Outcomes and Complications of Limbal Stem Cell Allograft Transplantation: A Report by the American Academy of Ophthalmology.

PURPOSE: To review the published literature on the safety and outcomes of keratolimbal allograft (KLAL) transplantation and living-related conjunctival limbal allograft (lr-CLAL) transplantation for bilateral severe/total limbal stem cell deficiency (LSCD). METHODS: Literature searches were last conducted in the PubMed database in February 2023 and were limited to the English language. They yielded 523 citations; 76 were reviewed in full text, and 21 met the inclusion criteria. Two studies were rated level II, and the remaining 19 studies were rated level III. There were no level I studies. RESULTS: After KLAL surgery, best-corrected visual acuity (BCVA) improved in 42% to 92% of eyes at final follow-up (range, 12-95 months). The BCVA was unchanged in 17% to 39% of eyes and decreased in 8% to 29% of eyes. Two of 14 studies that evaluated the results of KLAL reported a notable decline in visual acuity over time postoperatively. Survival of KLAL was variable, ranging from 21% to 90% at last follow-up (range, 12-95 months) and decreased over time. For patients undergoing lr-CLAL surgery, BCVA improved in 31% to 100% of eyes at final follow-up (range, 16-49 months). Of the 9 studies evaluating lr-CLAL, 4 reported BCVA unchanged in 30% to 39% of patients, and 3 reported a decline in BCVA in 8% to 10% of patients. The survival rate of lr-CLAL ranged from 50% to 100% at final follow-up (range, 16-49 months). The most common complications were postoperative elevation of intraocular pressure, persistent epithelial defects, and acute allograft immune rejections. CONCLUSIONS: Given limited options for patients with bilateral LSCD, both KLAL and lr-CLAL are viable choices that may provide improvement of vision and ocular surface findings. The studies trend toward a lower rejection rate and graft failure with lr-CLAL. However, the level and duration of immunosuppression vary widely between the studies and may impact allograft rejections and long-term graft survival. Complications related to immunosuppression are minimal. Repeat surgery may be needed to maintain a viable ocular surface. Reasonable long-term success can be achieved with both KLAL and lr-CLAL with appropriate systemic immunosuppression. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells.

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Limbal stem cell therapy.

PURPOSE OF REVIEW: To highlight the progress and future direction of limbal stem cell (LSC) therapies for the treatment of limbal stem cell deficiency (LSCD). RECENT FINDINGS: Direct LSC transplantation have demonstrated good long-term outcomes. Cultivated limbal epithelial transplantation (CLET) has been an alternative to treat severe to total LSCD aiming to improve the safety and efficacy of the LSC transplant. A prospective early-stage uncontrolled clinical trial shows the feasibility and safety of CLET manufactured under xenobiotic free conditions. Other cell sources for repopulating of the corneal epithelium such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells are being investigated. The first clinical trials of using MSCs showed short-term results, but long-term efficacy seems to be disappointing. A better understanding of the niche function and regulation of LSC survival and proliferation will lead to the development of medical therapies to rejuvenate the residual LSCs found in a majority of eyes with LSCD in vivo. Prior efforts have been largely focused on improving LSC transplantation. Additional effort should be placed on improving the accuracy of diagnosis and staging of LSCD, and implementing standardized outcome measures which enable comparison of efficacy of different LSCD treatments for different severity of LSCD. The choice of LSCD treatment will be customized based on the severity of LSCD in the future. SUMMARY: New approaches for managing different stages of LSCD are being developed. This concise review summarizes the progresses in LSC therapies for LSCD, underlying mechanisms, limitations, and future areas of development.

Severity Classification of Limbal Stem Cell Failure Due to Steven Johnson Syndrome in the Light of the Classification Consensus of Limbal Stem Cell Deficiency.

OBJECTIVES: To examine and to understand the limbal stem-cell deficiency (LSCD) because of Steven-Johnson syndrome (SJS) in line with the new classification system for the first time in the literature. METHODS: Medical records of patients with LSCD because of SJS were reviewed retrospectively. In addition to demographic data and ophthalmologic or systemic findings, anterior segment photographs of the patients were reviewed retrospectively. Limbal stem-cell deficiency severity was graded according to the classification published by the Limbal Stem Cell Working Group. RESULTS: Twenty-four eyes of 14 patients with eye involvement secondary to SJS were included in the study. The mean age of the patients was 36.09±16.70 (9-58) years and the female-to-male ratio was 11:3. The anterior segment photographs of the patients were evaluated by two independent masked observers. Limbal stem-cell deficiency severity was graded according to the classification published by Deng et al. Corneal opacity was divided into three stages according to the area of involvement. Corneal opacity was classified as Stage I if the central 5 mm region of the cornea was not affected, as Stage II if the central 5 mm region of the cornea was affected, and as Stage III if the entire corneal surface was affected. Limbal involvement was classified as Stage A if it was below 50%, as Stage B if it was between 50% and 100%, and as Stage C if it was 100%. CONCLUSION: This is the first study in the literature to describe and classify LSCD because of SJS, according to the new LSCD classification. Consistent with the results, LSCD follows a bimodal distribution. Most patients demonstrated severe (Stage III-32.14%) or mild (Stage IA-21.42%) LSCD.

rAAV-PHP.B escapes the mouse eye and causes lethality whereas rAAV9 can transduce aniridic corneal limbal stem cells without lethality.

