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Purpose: Corneal epithelial defects from trauma or surgery heal as new epithelial cells grow centripetally from the limbus and replenish the epithelium. Corneal wound healing requires cell signalling molecules. However, a topical treatment with these components is not available. Human breast milk (HBM) offers a potential, novel treatment as it contains bioactive molecules important in epithelial cell healing. This study seeks to investigate the potential of HBM in cornea wound healing. Methods: Balb/c mice, 8–12 weeks old, were anesthetized prior to creating a 2 mm central cornea epithelial defect. Mice were randomly assigned to a treatment group: HBM, ophthalmic ointment containing neomycin, polymyxin B, dexamethasone (RxTx), or saline and treated 4x/day for 2 days. Wound area was quantified by fluorescein and ImageJ at 0, 8, 24, and 48 h post wounding and eyes used for histology, RT-qPCR, and ELISA. Results: Wounded corneas treated with HBM demonstrated increased re-epithelialization at 8 h post injury compared to saline treatments. ELISA showed significantly higher Ki67 in HBM treated eyes vs. saline control at 8 h (p = 0.0278). Additionally, immunohistology revealed more Ki67 positive cells in the HBM group compared to saline at 8 h and 24 h (p = 0.0063 8 h; p = 0.0007 24 h). For inflammatory analysis, HBM group IL-1β levels were similar to the saline group, and higher than RxTx treated eyes (p < 0.05). Immunohistochemical staining for CD11b (macrophage marker) revealed HBM-treated eyes had significantly more positive cells vs. saline. RT-qPCR of limbal stem cell markers (LESCs) revealed upregulation of Integrin αV at 8 h with HBM vs. saline. Conclusions: HBM treatment on corneas with debridement of epithelium demonstrated improved healing, cellular proliferation, and upregulation of the LESC gene transcript, integrin αV, after wounding. Future studies could investigate LESC response to different signalling molecules in HBM to better understand the efficacy of this potential therapy. [ABSTRACT FROM AUTHOR]

Introduction: In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. Methods: Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. Results: In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). Conclusions: The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media. [ABSTRACT FROM AUTHOR]

In this article, site‐specific functionalized keratoprosthesis (KPro) was accomplished to meet epithelial on‐growth on the anterior optical surface, inhibit cell adhesion on the posterior optic surface, and promote cell ingrowth at the peripheral tissue‐haptic‐flange interface toward tissue integration. Gelatin immobilization on PC4 hydrogel surfaces was achieved through acid hydrolysis followed by EDC/NHS chemistry, and polyethylene glycol‐diacrylate (PEGDA)‐modified inter‐penetrating network (IPN) surfaces were prepared using photo‐polymerization of ethylene glycol‐diacrylate (EGDA) for enhancing and inhibiting cellular growth, respectively. The surface chemical structures of modified hydrogels were characterized using attenuated total reflectance‐infrared (ATR‐IR) spectroscopy and a bicinchoninic acid (BCA) protein adsorption assay. Further, suitable differentially functionalized surfaces were prepared and characterized using the universal testing machine (UTM), refractometer, atomic force microscopy (AFM), and contact angle measurements to understand mechanical, optical, surface morphological, and wettability properties, respectively. Finally, in vitro site‐specific cell adhesion, cell inhibition, and cell ingrowth functionalities were tested using isolated goat corneal limbal epithelial stem cells and corneal stromal fibroblast cells, respectively. Overall, the two connective articles represent a successful endeavor to develop a more esthetically pleasing, mechanically stable, tissue‐free hydrogel KPro with site‐specific surface functionality nearly mimicking the cornea's functionality. A further pre‐clinical investigation is necessary to advance this functional novel KPro to reality, especially the tissue integration aspect. [ABSTRACT FROM AUTHOR]

The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self‐renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re‐epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non‐human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment. [ABSTRACT FROM AUTHOR]

