1 week after BMT, although almost all vessels disappeared at 2 weeks after BMT. In addition, we revealed that infiltration of globular cells, and tortuosity and branching of nerves in the cornea occurred in both cGVHD mice and human cGVHD patients. Thus, we consider that cGVHD mouse model study by IVCM reproduces the state of ocular GVHD and may contribute to elucidating the pathological mechanism for ocular GVHD.

MSCs Become Collagen-Type I Producing Cells with Different Phenotype in Allogeneic and Syngeneic Bone Marrow Transplantation.

Mesenchymal stem cells (MSCs) have been widely used in therapeutic applications for many decades. However, more and more evidence suggests that factors such as the site of origin and pre-implantation treatment have a crucial impact on the result. This study investigates the role of freshly isolated MSCs in the lacrimal gland after allogeneic transplantation. For this purpose, MSCs from transgenic GFP mice were isolated and transplanted into allogeneic and syngeneic recipients. While the syngeneic MSCs maintained a spherical shape, allogeneic MSCs engrafted into the tissue as spindle-shaped cells in the interstitial stroma. Furthermore, the MSCs produced collagen type I in more than 85% to 95% of the detected GFP+ MSCs in the recipients of both models, supposedly contributing to pathogenic fibrosis in allogeneic recipients compared to syngeneic models. These findings indicate that allogeneic MSCs act completely differently from syngeneic MSCs, highlighting the importance of understanding the exact mechanisms behind MSCs.

Analysis of the Association between Galectin-3 Concentration in Tears and the Severity of Dry Eye Disease: A Case-Control Study.

This study aimed to investigate the relationship between the severity of dry eye disease (DED) and galectin-3 concentration (gal-3) and its cleavage (gal-3C) in tear fluid. Twenty-eight DED patients and 14 controls were recruited at Keio University Hospital. The lissamine green conjunctival staining (LG) score, fluorescein corneal staining (FL) score, tear film break-up time (TBUT), Schirmer's test, and ocular symptoms questionnaire score (dry eye questionnaire score, DEQS) were evaluated. Furthermore, the correlation between these parameters and the concentrations of gal-3 in tears (ng/µg) and the detection rate of gal-3C (%) were analyzed. Gal-3 concentration in tears was positively correlated with the LG score (R = 0.60, p < 0.01), FL score (R = 0.49, p < 0.01), and DEQS (R = 0.45, p < 0.01), and negatively correlated with the TBUT score (R = -0.40, p < 0.01) and Schirmer's I value (R = -0.36, p < 0.01). The detection rate of gal-3C in tears was significantly associated with the severity of DED, especially with the LG (p < 0.01) and FL (p < 0.01) scores. Therefore, the concentration of gal-3 and the detection rate of gal-3C in tears had a significant relationship with the severity of ocular surface barrier disruption.

Human corneal limbal organoids maintaining limbal stem cell niche function.

Corneal epithelial stem cells reside in the limbal area between the cornea and conjunctiva. We examined the potential use of limbal organoids as a source of transplantable limbal stem cells. After treating tissue with collagenase, limbal cells were seeded onto Matrigel and cultivated using limbal phenotype maintenance medium. After 1-month, approximately 500 organoids were formed from one donor cornea. Organoids derived from vertical sites (superior and inferior limbus) showed large colony forming efficiency, a higher ratio of slow cycling cells and N-cadherin-expressing epithelial cells compared to horizontal sites. The progenitor markers Keratin (K) 15 and p63 were expressed in epithelial sheets engineered form a single organoid. Organoids transplanted in the limbus of a rabbit limbal deficiency model confirmed the presence of organoid-derived cells extending on to host corneas by immunohistochemistry. Our data show that limbal organoids with a limbal phenotype can be maintained for up to 1 month in vitro which can each give rise to a fully stratified corneal epithelium complete with basal progenitor cells. Limbal organoids were successfully engrafted in vivo to provide epithelial cells in a rabbit limbal deficiency model, suggesting that organoids may be an efficient cell source for clinical use.

