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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.

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.

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.

The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.

The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.

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.

ObjectiveUsing anterior segment optical coherence tomography (AS-OCT), we investigated the epithelial thickness (ET) of the central cornea and limbal regions in patients with limbal stem cell deficiency (LSCD) as a diagnostic and staging parameter.DesignProspective, cross-sectional study.MethodsThe central corneal epithelium thickness (CET) and maximum limbal epithelium thickness (mLET) were measured in the superior, inferior, nasal, and temporal limbus on AS-OCT images of the normal and eyes with LSCD. CET was obtained by 1-point (OCT-CET1) and 3-point measurement (OCT-CET3). The values of OCT-CET1 and OCT-CET3 were compared to the CET obtained with in vivo confocal microscopy (IVCM-CET).ResultsSixty-eight eyes of 50 patients with LSCD and 52 eyes of 34 normal subjects were included. The mean (±standard deviation) OCT-CET3 was 55.0 ± 3.0 μm (range, 50.6-62.0 μm) in the control group and 41.6 ± 10.8 μm (range, 0-56.3 μm) in the LSCD group (P < .001). OCT-CET3 had a better correlation with IVCM-CET (r = 0.91) than did OCT-CET1 (r = 0.87, P = .001). The degree of reduction in OCT-CET3 increased in more advanced clinical stages of LSCD (all P < .001). The OCT-CET3 cutoff value that suggests LSCD was 46.6 μm. Compared with the control group, the LSCD group had decreases in mLET in all 4 limbal regions (all P < .001). The sensitivity and specificity of OCT-CET3 is the highest among all mLET in detecting LSCD.ConclusionsBoth CET and mLET were thinner in patients with LSCD than in normal subjects. OCT-CET3 appears to be a reliable parameter to confirm LSCD when there is clinical suspicion.

An accurate diagnosis of limbal stem cell deficiency (LSCD) is the premise of an appropriate treatment; however, there is no consensus about the diagnostic criteria for LSCD. We performed a systematic literature search of the peer-reviewed articles on PubMed, Medline, and Ovid to investigate how LSCD was diagnosed before surgical intervention. The methods used to diagnose LSCD included clinical presentation, impression cytology, and in vivo confocal microscopy. Among 131 eligible studies (4054 eyes), 26 studies (459 eyes, 11.3%) did not mention the diagnostic criteria. In the remaining 105 studies, the diagnosis of LSCD was made on the basis of clinical examination alone in 2398 eyes (62.9%), and additional diagnostic tests were used in 1047 (25.8%) eyes. Impression cytology was used in 981 eyes (24.2%), in vivo confocal microscopy was used in 29 eyes (0.7%), and both impression cytology and in vivo confocal microscopy were used in 37 eyes (0.9%). Our findings suggest that only a small portion of patients underwent a diagnostic test to confirm the diagnosis of LSCD. Treating physicians should be aware of the limitations of clinical examination in diagnosing LSCD and perform a diagnostic test whenever possible before surgical intervention.

The application of human amniotic membrane (AM) has a wide spectrum of indications in the treatment of ocular surface disorders. Transplantation of AM has been incorporated routinely as a component of ocular surface reconstruction in a variety of ocular pathologies. The application of human AM can be combined with nearly all types of limbal transplantation in treating limbal stem cell deficiency (LSCD). AM provides support and possible protection to the transplanted limbal tissues and limbal stem cells owing to its mechanical and biological properties, and these properties are thought to enhance the success rate of LSC transplantation. This paper reviews the current literature on the applications of AM in the surgical management of LSCD and summarizes the outcome of different surgical approaches. The current literature contains mostly low-level evidences in supporting the role of AM. The efficacy of AM in LSC transplantation needs to be confirmed by randomized controlled clinical trials.