Introduction: Conjunctival and limbal stem cell deficiency (LSCD) cause decompensation of the ocular surface. Besides causing poor vision and chronic pain, these conditions remain a challenge to rehabilitate. Areas covered: The goal of this review was to describe surgical techniques to aid in reconstructing the ocular surface. Acute treatments (i.e. amniotic membrane transplantation, tenonplasty) are intended to minimize inflammatory damage and prevent sequelae. Partial LSCD may be initially treated with a sectorial conjunctival epitheliectomy or ipsilateral limbal translocation. Total/near-total LSCD often requires an ocular surface stem cell transplantation (OSST) to restore the ocular surface. A mucous membrane graft or other tissue graft can help reconstruct the fornix and address dense symblephara/ankyloblephara prior to OSST. Expert opinion: There has been a burgeoning of different variations on the main OSST types with a trend toward combined procedures, harvesting smaller amounts of either limbal or mucosal tissue, and utilizing autologous non-limbal stem cell (i.e. oral mucosal conjunctival) tissue on the corneal surface. We are cautiously optimistic as we await intermediate-term and long-term results to be published. While we note resistance among corneal specialists to perform these treatments, we hope that increased exposure in training and knowledge of these procedures' success will increase adoption. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions This study aims to characterize the clinical outcomes after allogeneic simple limbal epithelial transplantation (alloSLET) utilizing tissue from cadaveric donor eyes to address persistent corneal epithelial defects caused by limbal stem cell deficiency.We conducted a retrospective analysis of medical records from 20 patients, encompassing 24 eyes, who underwent alloSLET at least 2 years prior. The primary endpoint was the achievement of complete epithelialization of the corneal surface by corneal epithelium. Secondary endpoints included corrected distance visual acuity (CDVA) and postoperative adverse events.The median postoperative follow‐up period was 36 months (range, 24–74 months). At 1, 3 and 6 months post‐surgery, 96% of eyes demonstrated epithelialized corneal surfaces, which declined to 71% at 12 months, to 54% at 24 and 36 months after surgery, and to 50% thereafter. There were no significant differences in graft survival between alloSLET performed alone versus in combination with penetrating keratoplasty. However, instances of graft failure were associated with postoperative elevated intraocular pressure (IOP) and a history of multiple amniotic membrane and corneal graft transplants.AlloSLET emerges as a viable mid‐term intervention for limbal stem cell deficiency‐associated non‐healing corneal epithelial defects in the absence of autologous limbal tissue. Our findings underscore the increased risk of graft failure in patients with elevated IOP and a background of multiple previous amniotic membrane and corneal graft procedures. [ABSTRACT FROM AUTHOR]

Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5− cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues. [ABSTRACT FROM AUTHOR]

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). 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. 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. 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. Proprietary or commercial disclosure may be found after the references. [ABSTRACT FROM AUTHOR]

Corneal injury is one of the substantial and challenging global concerns regarding vision loss. Corneal transplantation is the gold standard for restoring vision to a blurred cornea; however, suture-related complications, corneal haziness, and graft failure have led to ongoing research to improve graft efficiency. In this study, a highly suture-able hybrid corneal scaffold composed of decellularized corneas injected with GelMa/PEGDA in situ was fabricated to preserve the structural integrity and transparency of the injured corneas. First, GelMA/PEGDA hydrogels with different ratios (3/7, 1/1, and 7/3) were evaluated in terms of microstructure, degradation rate, cytocompatibility, and equilibrium water content. GelMa/PEGDA 7/3 was chosen based on the cytocompatibility assay, which confirmed the attachment, maintenance, and proliferation of limbal stem cells with an elongated morphology. Next, the explanted rabbit cornea was decellularized successfully to preserve its extracellular components and transparency with less than 1% DNA content. Thereafter, a hybrid cornea was developed by incorporating GelMA/PEGDA hydrogel (7/3) into the decellularized rabbit cornea under ultraviolet light. This hybrid cornea showed improved sutureability and possessed appropriate transparency. In addition, ex vivo examinations of the enucleated eyeballs of rabbits showed that this hybrid cornea effectively adhered to the corneal stromal and surrounding tissues by applying GelMA/PEGDA hydrogel, which is favorable for corneal transplantations. Based on these findings, the introduced hybrid cornea could be a potential candidate for the in situ repair of deep anterior corneal defects. [ABSTRACT FROM AUTHOR]

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their niche involving numerous signaling pathways, such as Wnt. Wnt proteins are secreted morphogens that play critical roles in embryonic development, stem cell proliferation, self-renewal, tissue regeneration, and remodeling in adults. It has been shown that a small molecule Wnt mimic could improve LSCs expansion ex vivo. Damage to the LSCs and/or their niche can lead to limbal stem cell deficiency (LSCD), a condition that can cause corneal blindness and is difficult to treat. This study explored if repopulating residual LSCs in partial LSCD through Wnt activation could be a novel therapeutic approach. To mimic LSCD due to a chemical injury, single cultured LSCs were exposed to various concentrations of sodium hydroxide. A progressive loss of the LSCs phenotype was observed: the percentage of p63bright cells and cytokeratin (K)14+ cells decreased while the percentage of K12+ increased. Wnt activation was attained by treating the LSCs with lithium chloride (LiCl) and a small-molecule Wnt mimic, respectively. After 18 h of treatment, LSCs proliferation was increased, and the LSCs phenotype was recovered, while the untreated cells did not proliferate and lost their phenotype. The percentage of p63bright cells was significantly higher in the Wnt mimic-treated cells compared with untreated cells, while the percentage of K12+ cells was significantly lower. These findings suggest that local Wnt activation may rescue LSCs upon alkaline injury.