Recently safety concerns have been raised in connection with high doses of recombinant adeno-associated viruses (rAAV). Therefore, we undertook a series of experiments to test viral capsid (rAAV9 and rAAV-PHP.B), dose, and route of administration (intrastromal, intravitreal, and intravenous) focused on aniridia, a congenital blindness that currently has no cure. The success of gene therapy for aniridia may depend on the presence of functional limbal stem cells (LSCs) in the damaged aniridic corneas and whether rAAV can transduce them. Both these concerns were unknown, and thus were also addressed by our studies. For the first time, we report ataxia and lethality after intravitreal or intrastromal rAAV-PHP.B virus injections. We demonstrated virus escape from the eye and transduction of non-ocular tissues by rAAV9 and rAAV-PHP.B capsids. We have also shown that intrastromal and intravitreal delivery of rAAV9 can transduce functional LSCs, as well as all four PAX6-expressing retinal cell types in aniridic eye, respectively. Overall, lack of adverse events and successful transduction of LSCs and retinal cells makes it clear that rAAV9 is the capsid of choice for future aniridia gene therapy. Our finding of rAAV lethality after intraocular injections will be impactful for other researchers developing rAAV-based gene therapies.

Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency.

PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Development and validation of a reliable rabbit model of limbal stem cell deficiency by mechanical debridement using an ophthalmic burr.

A simple and reproducible method is necessary to generate reliable animal models of limbal stem cell deficiency (LSCD) for assessing the safety and efficacy of new therapeutic modalities. This study aimed to develop and validate a rabbit model of LSCD through mechanical injury. The corneal and limbal epithelium of New Zealand White rabbits (n = 18) were mechanically debrided using an ophthalmic burr (Algerbrush II) with a 1.0-mm rotating head after 360° conjunctival peritomy. The debrided eyes were serially evaluated for changes in corneal opacity, neo-vascularization, epithelial defect and corneal thickness using clinical photography, slit lamp imaging, fluorescein staining, and anterior segment optical coherence tomography scanning (AS-OCT). Following this, an assessment of histopathology and phenotypic marker expression of the excised corneas was conducted. The experimental eyes were grouped as mild (n = 4), moderate (n = 10), and severe (n = 4) based on the grade of LSCD. The moderate group exhibited abnormal epithelium, cellular infiltration in the stroma, and vascularization in the central, peripheral, and limbal regions of the cornea. The severe group demonstrated central epithelial edema, peripheral epithelial thinning with sparse goblet cell population, extensive cellular infiltration in the stroma, and dense vascularization in the limbal region of the cornea. A significant decrease in the expression of K12 and p63 (p < 0.0001) was observed, indicating the loss of corneal epithelium and limbal epithelial stem cells in the LSCD cornea. This study demonstrates that the Alger brush-induced mechanical debridement model provides a reliable model of LSCD with comprehensive clinic-pathological features and that is well suited for evaluating novel therapeutic and regenerative approaches.

Autologous Serum Eye Drops in the Management of Limbal Stem Cell Deficiency Associated With Glaucoma Surgery.

PURPOSE: To evaluate safety and efficacy of autologous serum eye drops (AS) in the treatment of limbal stem cell deficiency (LSCD) associated with glaucoma surgery. METHODS: Retrospective case series of eyes with glaucoma surgery-induced LSCD treated with AS. Diagnosis of LSCD was confirmed by anterior segment optical coherence tomography, in vivo confocal microscopy, and/or impression cytology. Limbal stem cell deficiency severity was staged using a clinical scoring system (2-10 points). Outcome measures were changes (≥2 points) of the LSCD score and best-corrected visual acuity (BCVA) from the baseline to the last follow-up. RESULTS: Thirteen eyes of 12 consecutive patients treated with 50% AS for at least 3 months were included. The mean age was 78.9±7.5 years and the mean duration of AS use was 20.9±16.8 months. Indications of AS included LSCD progression in eight eyes (61.5%) and visual axis threatening in five eyes (38.5%). The mean LSCD score at baseline (6.7±1.6) was similar to that at last follow-up (6.5±2.2, P =0.625). Two eyes (15.4%) showed improvement, nine eyes (69.2%) were stable, and two eyes (15.4%) worsened. The mean baseline BCVA (0.89±0.64 logMAR) was similar to the mean final BCVA (1.05±0.63 logMAR, P =0.173). There were no serious adverse complications related to AS. CONCLUSION: AS appears to be well tolerated and may stabilize the progression of LSCD with limited effects. A larger study is necessary to confirm the findings.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Derivation of Limbal Stem Cells from Human Adult Mesenchymal Stem Cells for the Treatment of Limbal Stem Cell Deficiency.