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

PurposePathologic corneal neovascularization is a major cause of blindness worldwide, and treatment options are currently limited. VEGF is one of the critical mediators of corneal neovascularization but current anti-VEGF therapies have produced limited results in the cornea. Thus, additional therapeutic agents are needed to enhance the antiangiogenic arsenal. Our group previously demonstrated epithelial membrane protein-2 (EMP2) involvement in pathologic angiogenesis in multiple cancer models including breast cancer and glioblastoma. In this paper, we investigate the efficacy of anti-EMP2 immunotherapy in the prevention of corneal neovascularization.MethodsAn in vivo murine cornea alkali burn model was used to study pathologic neovascularization. A unilateral corneal burn was induced using NaOH, and subconjunctival injection of either anti-EMP2 antibody, control antibody, or sterile saline was performed after corneal burn. Neovascularization was clinically scored at 7 days postalkali burn, and eyes were enucleated for histologic analysis and immunostaining including VEGF, CD31, and CD34 expression.ResultsAnti-EMP2 antibody, compared to control antibody or vehicle, significantly reduced neovascularization as measured by clinical score and central cornea thickness, as well as by histologic reduction of neovascularization, decreased CD34 staining, and decreased CD31 staining. Incubation of corneal limbal cells in vitro with anti-EMP2 blocking antibody significantly decreased EMP2 expression, VEGF expression and secretion, and cell migration.ConclusionsThis work demonstrates the effectiveness of EMP2 as a novel target in pathologic corneal neovascularization in an animal model and supports additional investigation into EMP2 antibody blockade as a potential new therapeutic option.

PurposeA chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).MethodsLSCs were isolated from human donor eyes and cultured as single cells for 12 to 14 days with the following small molecules: IIIC3, an antagonist of the Wnt signaling inhibitor Dickkopf (DKK), and IC15, a Wnt signaling inhibitor. Proliferation of LSCs in the presence of IIIC3 and IC15 was determined by the number of cells and colonies established. Maintenance of stemness was determined by p63α, cytokeratin (K)12, and K14 expression.ResultsActivation of Wnt, through IIIC3-mediated DKK inhibition, resulted in similar colony forming efficiency (CFE) as in the untreated LSCs, but significantly increased the number of cultivated cells 7.21% with 5 μM. Inhibition of Wnt with IC15 significantly reduced the CFE (P ≤ 0.01) and the number of cultivated cells by 16% to 29%. Percentage of cells expressing high levels of p63α (p63αbright) and quantity of small cells (≤12 μm), which contain the LSCs, increased 4.71% and 11.26% (both P < 0.05), respectively, with 5 μM IIIC3. All concentrations of IIIC3 and IC15 retained the K14 undifferentiated marker (97%), while differentiation, as detected by expression of K12, was found in up to 2% of cells in 1 μM IIIC3, 1 μM IC15, or 5 μM IIIC3.ConclusionsWnt signaling is required in LSC proliferation and maintenance of an undifferentiated state. The current study is a proof of concept that the Wnt pathway could be modulated in LSCs to enhance or decrease the efficiency of human LSC expansion.

PurposeTo grade the severity of limbal stem cell deficiency (LSCD) based on the extent of clinical presentation and central corneal basal epithelial cell density (BCD).MethodsThis is a retrospective observational comparative study of 48 eyes of 35 patients with LSCD and 9 eyes of 7 normal subjects (controls). Confocal images of the central cornea were acquired. A clinical scoring system was created based on the extent of limbal and corneal surface involvement. LSCD was graded as mild, moderate, and severe stages based on the clinical scores. The degree of BCD reduction was given a score of 0 to 3.ResultsCompared with BCD in controls, BCD decreased by 23.0%, 40.4%, and 69.5% in the mild, moderate, and severe stages of LSCD classified by the clinical scoring system, respectively. The degree of BCD reduction was positively correlated with larger limbal and corneal surface involvement and when the central visual axis was affected (all P ≤ 0.0005). Mean corrected distance visual acuity logarithm of the minimum angle of resolution was 0.0 ± 0.0 in control eyes, 0.2 ± 0.5 in mild LSCD, 0.6 ± 0.4 in moderate LSCD, and 1.6 ± 1.1 in severe LSCD (P < 0.0001). There was a significant correlation between a higher clinical score and corrected distance visual acuity logarithm of the minimum angle of resolution (rho = 0.82; P < 0.0001) and a greater decrease in BCD (rho = -0.78; P < 0.0001).ConclusionsA clinical scoring system was developed to assess the extent of clinical presentation of LSCD. A classification system to grade the severity of LSCD can be established by combining the BCD score with the clinical score.

Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.