Abstract 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.

The cornea is the outermost layer of the eye and plays an essential role in our visual system. Limbal epithelial stem cells (LESCs), which are localized to a highly regulated limbal niche, are the master conductors of corneal epithelial regeneration. Damage to LESCs and their niche may result in limbal stem cell deficiency (LSCD), a disease confused ophthalmologists so many years and can lead to corneal conjunctivalization, neovascularization, and even blindness. How to restore the LESCs function is the hot topic for ocular scientists and clinicians around the world. This review introduced LESCs and the niche microenvironment, outlined various techniques for isolating and culturing LESCs used in LSCD research, presented common diseases that cause LSCD, and provided a comprehensive overview of both the diagnosis and multiple treatments for LSCD from basic research to clinical therapies, especially the emerging cell therapies based on various stem cell sources. In addition, we also innovatively concluded the latest strategies in recent years, including exogenous drugs, tissue engineering, nanotechnology, exosome and gene therapy, as well as the ongoing clinical trials for treating LSCD in recent five years. Finally, we highlighted challenges from bench to bedside in LSCD and discussed cutting-edge areas in LSCD therapeutic research. We hope that this review could pave the way for future research and translation on treating LSCD, a crucial step in the field of ocular health. [ABSTRACT FROM AUTHOR]

Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders. [ABSTRACT FROM AUTHOR]

Belantamab mafodotin is a new therapy for advanced multiple myeloma that consists of a fusion of monoclonal antibodies and a chemotherapeutic drug. One potential side effect of this therapy is the development of microcyst-like epithelial changes (MECs) in the cornea, which can cause blurred vision and dry eye syndrome. Around 70% of patients treated with Belantamab develop MECs, but these changes are typically reversible. Close collaboration between oncologists and ophthalmologists is necessary to monitor and manage these corneal changes. The mechanism behind the development of MECs is not fully understood, but it may involve the uptake of the drug by corneal cells and the induction of apoptosis. This case report describes the dynamics of MECs in a patient receiving Belantamab therapy and highlights the importance of individual factors in the duration and resolution of these changes. It also discusses the implications for clinical management and the need for ophthalmological monitoring in patients receiving antibody-drug conjugate therapy. [Extracted from the article]

Deficits in corneal innervation lead to neurotrophic keratopathy (NK). NK is frequently associated with facial palsy, and corneal damage can be accelerated by facial palsy deficits. Corneal nerves are important regulators of limbal stem cells, which play a critical role in epithelial maintenance and healing. Nonsurgical treatments of NK have undergone recent innovation, and growth factors implicated in corneal epithelial renewal are a promising therapeutic avenue. However, surgical intervention with corneal neurotization (CN) remains the only definitive treatment of NK. CN involves the transfer of unaffected sensory donor nerve branches to the affected cornea, and a variety of donor nerves and approaches have been described. CN can be performed in a direct or indirect manner; employ the supraorbital, supratrochlear, infraorbital, or great auricular nerves; and utilize autograft, allograft, or nerve transfer alone. Unfortunately, comparative studies of these factors are limited due to the procedure's novelty and varied recovery timelines after CN. Regardless of the chosen approach, CN has been shown to be a safe and effective procedure to restore corneal sensation and improve visual acuity in patients with NK. [ABSTRACT FROM AUTHOR]

Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin–eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype. [ABSTRACT FROM AUTHOR]

Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive eye disease. GDLD is characterized by the loss of barrier function in corneal epithelial cells (CECs) and amyloid deposition due to pathogenic variants in the TACSTD2 gene. Limbal stem cell transplantation (LSCT) has been suggested as an effective therapeutic alternative for patients with GDLD. However, despite LSCT, amyloid deposition recurs in some patients. The pathogenesis of recurrence is poorly studied. We present the case of a patient with GDLD. Genetic analysis revealed a homozygous deletion, NM_002353.3:c.653del, in the TACSTD2 gene. Functional analysis in a cell model system revealed the loss of the transmembrane domain and subcellular protein mislocalization. The patient with GDLD underwent direct allogeneic LSCT with epithelial debridement followed by deep anterior lamellar keratoplasty 10 months later due to amyloid deposition and deterioration of vision. Taken together, the results of transcriptome analysis and immunofluorescence staining of post-LSCT corneal sample with amyloid deposits obtained during keratoplasty demonstrated complete restoration of wild-type TACSTD2 expression, indicating that donor CECs replaced host CECs. Our study provides experimental evidence that amyloid deposition can recur after LSCT despite complete restoration of wild-type TACSTD2 expression. Recurrent amyloid deposition post-LSCT: TACSTD2 gene deletion case study Gelatinous drop-like corneal dystrophy is a rare eye disease passed down through families, causing vision loss due to a protein called amyloid building up in the cornea, the clear front surface of the eye. This study looks into the genes related to GDLD, focusing on a specific gene variant in TACSTD2, which is involved in the disease. Through sequencing of a patient's DNA, the team found a variant in TACSTD2 linked to the protein buildup in GDLD. Results showed that despite successful LSCT, the protein built up again, indicating that replacing damaged cells with healthy ones might not prevent recurrence. The study concludes that the TACSTD2 variant contributes to GDLD by disrupting normal protein function, leading to protein buildup. This insight improves our understanding of GDLD and suggests that future treatments need to address the genetic causes. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Organoid technology provides a versatile platform for simulating organogenesis, investigating disease pathogenesis, and exploring therapeutic interventions. Among various types of organoids that have been developed, corneal limbal organoids, the three-dimensional miniaturized corneas which are derived from either pluripotent stem cells or limbal epithelial stem cells, are particularly promising for clinical translation. This narrative review summarized the state-of-the-art in corneal limbal organoids research including the cultivation methods, clinical relevance and its limitations and challenges. The potential of corneal limbal organoids in mimicking corneal development, disease modelling, drug screening, and regenerative medicine was discussed. Technical improvements in cultivation techniques, imaging modalities, and gene editing tools are anticipated to overcome current limitations and further promote its clinical potential. Despite challenges and difficulties, the development of corneal limbal organoids opens a new era of regenerative medicine and provides a potential source of stem cell replacement therapies for challenging corneal diseases with the establishment of an in vitro corneal limbal organoid bank. [ABSTRACT FROM AUTHOR]

Methods: Herein, we obtained and characterized deltaN p63- and adenosine triphosphate-binding cassette subfamily G member 2-expressing limbal stem cells (LSCs). Chitosan and carboxymethyl chitosan (CTH) were cross-linked to be an in situ thermosensitive hydrogel (ACH), which was printed through fourdimensional (4D) printing to obtain a porous carrier with uniform pore diameter (4D-CTH). Rabbits were injected with alloxan to induce diabetes mellitus (DM). Following this, the LSC-carrying hydrogel was spread on the surface of the cornea of the diabetic rabbits to cure corneal epithelium injury. Results: Compared with the control group (LSCs only), rapid wound healing was observed in rabbits treated with LSC-carrying 4D-CTH. Furthermore, the test group also showed better corneal nerve repair ability. The results indicated the potential of LSC-carrying 4D-CTH in curing corneal epithelium injury. Conclusion: 4D-CTH holds potential as a useful tool for studying regenerative processes occurring during the treatment of various diabetic corneal epithelium pathologies with the use of stem cell-based technologies. [ABSTRACT FROM AUTHOR]

Background/Objectives: Aniridia-associated keratopathy (AAK) is a potentially vision-threatening pathology in congenital aniridia, for which both the underlying etiopathogenesis and effective treatment remain unclear. Methods:This prospective study was conducted to assess and compare the short-term outcome after superficial keratectomy (SK) alone or in a combination with an amniotic membrane transplantation (AMT). Here, 76 eyes were enrolled in 76 patients with grade 4 AAK. In all eyes, in order to assess preoperatively the efficiency of the limbal epithelial stem cells (LESC), the presence of corneal epithelial cells in confocal microscopy was established. The analyses included: best corrected visual acuity (BCVA), the stage of AAK and the number of corneal quadrants involved in corneal neovascularization (CNV). Results: Six months after surgery, the mean BCVA was 0.05 and ranged from 0.002 up to 0.1 in both groups. Improvement in BCVA occurred in 94.29% patients when *SK alone* was performed, and in 92.68% when in combination with AMT. There were no statistically significant differences in the effect of therapy depending on the type of surgery, regarding BCVA, stage of AAK and the number of quadrants with CNV. Conclusions: SK alone is an effective procedure in short outcomes limited to six months for advanced AAK in association with LESC partial efficiency. [ABSTRACT FROM AUTHOR]

