Latent diffusion augmentation enhances deep learning analysis of neuro-morphology in limbal stem cell deficiency.

Introduction: Limbal Stem Cell Deficiency (LSCD) is a blinding corneal disease characterized by the loss of function or deficiency in adult stem cells located at the junction between the cornea and the sclera (i.e., the limbus), namely the limbal stem cells (LSCs). Recent advances in in vivo imaging technology have improved disease diagnosis and staging to quantify several biomarkers of in vivo LSC function including epithelial thickness measured by anterior segment optical coherence tomography, and basal epithelial cell density and subbasal nerve plexus by in vivo confocal microscopy. A decrease in central corneal sub-basal nerve density and nerve fiber and branching number has been shown to correlate with the severity of the disease in parallel with increased nerve tortuosity. Yet, image acquisition and manual quantification require a high level of expertise and are time-consuming. Manual quantification presents inevitable interobserver variability. Methods: The current study employs a novel deep learning approach to classify neuron morphology in various LSCD stages and healthy controls, by integrating images created through latent diffusion augmentation. The proposed model, a residual U-Net, is based in part on the InceptionResNetV2 transfer learning model. Results: Deep learning was able to determine fiber number, branching, and fiber length with high accuracy (R2 of 0.63, 0.63, and 0.80, respectively). The model trained on images generated through latent diffusion on average outperformed the same model when trained on solely original images. The model was also able to detect LSCD with an AUC of 0.867, which showed slightly higher performance compared to classification using manually assessed metrics. Discussion: The results suggest that utilizing latent diffusion to supplement training data may be effective in bolstering model performance. The results of the model emphasize the ability as well as the shortcomings of this novel deep learning approach to predict various nerve morphology metrics as well as LSCD disease severity.

Wnt activation as a potential therapeutic approach to treat partial limbal stem cell deficiency.

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.

Ocular Surface Regeneration by Limbal Stem Cells Therapies: State of the Art, Challenges, and Perspectives.

Limbal stem cells (LSCs) are adult stem cells located at the limbus ensuring the continuous renewal of the corneal epithelium, critical to maintain an optimal visual function. Damages to the LSCs or their niche microenvironment lead to limbal stem cell deficiency (LSCD), a potentially blinding disease. Transplantation of LSCs as a treatment for severe to total LSCD has gained popularity since 1980s, owing to the clinical success of the first direct limbal autograft transplantation. Recent advances in the understanding of the LSCs' molecular identity and regulation have enabled preclinical and clinical advancements of promising LSCs therapies. However, lack of standardization of the diagnostic methods, staging of the disease severity, manufacturing process, and clinical outcome measures have hindered the advancement of the therapy. To move these therapies to the clinic, optimization and standardization of the diagnostic strategy, cell product manufacturing, and assessment of clinical efficacy with potency assays are key points to the development of customized therapies. Recent findings suggest that residual LSCs exist in eyes presenting with clinical signs of total LSCD, which opens new therapeutic strategies for eyes with partial LSCD. Prospective, randomized, multicentric controlled clinical trials are necessary to determine the efficacy of different LSCs therapies for different stages of LSCD using a set of standardized outcome measures.

Descemet membrane endothelial keratoplasty in eyes with glaucoma.

Endothelial keratoplasty has become the standard for the treatment of endothelial dysfunction. In Descemet membrane endothelial keratoplasty (DMEK), only the endothelium and Descemet membrane are transplanted, providing superior outcomes compared to Descemet stripping endothelial keratoplasty (DSEK). A substantial subset of patients who require DMEK have comorbid glaucoma. Even in eyes with complex anterior segment such as eyes with previous trabeculectomy or tube shunts, DMEK can restore meaningful vision and outperforms DSEK in terms of visual recovery, decreased rejection rate, and the need for high dose of topical steroids. However, accelerated endothelial cell loss and secondary graft failure have been described in eyes with previous glaucoma surgery, namely trabeculectomy and drainage device. During DMEK and DSEK procedures, raised intraocular pressure is required to attach the graft, which could worsen preexisting glaucoma or cause de novo glaucoma. Mechanisms of postoperative ocular hypertension include delayed air clearance, pupillary block, steroid response, and damage to angle structures. Medically treated glaucoma has increased risk for postoperative ocular hypertension. By understanding these additional complications and making appropriate modifications in surgical techniques and postoperative management, DMEK can be performed successfully and achieve very good visual outcome in eyes with glaucoma. Such modifications include precisely controlled unfolding technique, iridectomies that can help avoid pupillary block, tube shunts that can be trimmed to facilitate graft unfolding, air fill tension that can be adjusted, and postoperative steroid regimens that can be modified to decrease the risk for steroid response. Long-term survival of the DMEK graft, however, is shorter in eyes with previous glaucoma surgery than those without, as observed after other types of keratoplasty.