To quantify corneoscleral junction (CSJ) topography in soft contact lens (SCL) wearers, and assess the association between the CSJ and SCL performance and subjective comfort, forty-four adapted SCL wearers (16 Asians, 16 Caucasians, 12 Latinos) were recruited for the present study. Corneal topography was taken with a Medmont E300 (Medmont International, Pty Ltd.). CSJ images were taken with a commercial OCT (Bioptigen, Inc.). Our published CSJ image analysis technique was used to describe the geometric properties of the CSJ using the sum of squared orthogonalized residuals (SSRo). Multivariable mixed effects models were employed to examine associations between SSRo and subject demographics, ocular characteristics, SCL fit and performance, and comfort. The SSRo was significantly related to quadrant (p

PurposeTo report a case of corneal neovascularization misdiagnosed as total limbal stem cell deficiency (LSCD).MethodsThis is a case report of a 61-year-old woman who has a history of bilateral idiopathic scleritis, keratitis, and uveitis for more than 20 years. She was diagnosed with total LSCD in her left eye based on clinical presentation alone and was confirmed as a candidate for limbal transplantation at several major tertiary eye care centers in the United States. After referral to the Stein Eye Institute, in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) were performed to clarify the diagnosis.ResultsSlit-lamp examination of the left eye revealed 360-degree severe thinning at the limbus and peripheral corneal pannus and neovascularization that spared the central cornea, a smooth epithelium without fluorescein staining at the central cornea, an uneven surface, and pooling of fluorescein at the peripheral cornea accompanied by minimal fluorescein staining of the sectoral peripheral epithelium. IVCM showed that epithelial cells in the central cornea exhibited a corneal phenotype and that the morphology of the epithelium in all limbal regions except the nasal limbus was normal. Epithelial cellular density and thickness were within the normal range. AS-OCT showed severe thinning in the limbus and a normal epithelial layer in the cornea and limbus. Based on the findings of IVCM and AS-OCT, we concluded that the patient had minimal LSCD, and limbal stem cell transplantation was not recommended.ConclusionsClinical presentation alone is insufficient to correctly diagnose LSCD in complex cases. Additional diagnostic tests, such as IVCM, are necessary to confirm the diagnosis before any surgical intervention.

Limbal stem cells (LSCs) maintain the normal homeostasis and wound healing of corneal epithelium. Limbal stem cell deficiency (LSCD) is a pathologic condition that results from the dysfunction and/or an insufficient quantity of LSCs. The diagnosis of LSCD has been made mainly based on medical history and clinical signs, which often are not specific to LSCD. Methods to stage the severity of LSCD have been lacking. With the application of newly developed ocular imaging modalities and molecular methods as diagnostic tools, standardized quantitative criteria for the staging of LSCD can be established. Because of these recent advancements, effective patient-specific therapy for different stages of LSCD may be feasible.

Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.

BackgroundThe aims of the study were to investigate limbal epithelial thickness in subjects with limbal stem cell deficiency and to evaluate the correlation between the palisades of Vogt and limbal epithelial thickness.DesignCross-sectional observational study.ParticipantsTwenty-four subjects (39 eyes) with limbal stem cell deficiency and 20 normal controls (20 eyes).MethodsAnterior segment optical coherence tomography and laser scanning confocal microscopy were performed to assess each quadrant of the limbus.Main outcome measuresLimbal epithelial thickness and palisades of Vogt morphology in each quadrant were characterized. The correlation between limbal epithelial thickness and palisades of Vogt was analysed.ResultsThe average limbal epithelial thicknesses in eyes with limbal stem cell deficiency were 19.9%, 23.4%, 13.8% and 13.5% less than normal controls at superior, inferior, nasal and temporal limbus (P = 0.008, 0.006, 0.014 and 0.011, respectively). Limbal epithelial thicknesses within limbal quadrants with palisades of Vogt were similar to those measured in the same quadrants in normal controls, whereas limbal epithelial thicknesses in the superior, inferior, nasal and temporal quadrants without palisades of Vogt were 27.8%, 29.8%, 14.7% and 15.6% less than the limbal epithelial thickness in corresponding regions of normal eyes (superior and inferior: P