AimsTo evaluate the clinical and visual outcomes of fluid-filled scleral lens devices (SL) wear in patients with limbal stem cell deficiency (LSCD).DesignRetrospective consecutive case series.Methods27 eyes with LSCD confirmed by in vivo confocal microscopy at the Stein Eye Institute and fitted with SL were included. Correlations between corrected distance visual acuity (CDVA) and LSCD stage determined by clinical grading were performed between baseline (after the SL fit) and the last follow-up (the time of discontinuation of SL wear or the last visit in eyes in which SL were continued). In a subset of patients that had worsened LSCD while using SL, anterior segment optical coherence tomography (AS-OCT) and anterior segment fluorescein angiogram (AS-FA) were performed.ResultsBaseline LSCD grading was stage I in 12 eyes (44.4%), stage 2 in 12 eyes (44.4%), and stage III in 3 eyes (11.1%). At the last follow-up, CDVA was improved in 7 eyes (25.9%), remained stable in 13 eyes (48.1%) and decreased in 7 eyes (25.9%, P = 0.16). The LSCD stage was improved in 7 eyes (25.9%), remained stable in 8 eyes (29.6%) and worsened in 12 eyes (44.4%, P = 0.10). AS-OCT and AS-FA, performed in 5 eyes, showed limbal compression and delayed fluorescein filling.ConclusionSL can improve visual acuity and maintain the ocular surface in the majority of eyes. Worsening of the ocular surface might be a result of limbal hypoxia. Close monitoring of SL fit is necessary in these compromised eyes.

BackgroundDysfunction or loss of limbal stem cells can result in limbal stem cell deficiency (LSCD), a disease that cause corneal opacity, pain, and loss of vision. Cultivated limbal epithelial transplantation (CLET) can be used to restore stem cell niche homeostasis and replenish the progenitor pool. Transplantation has been reported with high success rate, but there is an unmet need of prognostic markers that correlate with clinical outcomes. To date, the progenitor content in the graft is the only parameter that has been retrospectively linked to success.MethodsIn this study, we investigate extracellular micro RNAs (miRNAs) associated with stem/progenitor cells in cultivated limbal epithelial cells (cLECs). Using micro RNA sequencing and linear regression modelling, we identify a miRNA signature in cultures containing high proportion of stem/progenitor cells. We then develop a robust RNA extraction workflow from culture media to confirm a positive miRNA correlation with stem/progenitor cell proportion.ResultsmiR-6723-5p is associated with cultures containing high proportion of stem/progenitor cells, and is detected in the basal layer of corneal epithelium.ConclusionsThese results indicate that miR-6723-5p could potentially serve as a stem/progenitor cell marker in cLECs.

Background: Corneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6's influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment. Methods: To investigate the PAX6/CXCL14 regulatory axis's role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis's function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis's mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries. Results: Our study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14's receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14's therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models. Conclusion: CCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries. [ABSTRACT FROM AUTHOR]

Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs. [ABSTRACT FROM AUTHOR]

This document provides an update on the management of ocular chemical injuries and limbal stem cell deficiency (LSCD). It discusses the treatment options for both acute and late-stage management of chemical injuries, including irrigation, removal of chemical particles, and the use of topical medications. Surgical techniques such as autografts and allografts are also explored for the treatment of LSCD. Additionally, the document discusses alternative treatments such as cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET), and cultivated oral mucosal epithelial transplantation (COMET). The references provided cover a range of topics related to the treatment of ocular surface disorders and LSCD. [Extracted from the article]

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche. [ABSTRACT FROM AUTHOR]

Corneal diseases are a major cause of vision loss worldwide. Traditional methods like corneal transplants from donors are effective but face challenges like limited donor availability and the risk of graft rejection. Therefore, new treatment methods are essential. This review examines the growing field of bioprinting and biofabrication in corneal tissue engineering. We begin by discussing various bioprinting methods such as stereolithography, inkjet, and extrusion printing, highlighting their strengths and weaknesses for eye-related uses. We also explore how biological tissues are made suitable for bioprinting through a process called decellularization, which can be achieved using chemical, physical, or biological methods. The review then looks at natural materials, known as bioinks, used in bioprinting. We focus on materials like gelatin, collagen, fibrin, chitin, chitosan, silk fibroin, and alginate, examining their mechanical and biological properties. The importance of hydrogel scaffolds, particularly those based on collagen and other materials, is also discussed in the context of repairing corneal tissue. Another key area we cover is the use of stem cells in corneal regeneration. We pay special attention to limbal epithelial stem cells and mesenchymal stromal cells, highlighting their roles in this process. The review concludes with an overview of the latest advancements in corneal tissue bioprinting, from early techniques to advanced methods of delivering stem cells using bioengineered materials. In summary, this review presents the current state and future potential of bioprinting and biofabrication in creating functional corneal tissues, highlighting new developments and ongoing challenges with a view towards restoring vision. [ABSTRACT FROM AUTHOR]