Future regenerative therapies for corneal disease.

PURPOSE OF REVIEW: To highlight the progress and future direction of stem-cell based regenerative therapies for the treatment of corneal disease. RECENT FINDINGS: Corneal stem cell-based therapies, such as limbal stem cell transplantation, corneal stromal stem cell transplantation, endothelial stem cell transplantation, and stem cell-derived extracellular vesicles have demonstrated promising results in the laboratory. Although most are still in preclinical development or early phase clinical trials, these stem cell-based therapies hold potential to facilitate tissue regeneration, restore native function, and inhibit pathologic disease processes such as fibrosis, inflammation, and neovascularization. SUMMARY: Stem cell-based therapy offers a promising therapeutic option that can circumvent several of the challenges and limitations of traditional surgical treatment. This concise review summarizes the progress in stem-cell based therapies for corneal diseases along with their history, underlying mechanisms, limitations, and future areas for development.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

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.

Clinical outcomes and complications of fluid-filled scleral lens devices for the management of limbal stem cell deficiency.

AIMS: To evaluate the clinical and visual outcomes of fluid-filled scleral lens devices (SL) wear in patients with limbal stem cell deficiency (LSCD). DESIGN: Retrospective consecutive case series. METHODS: 27 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. RESULTS: Baseline 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. CONCLUSION: SL 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.

Stability and Function of Extracellular Vesicles Derived from Immortalized Human Corneal Stromal Stem Cells: A Proof of Concept Study.

With significant advancement and development of extracellular vesicle (EV)-based therapies, there is a growing need to understand how their storage affects their physical and functional characteristics. EVs were isolated from the conditioned medium of a corneal stromal stem cell line (imCSSC) using Total Exosome isolation kit (TEI) and ultracentrifugation (UC) combined protocol. Purified EVs were stored at 4°C, - 80°C, room temperature (RT) after lyophilization with or without trehalose for 4 weeks. EVs stored at - 80°C and RT (lyophilization with trehalose) demonstrated a comparable morphology, while the freeze-dried samples without trehalose showed aggregation and degradation under a transmission electron microscope (TEM). Lyophilized samples without trehalose demonstrated a decreased particle concentration, recovery rate and protein concentration, which was remediated by the addition of trehalose. EVs stored at - 80℃ showed no change in the protein expression of CD9, CD63, and CD81. Regardless of the storage condition, all EV samples investigated reduced inflammation, as well as inhibited expression of fibrotic markers in vitro. Lyophilization of EVs with trehalose was a feasible storage method that retained the physical property and in vitro biological activities of EVs after 4 weeks of storage, while - 80°C offered the best retention of imCSSC-derived EV physical properties. For the first time, this data demonstrated a practical and translatable method for the storage of CSSC-derived EVs for clinical use.

Generation of Functional Immortalized Human Corneal Stromal Stem Cells.

In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.

650 GHz imaging as alignment verification for millimeter wave corneal reflectometry.

A system concept for online alignment verification of millimeter-wave, corneal reflectometry is presented. The system utilizes beam scanning to generate magnitude-only reflectivity maps of the cornea at 650 GHz and compares these images to a precomputed/measured template map to confirm/reject sufficient alignment. A system utilizing 5 off-axis parabolic mirrors, a thin film beam splitter, and 2-axis galvanometric mirror was designed, simulated, and evaluated with geometric and physical optics. Simulation results informed the construction of a demonstrator system which was tested with a reference reflector. Similarity metrics computed with the aligned template and 26 misaligned positions, distributed on a 0.5 mm x 0.5 mm x 0.5 mm mesh, demonstrated sufficient misalignment detection sensitivity in 23 out of 26 positions. The results show that positional accuracy on the order of 0.5 mm is possible using 0.462 mm wavelength radiation due to the perturbation of coupling efficiency via beam distortion and beam walk-off.

Infectious Keratitis Isolates and Susceptibility in Southern California.