PurposeTo quantify the changes in the subbasal nerve plexus in patients with limbal stem cell deficiency (LSCD) using in vivo laser scanning confocal microscopy.MethodsIn this retrospective cross-sectional comparative study, confocal images of 51 eyes of 37 patients with LSCD collected between 2010 and 2015 by the Heidelberg Retina Tomograph III Rostock Corneal Module Confocal Microscope were analyzed. Two independent observers evaluated the scans of the central cornea. Seventeen normal eyes of 13 subjects served as controls. Total subbasal nerve density (SND), density of long nerves (ie, nerves 200 μm or longer), and the degree of tortuosity were quantified.ResultsThe mean (±SD) total SND and long nerve density were 48.0 ± 34.2 and 9.7 ± 10.9 nerves/mm, respectively, in all eyes with LSCD and 97.3 ± 29.9 and 35.3 ± 25.3 nerves/mm, respectively, in eyes of the control group (P < 0.001 for both comparisons). Compared with SND in control subjects, SND was reduced by 34.9% in the early stage, 54.0% in the intermediate stage, and 73.5% in the late stage of LSCD. The degrees of nerve tortuosity were significantly greater in patients with LSCD than in control subjects and differed among the early, intermediate, and late stages of LSCD. Reductions in total SND and long nerve density were positively correlated with the severity of LSCD.ConclusionsReductions in total SND and long nerve density were accompanied by increases in nerve tortuosity in eyes with LSCD. These parameters could be used as quantifiable measures of LSCD severity.

The most efficient method to expand limbal stem cells (LSCs) in vitro for clinical transplantation is to culture single LSCs directly on growth-arrested mouse fibroblast 3T3 cells. To reduce possible xenobiotic contamination from 3T3s, primary human adipose-derived stem cells (ASCs) were examined as feeder cells to support the expansion of LSCs in vitro. To optimize the ASC-supported culture, freshly isolated limbal epithelial cells in the form of single cells (SC-ASC) or cell clusters (CC-ASC) were cultured using three different methods: LSCs seeded directly on feeder cells, a 3-dimensional (3D) culture system and a 3D culture system with fibrin (fibrin 3D). The expanded LSCs were examined at the end of a 2-week culture. The standard 3T3 culture served as control. Expansion of SC-ASC showed limited proliferation and exhibited differentiated morphology. CC-ASC generated epithelial cells with undifferentiated morphology in all culture methods, among which CC-ASC in 3D culture supported the highest cell doubling (cells doubled 9.0 times compared to cells doubled 4.9 times in control) while maintained the percentage of putative limbal stem/progenitor cells compared to the control. There were few cell-cell contacts between cultured LSCs and ASCs in 3D CC-ASC. In conclusion, ASCs support the growth of LSCs in the form of cell clusters but not in single cells. 3D CC-ASC could serve as a substitute for the standard 3T3 culture to expand LSCs.

PurposeTo report the presence of normal limbal epithelium detected by in vivo confocal laser scanning microscopy (IVCM) in 3 cases of clinically diagnosed total limbal stem cell deficiency (LSCD).MethodsThis is a retrospective case report consisting of 3 patients who were diagnosed with total LSCD based on clinical examination and/or impression cytology. Clinical data including ocular history, presentation, slit-lamp examination, IVCM, and impression cytology were reviewed.ResultsThe etiology was chemical burn in 3 cases. One patient had 2 failed penetrating keratoplasties. Another had allogeneic keratolimbal transplantation, but the graft failed 1 year after surgery. The third patient had failed amniotic membrane transplantation. These 3 patients presented with signs of total LSCD including the absence of normal Vogt palisades, complete superficial vascularization of the peripheral cornea, nonhealing epithelial defects, and corneal scarring. Impression cytology was performed in 2 cases to confirm the presence of goblet cells. However, each patient still had distinct areas of corneal and/or limbal epithelial cells detected by IVCM.ConclusionsResidual normal limbal epithelial cells could be present in eyes with clinical features of total LSCD. IVCM seems to be a more accurate method to evaluate the degree of LSCD.