This article provides information on the management of corneal abrasions following intravitreal injection. It discusses the diagnosis, treatment, and follow-up of a patient who experienced a corneal abrasion after the injection. The article also includes education guidelines and learning objectives for understanding corneal abrasions. Additionally, the document includes references related to corneal wound healing, the use of topical analgesics for pain relief, and the efficacy and safety of bandage contact lenses in treating various eye conditions. It serves as a comprehensive resource for library patrons conducting research on these specific topics. [Extracted from the article]

Simple Summary: The corneal epithelium is a protective barrier and refractive structure in the eye maintained through a complex regenerative process involving the lacrimal gland, tear film, and corneal nerves. This review explores the insulin-like growth factor (IGF) system and its role in corneal epithelium homeostasis. Emphasis is placed on the significance of limbal epithelial stem cells and potential therapeutic applications targeting the system components. The corneal epithelium, comprising three layers of cells, represents the outermost portion of the eye and functions as a vital protective barrier while concurrently serving as a critical refractive structure. Maintaining its homeostasis involves a complex regenerative process facilitated by the functions of the lacrimal gland, tear film, and corneal nerves. Crucially, limbal epithelial stem cells located in the limbus (transitional zone between the cornea and the conjunctiva) are instrumental for the corneal epithelium integrity by replenishing and renewing cells. Re-epithelialization failure results in persistent defects, often associated with various ocular conditions including diabetic keratopathy. The insulin-like growth factor (IGF) system is a sophisticated network of insulin and other proteins essential for numerous physiological processes. This review examines its role in maintaining the corneal epithelium homeostasis, with a special focus on the interplay with corneal limbal stem cells and the potential therapeutic applications of the system components. [ABSTRACT FROM AUTHOR]

This document summarizes a study conducted in India to evaluate the effectiveness of conjunctival autograft transplantation in treating primary pterygium. The study included 50 patients and found that most patients experienced improved visual acuity after the surgery. The study also identified a correlation between graft edema and graft stability, as well as between suture loosening and recurrence. The findings contribute to the existing knowledge on pterygium treatment, but further research with larger sample sizes and more experienced surgeons is needed. [Extracted from the article]

The corneal epithelium is renowned for high regenerative potential, which is dependent on the coordinated function of its diverse progenitor subpopulations. However, the molecular pathways governing corneal epithelial progenitor differentiation are incompletely understood. Here, we identify a highly proliferative limbal epithelial progenitor subpopulation characterized by expression of basal cell adhesion molecule (BCAM) that is capable of holocone formation and corneal epithelial sheet generation. BCAM-positive cells can be found among ABCB5-positive limbal stem cells (LSCs) as well as among ABCB5-negative limbal epithelial cell populations. Mechanistically, we show that BCAM is functionally required for cellular migration and differentiation and that its expression is regulated by the transcription factor p63. In aggregate, our study identifies limbal BCAM expression as a marker of highly proliferative corneal epithelial progenitor cells and defines the role of BCAM as a critical molecular mediator of corneal epithelial differentiation.

PurposeMiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing.MethodsOur previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs.ResultsOverexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a.ConclusionOur study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.

Background: Corneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs. Methods: Prepare solutions of chitosan acetate, carboxymethyl chitosan, and β-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections. Results: Using the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01). Conclusion: In summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects. [ABSTRACT FROM AUTHOR]

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5β). Several studies have shown that the ABCB5β homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5β/B6 and ABCB5β/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5β in melanocytes and melanoma. [ABSTRACT FROM AUTHOR]

Corneal stromal stem cells (CSSCs) are of particular interest in regenerative ophthalmology, offering a new therapeutic target for corneal injuries and diseases. This review provides a comprehensive examination of CSSCs, exploring their anatomy, functions, and role in maintaining corneal integrity. Molecular markers, wound healing mechanisms, and potential therapeutic applications are discussed. Global corneal blindness, especially in more resource-limited regions, underscores the need for innovative solutions. Challenges posed by corneal defects, emphasizing the urgent need for advanced therapeutic interventions, are discussed. The review places a spotlight on exosome therapy as a potential therapy. CSSC-derived exosomes exhibit significant potential for modulating inflammation, promoting tissue repair, and addressing corneal transparency. Additionally, the rejuvenation potential of CSSCs through epigenetic reprogramming adds to the evolving regenerative landscape. The imperative for clinical trials and human studies to seamlessly integrate these strategies into practice is emphasized. This points towards a future where CSSC-based therapies, particularly leveraging exosomes, play a central role in diversifying ophthalmic regenerative medicine. [ABSTRACT FROM AUTHOR]