Approximately 10 million individuals have blindness due to limbal stem cell (LSCs) deficiency, one of the most challenging problems in ophthalmology. To replenish the LSC pool, an autologous extraocular cell source is appropriate, thereby avoiding the risk of immune rejection, the need for immunosuppression and the risk of damaging the contralateral eye. In recent years, adipose-derived mesenchymal stem cells (ADSCs) have been a key element in ocular regenerative medicine. In this study, we developed a protocol for deriving human LSCs from ADSCs compatible with the standard carrier human amniotic membrane, helping provide a stem cell pool capable of maintaining proper corneal epithelial homeostasis. The best protocol included an ectodermal induction step by culturing ADSCs with media containing fetal bovine serum, transforming growth factor-ß inhibitor SB-505124, Wnt inhibitor IWP-2 and FGF2 for 7 days, followed by an LSC induction step of culture in modified supplemental hormonal epithelial medium supplemented with pigment epithelium-derived factor and keratinocyte growth factor for 10 additional days. The optimal differentiation efficiency was achieved when cells were cultured in this manner over vitronectin coating, resulting in up to 50% double-positive αp63/BMI-1 cells. The results of this project will benefit patients with LSC deficiency, aiding the restoration of vision.

Early Clinical Outcomes of the First Commercialized Human Autologous Ex Vivo Cultivated Oral Mucosal Epithelial Cell Transplantation for Limbal Stem Cell Deficiency: Two Case Reports and Literature Review.

The first product in the world for ex vivo cultivated oral mucosal epithelial cell transplantation (COMET) to treat limbal stem cell deficiency (LSCD), named Ocural®, was launched in June 2021 in Japan. COMET was performed on two patients, including the first case in the post-marketing phase of Ocural®. Pathological and immunohistochemical examinations were also carried out using specimens obtained before and after COMET and the spare cell sheet. In case 1, the ocular surface remained free from epithelial defects for approximately six months. In case 2, although defect of the cornea-like epithelia was observed after COMET for one month, it was resolved after the insertion of lacrimal punctal plugs. In case 1, adjuvant treatment was interrupted due to an accident during the second month after COMET, resulting in conjunctival ingrowth and corneal opacity. Eventually, a lamellar keratoplasty was required at six months after COMET. Immunohistochemistry revealed the presence of markers for stem cells (p63, p75), proliferation (Ki-67), and differentiation (Keratin-3, -4, and -13) in both the cornea-like tissue after COMET and a cultivated oral mucosal epithelial cell sheet. In conclusion, Ocural® can be accomplished without major complications, and the stem cells derived from oral mucosa might be successfully engrafted.

Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency.

Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.

Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study.

PURPOSE: Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. METHODS: LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30 s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15 s, 30 s, 60 s, 90 s, and 120 s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. RESULTS: In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). CONCLUSIONS: Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5 days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.

[Research progress on imagine diagnosis of limbal stem cell deficiency].

Limbal stem cell deficiency (LSCD) is an ocular surface disease resulting from a reduction and/or dysfunction of limbal stem cells. The symptoms of LSCD are non-specific and can be difficult to distinguish from other ocular surface diseases through slit-lamp examination. Impression cytology is currently considered the gold standard for LSCD diagnosis; however,it is a qualitative method with low sensitivity. Nonetheless,emerging imaging techniques offer quantitative diagnosis and staging of LSCD. This review article examines four imaging methods and their associated parameters for diagnosing LSCD: optical coherence tomography,which measures corneal epithelial thickness; optical coherence tomography angiography,which detects corneal neovascularization; in vivo confocal microscopy,which measures corneal epithelial thickness,subbasal nerve density,and corneal basal cell density; and future applications of full-field/spectral-domain optical coherence tomography.

[Limbal stem cell-sparing lamellar keratoplasty for advanced keratoglobus in a patient with osteogenesis imperfecta: a case report].

This 15-year-old male patient has been diagnosed with osteogenesis imperfecta through genetic testing after birth and has poor vision. His full corneas in both eyes are unevenly thinned and bulging in a spherical shape, with the right eye being more severe. He underwent a limbal stem cell-sparing lamellar keratoplasty in the right eye, resulting in improved vision with a corrected visual acuity of 0.5, a decrease in corneal curvature, and a significant increase in corneal thickness. The surgery had a satisfactory outcome. The condition of the left eye is progressing and will require further surgical treatment.