The standard method of cultivating limbal epithelial progenitor/stem cells (LSCs) on a monolayer of mouse 3T3 feeder cells possesses the risk of cross-contamination in clinical applications. Human feeder cells have been used to eliminate this risk; however, efficiency from xenobiotic-free cultures on a monolayer appears to be lower than in the standard method using 3T3 cells. We investigated whether bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells for the expansion of LSCs in the 3-dimensional (3D) system. Primary single human LSCs on a monolayer of 3T3s served as the control. Very poor growth was observed when single LSCs were cultured on BMSCs. When LSC clusters were cultured on a BMSC monolayer (CC-BM), 3D culture system (3D CC-BM) and fibrin 3D system (fibrin 3D CC-BM), the 3D CC-BM method supported a greater LSC expansion. The 3D CC-BM system produced a 2.5-fold higher cell growth rate than the control (p0.05), whereas the proportion of K12(+) cells was lower (p

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

PurposeTo investigate changes in limbal basal epithelial cell density in eyes with limbal stem cell deficiency (LSCD) using in vivo confocal laser scanning microscopy.DesignRetrospective observational comparative study.MethodsA total of 43 eyes of 30 patients diagnosed with LSCD were included in the study. Ten eyes from normal subjects were included as control. Confocal imaging of the central cornea, and the superior, nasal, inferior and temporal limbus were collected using the Heidelberg Retina Tomograph III Rostock Corneal Module. Basal cell density in all locations was measured by 2 independent observers.ResultsThe mean basal cell density of the normal group was 9264 ± 598 cells/mm(2) in the cornea and 7120 ± 362 cells/mm(2) in the limbus. In the LSCD group, the mean basal cell density in the cornea decreased 31.0% (6389 ± 1820 cells/mm(2), P < .001) and in the limbus decreased 23.6% (5440 ± 1123 cells/mm(2), P < .001) compared to that in the control. There was a trend of basal cell density decline in more advanced stages of LSCD. The basal cell density declined in the unaffected regions at a similar degree as that in the affected region in sectoral LSCD (P > .05). The basal cell diameter increased by 24.6% in the cornea (14.7 μm) and by 15.7% in the limbus (15.5 μm) compared to the control.ConclusionsBasal cell density in both central cornea and limbus decreases in LSCD. Limbal stem cells (LSCs) are affected globally and basal cell density could be used as a parameter to measure LSC function at the early stages of the disease process.

PurposeWe tested the viability of human limbal mesenchymal cells (LMCs) to support the expansion of human corneal epithelial stem/progenitor cells (LSCs).MethodsHuman LMCs were isolated from sclerocorneal tissue using collagenase A. Primary limbal epithelial cells (LECs) in the form of single cell suspension or cell clusters were cocultured on a monolayer of either 3T3 cells (control) or LMCs (SC-LMC culture). The LEC clusters also were grown directly on LMCs (CC-LMC culture) and in an optimized 3-dimensional culture method (3D CC-LMC culture). Colony-forming efficiency (CFE) and LEC proliferation were analyzed. The phenotype of the cultured LECs was assessed by their expression level of putative stem cell markers and a differentiation marker by qRT-PCR and immunocytochemistry.ResultsThe LECs in the SC-LMC culture had a very limited growth and the stem/progenitor phenotype was lost compared to the control. Growth and cell morphology improved using the CC-LMC culture. The 3D CC-LMC culture method was the best to support the growth of the LSC population. Expression of ATP-binding cassette family G2 and ΔNp63 at the mRNA level was maintained or increased in CC-LMCs and 3D CC-LMC cultures compared to the control. The percentage of the K14(+) and K12(+) cells was comparable in these three cultures. There was no significant difference in the percentage of p63α high expressing cells in the control (21%) and 3D CC-LMC culture (17%, P > 0.05).ConclusionsHuman LMCs can substitute 3T3 cells in the expansion of LSCs using the 3-dimensional culture system.

PurposeTo develop and test a novel quantitative method of describing the corneoscleral junction, including metrics that reflect both the angle and the topography in this region of the ocular surface.MethodsForty-eight neophyte subjects were recruited (16 Asian, 16 white, and 16 Latino). Optical coherence tomography images of the nasal, temporal, superior, and inferior quadrants in both eyes were taken. Custom image analysis software was written in Matlab to allow the observer to select a point defining the center of the junction, from which 20 concentric circles were automatically drawn. The surface of the junction in the image was automatically located by edge-detection routines, and the circles intersecting this edge defined a series of points in the Cartesian plane. A linear regression was fit to these points, and a set of metrics based on the regression residuals was calculated.ResultsThe sum of the squared orthogonalized residuals (SSRo) was the most repeatable metric and had the advantage of being unaffected by the orientation of the image. The SSRo was significantly greater in the nasal quadrant (p < 0.001), reflecting a more pronounced angle and/or rougher surface. The flattest and smoothest topography was found in the temporal quadrant. Whites had significantly higher SSRo than Asians and Latinos (p < 0.001).ConclusionsThis study presents a novel metric for characterizing the angle and topography of the corneoscleral junction using optical coherence tomography and establishes differences among quadrants and between ethnic groups.