Descemet stripping and automated endothelial keratoplasty (DSAEK) versus non-Descemet stripping and automated endothelial keratoplasty (nDSAEK) for bullous keratopathy.

PURPOSE: To investigate the long-term results of Descemet stripping and automated endothelial keratoplasty (DSAEK) versus non-Descemet stripping and automated endothelial keratoplasty (nDSAEK) for non-Fuchs bullous keratopathy. STUDY DESIGN: Retrospective comparative study. METHODS: Twenty-nine eyes of 28 patients who underwent DSAEK and 60 eyes of 56 patients who underwent nDSAEK for bullous keratopathy were retrospectively analyzed in a clinical case-control study. All the operations were done using precut donor buttons prepared by overseas eye banks. The recipient Descemet membrane was stripped in DSAEK, whilst it was left intact in nDSAEK. Donor buttons were inserted through a 4.2-mm corneal incision using the pull-through technique. Air was injected into the anterior chamber at the end of the operation. We investigated the visual acuity, refraction, endothelial cell density, and complications. RESULTS: The average best spectacle-corrected visual acuity (BSCVA) was significantly improved at all time points measured after surgery in both the DSAEK and the nDSAEK groups, and no significant differences were found between the 2 groups after surgery. No significant differences in spherical equivalent were found between the groups before and after surgery. The endothelial cell density gradually decreased after the operation in both groups; however, no significant difference between the 2 groups was found at any of the time points measured. CONCLUSIONS: DSAEK and nDSAEK provided excellent refractive and reasonable visual outcomes in our long-term observation.

Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

Chronic graft-vs-host disease (cGVHD) is a multifactorial inflammatory disease that affects patients undergoing hematopoietic stem cell transplantation. Multiple organs, including the lacrimal glands (LGs), are negatively affected by cGVHD and lose function due to the resultant fibrosis. An abnormal immune response is thought to be a major factor in the development of chronic ocular GVHD, which is currently treated primarily with immunosuppressive therapies. However, all the treatments yield unsatisfactory outcomes, and additional treatment strategies are needed. To meet this unmet medical need, we aimed to elucidate an additional pathway of chronic ocular GVHD. Our findings suggest a potential association between chronic ocular GVHD pathogenesis and stress-induced cellular senescence through the senescence-associated secretory phenotype (SASP). Senescent cells produce cytokines and chemokines, such as IL-6 and CXCL9. Indeed, senescent cell accumulation was presumably associated with cGVHD development in LGs, as evidenced by the improvement in LGs after the selective elimination of senescent cells (senolysis) with ABT-263. Results in the sclerodermatous cGVHD mouse model suggest that inhibiting the major components of the SASP, including IL-6 and CXCL9, with senolytics is a potential novel strategy for treating cGVHD-affected LGs. Taken together, our results indicate a potential association between the SASP and cGVHD development in LGs and suggest that targeted senolytic treatment may be a new therapeutic option for this disease.

Conjunctival Epithelial Ingrowth After Penetrating Keratoplasty.

PURPOSE: To report a case of conjunctival epithelial ingrowth after penetrating keratoplasty. METHODS: A 57-year-old woman with herpetic corneal keratitis, endotheliitis, and bullous keratopathy underwent penetrating keratoplasty (PKP) and secondary cataract surgery. One month after cataract surgery, an epithelial ingrowth was observed at the 5 o'clock donor host junction. Ingrowth extended into the anterior chamber and along the iris surface by 9 months. Another PKP was performed, and the excised graft was submitted for histopathology. RESULTS: The graft showed CK13-positive and CK3-negative cells lining the endothelial surface, indicating the conjunctival origin of ingrown epithelium. Ten months postoperatively, no recurrence of ingrowth was observed. CONCLUSIONS: We experienced a rare case of conjunctival epithelial ingrowth after penetrating keratoplasty. There was no recurrence of the ingrowth after surgical removal, and the conjunctival origin may explain the relatively benign course of the complication.

Review: corneal endothelial cell derivation methods from ES/iPS cells.