PURPOSE: The purpose of this study was to determine longitudinal trends in prevalence and resistance profiles for infectious keratitis at referral centers in Southern California. METHODS: Cultured infectious keratitis cases from January 1, 2006, through December 31, 2009, and January 1, 2016, through December 31, 2019, at the University of California, Los Angeles, were evaluated. Outcome measures included microbial isolate prevalence and antibiotic/antifungal susceptibility and resistance patterns. RESULTS: One hundred thirty-nine and 315 culture-positive isolates were obtained between 2006-2009 and 2016-2019, respectively. Gram-positive organisms accounted for 65% (2006-2009) and 74% (2016-2019) of bacterial isolates ( P = 0.076). Staphylococcus infections, the most common gram-positive and bacterial isolate in both study epochs, demonstrated increased prevalence from 2006-2009 to 2016-2019 (41% vs. 53%, P = 0.019). Although coagulase-negative Staphylococcus (CoNS) increased from 40% to 58% ( P = 0.0012), the prevalence of methicillin-resistant Staphylococcus aureus was unchanged (28% vs. 28%, P = 0.99). Pseudomonas aeruginosa , the most common gram-negative organism, demonstrated decreased prevalence from 18% to 10% ( P = 0.027). Candida species comprised 3.5% of culture-positive isolates in both epochs. All gram-positive isolates were susceptible to vancomycin, and all Staphylococcus isolates were susceptible to linezolid. Pseudomonas aeruginosa remained susceptible to tested fluoroquinolones (>93%) and aminoglycosides (100%) over time. CONCLUSIONS: In southern California between 2006 and 2019, there was a shift toward Staphylococcus species, with increased CoNS, decreased methicillin-sensitive Staphylococcus aureus , and decreased prevalence of P. aeruginosa . Empiric therapy of vancomycin and a fluoroquinolone or aminoglycoside provides effective antibacterial coverage for predominant bacterial species when culture sensitivities are pending.

R-etodolac is a more potent Wnt signaling inhibitor than enantiomer, S-etodolac.

Etodolac is an FDA-approved nonsteroidal anti-inflammatory drug (NSAID) used to treat a variety of inflammatory diseases. The drug is administered as a racemate (50/50 mixture of R- and S- enantiomers), however, studies have shown that the two enantiomers have distinct biologic and pharmacokinetic differences. Wnt signaling, which plays key roles in cell proliferation, polarity, and differentiation, has been shown to be inhibited by R-etodolac; however, comparative analyses of R- and S-etodolac in this function have not been conducted. We used in silico molecular docking and TOPflash functional biologic assays to compare R- and S-enantiomers effect on Wnt signaling inhibition. Further, we used a cultivated limbal stem epithelial cell (cLSCs) model to investigate enantiospecific changes in the colony-forming efficiency (CFE) of cLSCs. The data shows that R-etodolac is a more potent inhibitor of Wnt signaling. In addition, consistently, while both enantiomers demonstrate a dose-dependent decrease in CFE of cLSCs, R-etodolac is a more potent inhibitor.

Rapid 3D bioprinting of a multicellular model recapitulating pterygium microenvironment.

Pterygium is an ocular surface disorder with high prevalence that can lead to vision impairment. As a pathological outgrowth of conjunctiva, pterygium involves neovascularization and chronic inflammation. Here, we developed a 3D multicellular in vitro pterygium model using a digital light processing (DLP)-based 3D bioprinting platform with human conjunctival stem cells (hCjSCs). A novel feeder-free culture system was adopted and efficiently expanded the primary hCjSCs with homogeneity, stemness and differentiation potency. The DLP-based 3D bioprinting method was able to fabricate hydrogel scaffolds that support the viability and biological integrity of the encapsulated hCjSCs. The bioprinted 3D pterygium model consisted of hCjSCs, immune cells, and vascular cells to recapitulate the disease microenvironment. Transcriptomic analysis using RNA sequencing (RNA-seq) identified a distinct profile correlated to inflammation response, angiogenesis, and epithelial mesenchymal transition in the bioprinted 3D pterygium model. In addition, the pterygium signatures and disease relevance of the bioprinted model were validated with the public RNA-seq data from patient-derived pterygium tissues. By integrating the stem cell technology with 3D bioprinting, this is the first reported 3D in vitro disease model for pterygium that can be utilized for future studies towards personalized medicine and drug screening.

Cytokeratin 13 Is a New Biomarker for the Diagnosis of Limbal Stem Cell Deficiency.

PURPOSE: The purpose of this study was to evaluate the expression of cytokeratin (K) 13 on the corneal surface and to validate its application in the diagnosis of limbal stem cell deficiency (LSCD). METHODS: This prospective comparative study included 26 corneal impression cytology (IC) specimens from patients diagnosed with LSCD. Twenty-three IC specimens from normal donors served as controls. K12 and K13 expression were detected on the IC specimens by immunohistochemistry study. The number of K12 + or K13 + cells in all areas of the IC was quantified using ImageJ software. RESULTS: The epithelial cells harvested from IC specimens from control corneas were all K12 + . In eyes with LSCD, K13 + and K12 + /K13 + cells accounted for 93.8% and 2.6%, respectively, in the cornea. In eyes with sectoral LSCD, the median number of K13 + cells in the clinically affected area was higher than that in the unaffected area (810.0 vs. 115.0 cells/mm 2 ; P < 0.001). No significant correlation was found between the LSCD severity and the number of K12 + cells (r = -0.284, P = 0.16) or K13 + cells (r = -0.011, P = 0.95). The presence of at least 16 K13 + cells/mm 2 was suggestive of LSCD. CONCLUSIONS: Identification of K13 + cells on IC specimens provides a simple and reliable method to detect conjunctival epithelial cells on the cornea. K13 is a marker for diagnosing LSCD and localizing the involved area in sectoral LSCD.