The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultured on opposite sides of a porous membrane. Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to standard culture or to a 3D sandwich culture method. The 3D sandwich method consistently yielded LSCs derived from cell clusters and tissue explants. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and stem cell phenotypes comparable to those of LSCs derived from the standard culture method. Limbal epithelial cell clusters cultured with the sandwich method had a significantly higher proliferation rate than did those cultured with the standard method. The 3D sandwich method did not favor the propagation of single LSCs. In summary, the 3D sandwich method permits complete separation between cultured cells and feeder cells, while providing an even and maximal proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.

Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of 10 Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was colocalized with the putative LSC markers including p63α, N-cadherin and keratin (K) 14, and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (Fz(KD)) using shRNA in primary LSCs, the expression of putative LSC markers ABCG2, ΔNp63α, and K14 was decreased significantly. The colony forming efficiency of the Fz7(KD) LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche, and Fz7 helps to maintain the undifferentiated state of LSCs.

Niche factors are important in the maintenance and regulation of stem cells. Limbal stromal cells are potentially a component of limbal stem cell (LSC) niche. We investigated the role of the limbal stromal cells in the ex vivo expansion of limbal stem/progenitor cells. Limbal epithelial cells were cultured as single-cell suspension and cell clusters from dispase II or collagenase A (ColA), or tissue explant. ColA isolated limbal stromal cells along with limbal epithelial cells. In the presence of limbal stromal cells, a higher absolute number of p63α(bright) cells (p

Corneal epithelial stem cells or limbal stem cells (LSCs) are responsible for the maintenance of the corneal epithelium in humans. The exact location of LSCs is still under debate, but the increasing need for identifying the biological processes in the limbus, where LSCs are located, is of great importance in the regulation of LSCs. In our current study we identified 146 preferentially expressed genes in the human limbus in direct comparison to that in the cornea and conjunctiva. The expression of newly identified limbal transcripts endomucin, fibromodulin, paired-like homeodomain 2 (PITX2) and axin-2 were validated using qRT-PCR. Further protein analysis on the newly identified limbal transcripts showed protein localization of PITX2 in the basal and suprabasal layer of the limbal epithelium and very low expression in the cornea and conjunctiva. Two other limbal transcripts, frizzled-7 and tenascin-C, were expressed in the basal epithelial layer of the limbus. Gene ontology and network analysis of the overexpressed limbal genes revealed cell-cell adhesion, Wnt and TGF-β/BMP signaling components among other developmental processes in the limbus. These results could aid in a better understanding of the regulatory elements in the LSC microenvironment.

PurposeTo elucidate the unique molecular factors and biological processes that are differentially expressed in the limbal stem cell microenvironment by comparing directly to that of its immediate adjacent structures, the cornea and conjunctiva.MethodsTotal RNA was isolated and amplified from the limbus, cornea, and conjunctiva. A gene expression profile of each tissue type was obtained by using microarray technique. The transcripts in which the expression level was at least twofold higher than that in the other two tissue types were identified. The expression levels of selected genes were confirmed by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Protein expression of selected genes were confirmed by an immunohistochemistry study in normal human ocular tissue.ResultsThere were 186 preferentially overexpressed transcripts in the limbus in direct comparison to that in the cornea and conjunctiva. Many signature genes in the cornea and conjunctiva were among the preferentially expressed transcripts obtained by the microarray data. In addition, a significant number of new genes were identified, and the expression level of all nine selected genes was verified by QRT-PCR. Protein expression levels of keratin 13, tenascin c, homeodomain only protein (HOP), and TP53 apoptosis effector (PERP) were confirmed in human ocular tissues. Functional analysis of the preferentially expressed genes in the limbus reviewed that melanin metabolism and cell-cell adhesion were among the noticeable biological processes. Chromosomal distribution of the overexpressed genes in the limbus was disproportional to that of all known human genes.ConclusionsThese findings may shed light on the unique molecular components and regulation of limbal stem cells and their niche.