[The diagnosis and treatment of contact lens-induced limbal stem cell deficiency].

Objective: To analyze the clinical features and treatment outcomes of eyes with contact lens-induced limbal stem cell deficiency (CL-iLSCD). Methods: This cross-sectional study involved patients diagnosed with CL-iLSCD at the Eye, Ear, Nose & Throat Hospital of Fudan University between October 2018 and September 2022. A total of 17 patients (25 eyes) with a mean age of (36.4±6.9) years were enrolled. Among them, 14 were females (82.4%). Corneal and limbal abnormalities, especially the range of epitheliopathy, were observed under a slit lamp biomicroscope with fluorescein staining. Anterior segment optical coherence tomography and in vivo laser scanning confocal microscopy were performed to obtain the central corneal epithelial thickness, density of basal epithelial cells and corneal nerve fiber length. The clinical features of CL-iLSCD, along with their treatment outcomes and related risk factors, were analyzed. Results: All patients wore soft contact lenses, with an average daily wearing time of (10.5±2.5) hours and a median wearing duration of 10 (4 to 30) years. Ocular symptoms, including decreased vision, ocular discomfort or pain, redness, and photophobia, were present in 22 eyes (88.0%). The most characteristic clinical sign of CL-iLSCD was comb-or whorl-pattern late fluorescein staining under cobalt blue light, which was most commonly seen at the superior limbus (25/25, 100.0%). Additionally, reductions in central corneal epithelial thickness, basal cell density, and corneal nerve fiber length were observed. A comprehensive score was assigned to each eye based on clinical findings and in vivo imaging biomarkers. LSCD was mild, moderate, and severe in 5, 11, and 8 eyes, respectively. A history of misdiagnosis was found in 20 eyes (80.0%). After discontinuing the use of contact lenses and receiving medical treatment, significant improvement was observed in all eyes, with 13 eyes fully recovered. Conclusions: The symptoms and clinical signs of CL-iLSCD can be subtle at the early stage. Discontinuing contact lens wear and medication are effective to treat CL-iLSCD.

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.

Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing.

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

A New Technique for Harvesting Limbal Stem Cell Tissue for Transplantation Using an Automated Microkeratome and a Novel Globe-Fixation System.

PURPOSE: To introduce an unprecedented technique, instrumentation, and setup for the superficial limbus harvest from the human cadaver donor whole globe. METHODS: We studied several superficial limbus harvest alternatives, developed a preferred approach with our available instruments, and optimized it on a handful of (seropositive) cadaver donors of whole globes. RESULTS/TECHNIQUE: The globe was pressurized to about normal intraocular pressure by viscoelastic injection through the optic nerve stump. The globe was then mounted on a dynamic globe fixator that maintained a negative pressure in its stabilizing socket. Exertion of the negative pressure effectively elevated globe intraocular pressure (to over 40 mm Hg) and made the corneoscleral wall tight. The socket was then held tilted to the left side for about 35 to 40 degrees to put the limbal zone horizontally and mildly "chin-up." Next, the microkeratome was put on the uppermost and exposed limbus of the globe and activated, and the socket was rotated clockwise under the microkeratome head and its oscillating blade, allowing an effective 360-degree revolution of the microkeratome head around the limbal belt (for a right-handed operator and a counterclockwise cut). CONCLUSIONS: We consistently succeeded in peeling intact 360-degree strips of the smooth superficial limbus by using blades with varying depths. Our method can be further equipped and optimized and be used by the eye banks and the surgeons for keratolimbal grafting as a more efficient limbal stem cell tissue harvest technique.

Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA sequencing.

A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development of hPSC-based therapeutics. In this study, single-cell RNA sequencing (scRNA-seq) was performed to decipher the heterogeneity during differentiation of three hPSC lines toward corneal limbal stem cells (LSCs). The scRNA-seq data revealed nine clusters encompassing the entire differentiation process, among which five followed the anticipated differentiation path of LSCs. The remaining four clusters were previously undescribed cell states that were annotated as either mesodermal-like or undifferentiated subpopulations, and their prevalence was hPSC line dependent. Distinct cluster-specific marker genes identified in this study were confirmed by immunofluorescence analysis and employed to purify hPSC-derived LSCs, which effectively minimized the variation in the line-dependent differentiation efficiency. In summary, scRNA-seq offered molecular insights into the heterogeneity of hPSC-LSC differentiation, allowing a data-driven strategy for consistent and robust generation of LSCs, essential for future advancement toward clinical translation.