With the development of regenerative medicine in ophthalmology, the identification of cells with high proliferative potential in the limbal area has attracted the attention of ophthalmologists and offered a new option for treatment in clinical practice. Limbal stem cell deficiency (LSCD) is an identified eye disease with a difficult and negative outcome, for which the traditional treatment is keratoplasty. This study sought to evaluate the efficacy of matrix-assisted cell transplantation consisting of in vitro-cultured autologous limbal stem cells (LSCs) and type I collagen for the treatment of LSCD in rabbits. LSCD was induced in 10 rabbits by a combination of mechanical limbectomy and alkali burns. Cells were cultured on a plate for 14 days before being transferred to a collagen-based matrix for another 7 days. Rabbits were divided into two groups as follows: the experimental group (five rabbits) received matrix-assisted cell transplantation, while the control group (five rabbits) received only conservative therapy with anti-inflammatory eye drops. During the postoperative period, all rabbits were examined using slit-lamp biomicroscopy with photo-registration and fluorescent staining, impression cytology and anterior segment optical coherence tomography (AS-OCT). Rabbits were euthanized at 30 and 120 days, and their corneas were processed for histology and immunohistochemistry. As a consequence, rabbits in the experimental group demonstrated the restoration of the corneal epithelium and transparency without epithelial defects. Moreover, goblet cells were absent in the central zone of the corneal epithelium. In conclusion, our new method of treatment enhanced the corneal surface and is an effective method of treatment for LSCD in rabbits. [ABSTRACT FROM AUTHOR]

Background: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans. [ABSTRACT FROM AUTHOR]

Simple Summary: Contact lens (CL)-induced limbal stem cell deficiency (LSCD) is not well-known and is one of the lesser-understood causes of LSCD. With increasing contact lens usage worldwide, its prevalence is bound to increase. This review will critically discuss the underlying risk factors, methods of diagnosis and treatment of CL-induced LSCD. Limbal stem cell deficiency (LSCD) is a pathologic condition caused by the dysfunction and destruction of stem cells, stem cell precursors and limbal cell niche in the corneal epithelium, leading to severe conjunctivalization of the cornea. Etiologies for LSCD span from congenital (aniridia), traumatic (chemical or thermal injuries), autoimmune (Stevens–Johnson syndrome) and iatrogenic disease to contact lens (CL) wear. Of these, CL wear is the least understood and is often a subclinical cause of LSCD. Even with recent advances in LSCD research, limitations persist in establishing the pathogenesis and treatment guidelines for CL-induced LSCD. A literature search was conducted to include original articles containing patients with CL-induced LSCD. This review will critically discuss the complex pathophysiology behind CL-induced LSCD, the underlying risk factors and epidemiology of the disease as well as methods to obtain a diagnosis. Various treatment options will be reviewed based on proposed treatment strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: This article aims to present the corneal tattooing method and how using a tattoo pen machine can improve aesthetic appearance in patients with corneal leukoma. Methods: In this study, 42 patients were evaluated who had no visual potential and who had undergone colored corneal tattooing using an automatic tattoo pen machine for aesthetic purposes. The procedure was conducted according to the principles of the Declaration of Helsinki. The commercially available tattoo ink that has traditionally been used on human skin (brown, green, and black) for years was used for all the patients in this study, and 252 corneal photographs (with a Topcon slit lamp imaging device at 16 magnifications, i.e., 16 ×) taken within the last 2 years were evaluated retrospectively. Red, green, and blue (RGB) and hue, saturation, and lightness (HSL) values of the tattooed areas, such as pupils and iris, in corneal photographs were determined online using the Color Code Finder program. The RGB and HSL values of the pupil and iris were compared before surgery on the first day and first week, first month, third month, and twelfth month after surgery. Results: In the first postoperative month, the mean pupil lightness (L) and iris L values were found to have increased by 10.7% and 5.7%, respectively. Between the first month and the first year, the L value of the mean pupil and that of the iris increased by 1.7% and 5.2%, respectively. The increase in the RGB value of the mean pupil in the first month was statistically significant (p = 0.02). The highest increase in RGB values of the iris was observed in the first week and first month (p = 0.113). This result shows that the majority of fading occurred in the first month. After the first month, the increase in the L value in the black-colored pupil was less than that in the brown- or green-colored iris. These results show that light colors fade faster and more. Conclusion: Esthetically, corneal leukoma causes severe psychological problems. Many patients are unable to use prosthetic contact lenses. Evisceration surgery has many complications, and limbal stem cells are used in evisceration surgery. Corneal tattooing using a tattoo pen machine is an easy, practical, and repeatable method used for aesthetic purposes. Successful results require the use of appropriate methods, ink, and ophthalmologist's experience. All patients in this study had a more aesthetic appearance than the preoperative white eye. Further studies are needed to develop a colored aesthetic tattooing method with a tattoo pen machine. [ABSTRACT FROM AUTHOR]

Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development, and aniridia-associated keratopathy (AAK). This progressive corneal opacification resembles late-onset limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. The factors leading to AAK are not known and defects in native LSC differentiation and/or features leading to ocular surface dysfunction like inflammation and loss of innervation could contribute to development of AAK. Here, we produced induced pluripotent stem cells (hiPSC) from 3 AAK patients and examined whether PAX6 haploinsufficiency affects LSC lineage commitment. During LSC differentiation, characterization of the AAK lines showed lowered PAX6 expression as compared to wild type (WT) controls and expression peak of PAX6 during early phase of differentiation was detected only in the WT hiPSC lines. Whether it reflects developmental regulation remains to be studied further. Nevertheless, the AAK-hiPSCs successfully differentiated toward LSC lineage, in line with the presence of LSCs in young patients before cell loss later in life. In addition, patient-specific LSCs showed similar wound healing capacity as WT cells. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected without clear correlation to AAK. As development and maintenance of corneal epithelium involves an interplay between LSCs and their environment, the AAK-hiPSCs generated here can be further used to study the crosstalk between LSCs and limbal niche including, eg, corneal immune cells, stroma cells, and neurons. Graphical Abstract [ABSTRACT FROM AUTHOR]

Abstract Background Limbal stem/progenitor cells (LSPCs) play a crucial role in maintaining corneal health by regulating epithelial homeostasis. Although PM2.5 is associated with the occurrence of several corneal diseases, its effects on LSPCs are not clearly understood. Methods In this study, we explored the correlation between PM2.5 exposure and human limbal epithelial thickness measured by Fourier-domain Optical Coherence Tomography in the ophthalmologic clinic. Long- and short-term PM2.5 exposed-rat models were established to investigate the changes in LSPCs and the associated mechanisms. Results We found that people living in regions with higher PM2.5 concentrations had thinner limbal epithelium, indicating the loss of LSPCs. In rat models, long-term PM2.5 exposure impairs LSPCs renewal and differentiation, manifesting as corneal epithelial defects and thinner epithelium in the cornea and limbus. However, LSPCs were activated in short-term PM2.5-exposed rat models. RNA sequencing implied that the circadian rhythm in LSPCs was perturbed during PM2.5 exposure. The mRNA level of circadian genes including Per1, Per2, Per3, and Rev-erbα was upregulated in both short- and long-term models, suggesting circadian rhythm was involved in the activation and dysregulation of LSPCs at different stages. PM2.5 also disturbed the limbal microenvironment as evidenced by changes in corneal subbasal nerve fiber density, vascular density and permeability, and immune cell infiltration, which further resulted in the circadian mismatches and dysfunction of LSPCs. Conclusion This study systematically demonstrates that PM2.5 impairs LSPCs and their microenvironment. Moreover, we show that circadian misalignment of LSPCs may be a new mechanism by which PM2.5 induces corneal diseases. Therapeutic options that target circadian rhythm may be viable options for improving LSPC functions and alleviating various PM2.5-associated corneal diseases.

Limbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells (LESCs) which compromises corneal transparency, leading to blindness. It cannot be treated with pharmacological or corneal transplantation interventions, instead a specialized stem cell (SC) therapy is needed to restore eye health and sight. Herein, a native cornea‐derived biomaterial, a by‐product of a laser refractive surgical procedure called small incision lenticule extraction is identified as a new cell delivery matrix. Culture conditions are optimized to facilitate LESC attachment, expansion and stratification, and their identity is immunophenotyped. Using electron microscopy, bio‐constructs display stratification, similar to the architectural and cellular organization of a native mammalian cornea with formation of a basement membrane and an orderly array of collagen fibrils. Neuronal growth and depleted CD45+/CD14+ leukocytes on lenticules are also shown, suggesting that in transplantation experiments, they will re‐innervate and not trigger a host‐mediated immune response. Finally, human lenticules are geometrically customized to successfully fit them over a LSCD murine cornea ex vivo, during which they maintain curvature. The authors are poised to conduced similar studies in live mice using these and other carriers currently used in the clinic to compare SC therapy outcomes. [ABSTRACT FROM AUTHOR]