Cell Morphology as an In Vivo Parameter for the Diagnosis of Limbal Stem Cell Deficiency.

PURPOSE: The aim of this study was to investigate basal epithelial cell morphology (CM) in the central cornea and limbal areas of eyes with limbal stem cell deficiency (LSCD). METHODS: This was a prospective, cross-sectional comparative study. We developed a CM scoring system based on basal epithelial cell phenotypes graded from 0 (normal) to 3 (severe morphologic alterations); this system was evaluated by 2 independent masked observers. The CM score was compared with the LSCD clinical score, mean best-corrected visual acuity, and in vivo laser scanning confocal microscopy parameters used to stage LSCD (ie, basal epithelial cell density, basal epithelial thickness, and subbasal corneal nerve fiber length density). RESULTS: One hundred sixty-eight eyes with LSCD and 63 normal eyes were included. Compared with the control group, the LSCD group had significantly higher mean (±SD) CM scores in the central cornea (1.8 ± 0.7 vs. 0.5 ± 0.4, respectively; P = 0.01) and limbal areas (1.6 ± 0.2 vs. 1.3 ± 0.0, respectively; P < 0.05). The mean CM score in the central cornea was positively correlated with the clinical score ( P < 0.01, r = 0.66) and negatively correlated with the best-corrected visual acuity ( P < 0.01, r = 0.42). The CM scores were positively correlated with all other in vivo laser scanning confocal microscopy parameters in the central cornea and limbal areas (all P < 0.001). CONCLUSIONS: Basal epithelial CM is altered in the central cornea and limbus of eyes with LSCD and thus can be used to stage the clinical severity of the disease.

Biomarkers of in vivo limbal stem cell function.

PURPOSE: To evaluate in vivo parameters as biomarkers of limbal stem cell function and to establish an objective system that detects and stage limbal stem cell deficiency (LSCD). METHODS: A total of 126 patients (172 eyes) with LSCD and 67 normal subjects (99 eyes) were included in this observational cross-sectional comparative study. Slit-lamp biomicroscopy, in vivo laser scanning confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) were performed to obtain the following: clinical score, cell morphology score, basal cell density (BCD), central corneal epithelial thickness (CET), limbal epithelial thickness (LET), total corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), and tortuosity coefficient. Their potential correlations with the severity of LSCD were investigated, and cutoff values were determined. RESULTS: An increase clinical score correlated with a decrease in central cornea BCD, limbal BCD, CET, mean LET, maximum LET, CNFL, CNFD, CNBD, and tortuosity coefficient. Regression analyses showed that central cornea BCD, CET and CNFL were the best parameters to differentiate LSCD from normal eyes (Coef = 3.123, 3.379, and 2.223; all p < 0.05). The rank correlation analysis showed a similar outcome between the clinical scores and the central cornea BCD (ρ = 0.79), CET (ρ = 0.82), and CNFL (ρ = 0.71). A comprehensive LSCD grading formula based on a combination of these parameters was established. CONCLUSIONS: A comprehensive staging system combining clinical presentation, central cornea BCD, CET, and CNFL is established to accurately and objectively diagnose LSCD and stage its severity.

Regulation of Limbal Epithelial Stem Cells: Importance of the Niche.

Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFß. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.

Wnt6 plays a complex role in maintaining human limbal stem/progenitor cells.

The corneal epithelium is consistently regenerated by limbal stem/progenitor cells (LSCs), a very small population of adult stem cells residing in the limbus. Several Wnt ligands, including Wnt6, are preferentially expressed in the limbus. To investigate the role of Wnt6 in regulating proliferation and maintenance of human LSCs in an in vitro LSC expansion setting, we generated NIH-3T3 feeder cells to overexpress different levels of Wnt6. Characterization of LSCs cultured on Wnt6 expressing 3T3 cells showed that high level of Wnt6 increased proliferation of LSCs. Medium and high levels of Wnt6 also increased the percentage of small cells (diameter ≤ 12 µm), a feature of the stem cell population. Additionally, the percentage of cells expressing the differentiation marker K12 was significantly reduced in the presence of medium and high Wnt6 levels. Although Wnt6 is mostly known as a canonical Wnt ligand, our data showed that canonical and non-canonical Wnt signaling pathways were activated in the Wnt6-supplemented LSC cultures, a finding suggesting that interrelationships between both pathways are required for LSC regulation.