Globally, approximately 12.7 million people are awaiting a transplantation, while only 185,000 cases of corneal transplantation are performed in a year. Corneal endothelial dysfunction (bullous keratopathy) due to Fuchs' corneal endothelial dystrophy, or insults associated with intraocular surgeries, shared half of all indications for corneal transplantation. Regenerative therapy for corneal endothelium independent of eye bank eyes has great importance to solve the large supply-demand mismatching in corneal transplantation and reduce the number of worldwide corneal blindness. If corneal endothelial cells could be derived from ES or iPS cells, these stem cells would be the ideal cell source for cell therapy treatment of bullous keratopathy. Four representative corneal endothelial cell derivation methods were reviewed. Components in earlier methods included lens epithelial cell-conditioned medium or fetal bovine serum, but the methods have been improved and materials have been chemically more defined over the years. Conditioned medium or serum is replaced to recombinant proteins and small molecule compounds. These improvements enabled to open the corneal endothelial developmental mechanisms, in which epithelial-mesenchymal and mesenchymal-endothelial transition by TGF beta, BMP, and Wnt signaling have important roles. The protocols are gradually approaching clinical application; however, proof of efficacy and safety of the cells by adequate animal models are the challenges for the future.

Global Consensus on Definition, Classification, Diagnosis, and Staging of Limbal Stem Cell Deficiency.

PURPOSE: Despite extensive knowledge gained over the last 3 decades regarding limbal stem cell deficiency (LSCD), the disease is not clearly defined, and there is lack of agreement on the diagnostic criteria, staging, and classification system among treating physicians and research scientists working on this field. There is therefore an unmet need to obtain global consensus on the definition, classification, diagnosis, and staging of LSCD. METHODS: A Limbal Stem Cell Working Group was first established by The Cornea Society in 2012. The Working Group was divided into subcommittees. Four face-to-face meetings, frequent email discussions, and teleconferences were conducted since then to obtain agreement on a strategic plan and methodology from all participants after a comprehensive literature search, and final agreement was reached on the definition, classification, diagnosis, and staging of LSCD. A writing group was formed to draft the current manuscript, which has been extensively revised to reflect the consensus of the Working Group. RESULTS: A consensus was reached on the definition, classification, diagnosis, and staging of LSCD. The clinical presentation and diagnostic criteria of LSCD were clarified, and a staging system of LSCD based on clinical presentation was established. CONCLUSIONS: This global consensus provides a comprehensive framework for the definition, classification, diagnosis, and staging of LSCD. The newly established criteria will aid in the correct diagnosis and formulation of an appropriate treatment for different stages of LSCD, which will facilitate a better understanding of the condition and help with clinical management, research, and clinical trials in this area.

Immunological Properties of Neural Crest Cells Derived from Human Induced Pluripotent Stem Cells.

Collecting sufficient quantities of primary neural crest cells (NCCs) for experiments is difficult, as NCCs are embryonic transient tissue that basically does not proliferate. We successfully induced NCCs from human induced pluripotent stem cells (iPSCs) in accordance with a previously described method with some modifications. The protocol used in this study efficiently produced large amounts of iPSC-derived NCCs (iPSC-NCCs). Many researchers have recently produced large amounts of iPSC-NCCs and used these to examine the physiological properties, such as migratory activity, and the potential for medical uses such as wound healing. Immunological properties of NCCs are yet to be reported. Therefore, the purpose of this study was to assess the immunological properties of human iPSC-NCCs. Our current study showed that iPSC-NCCs were hypoimmunogenic and had immunosuppressive properties in vitro. Expression of HLA class I molecules on iPSC-NCCs was lower than that observed for iPSCs, and there was no expression of HLA class II and costimulatory molecules on the cells. With regard to the immunosuppressive properties, iPSC-NCCs greatly inhibited T cell activation (cell proliferation and production of inflammatory cytokines) after stimulation. iPSC-NCCs constitutively expressed membrane-bound TGF-ß, and TGF-ß produced by iPSC-NCCs played a critical role in T cell suppression. Thus, cultured human NCCs can fully suppress T cell activation in vitro. This study may contribute to the realization of using stem cell-derived NCCs in cell-based medicine.