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In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.

To investigate the stemness of limbal epithelial stem cell sheets in relation to the donor’s age. Human limbal explants from cadaveric donors were set on human amniotic membrane scaffolds with the xeno-free medium. We evaluated limbal epithelial sheet size, expression of stem/progenitor cell markers, and colony formation efficiency from donors of different age groups (age ≤ 45, age 45–65, and age > 65). Expression of the proliferation marker Ki67, stem/progenitor cell markers p63α and ABCG2, cornea specific marker PANCK, and differentiation marker CK12 were evaluated. To determine the effect of donor age on the storage period of limbal explant sheets, the limbal explant outgrowth sheets were stored in 4 °C for 2 days and analyzed for JC-1, p63α, and PANCK with FACS on each day. From days 6 to 12, the outgrowth area of the limbal epithelial stem cell sheet was significantly larger in the age ≤ 45 groups (296 ± 54.7 mm2, day 9) compared to the other two age groups [age 45–65 group (278 ± 62.6 mm2), age > 65 group (257 ± 44.0 mm2), day 9] (p 65) showed lower expression of stem/progenitor cell markers p63α and ABCG2 and decreased CFE compared to the other two groups. There were significantly more p63α+ cells in outgrowth cells in the age ≤ 45 group (18.2 ± 3.6%) compared to the age > 65 group (14.1 ± 4.6%; p 65 groups (25.7 ± 7.1%, p low). Cells from the age ≤ 45 group showed a higher clonogenic capacity than those from the other two age groups (45 65 group, positive cells of p63α on D0, 1, and 2 were significantly lower compared to those in the age ≤ 45 group on the storage period (p

Background Hyaluronan (HA) has previously been identified as an integral component of the limbal stem cell niche in vivo. In this study, we investigated whether a similar HA matrix is also expressed in vitro providing a niche supporting limbal epithelial stem cells (LESCs) during ex vivo expansion. We also investigated whether providing exogenous HA in vitro is beneficial to LESCs during ex vivo expansion. Method Human LESCs (hLESCs) were isolated from donor corneas and a mouse corneal epithelial progenitor cell line (TKE2) was obtained. The HA matrix was identified surrounding LESCs in vitro using immunocytochemistry, flow cytometry and red blood exclusion assay. Thereafter, LESCs were maintained on HA coated dishes or in the presence of HA supplemented in the media, and viability, proliferation, cell size, colony formation capabilities and expression of putative stem cell markers were compared with cells maintained on commonly used coated dishes. Results hLESCs and TKE2 cells express an HA-rich matrix in vitro, and this matrix is essential for maintaining LESCs. Further supplying exogenous HA, as a substrate and supplemented to the media, increases LESC proliferation, colony formation capabilities and the expression levels of putative limbal stem cell markers. Conclusion Our data show that both exogenous and endogenous HA help to maintain the LESC phenotype. Exogenous HA provides improved culture conditions for LESC during ex vivo expansion. Thus, HA forms a favorable microenvironment for LESCs during ex vivo expansion and, therefore, could be considered as an easy and cost-effective substrate and/or supplement for culturing LESCs in the clinic.

To conduct a systematic review of clinical research on the use of regenerative medicine for the cornea in human patients. A systematic literature search of MEDLINE and the Cochrane Library was performed in May 2020. Forty-two articles were identified. Thirty-eight of those articles focused on the treatment for limbal stem cell deficiency (LSCD), of which 17 articles involved autologous cultured limbal epithelial cell sheet transplantation (CLET), 13 involved allogeneic CLET, and 14 involved autologous cultured oral mucosal epithelial cell sheet transplantation (COMET). For autologous CLET, the median ocular surface reconstruction rate, visual recovery rate, incidence of immunologic rejection, infectious keratitis, and ocular hypertension/glaucoma were 74.1%, 54.5%, 0%, 4.6%, and 6.3%, respectively. For allogeneic CLET, they were 71.4%, 71.4%, 7.1%, 12.0%, and 7.1%, respectively. For autologous COMET, they were 66.7%, 66.7%, 0%, 5.3%, and 8.1%, respectively. Systemic immunosuppressants and steroid medications were predominantly used following allogeneic CLET, whereas they were not routinely used after autologous CLET. Three studies focused on the treatment of keratoconus using autologous adipose-derived adult stem cells and reported no marked adverse events. One study reported on the treatment of bullous keratopathy using allogeneic cultured corneal endothelial cells. All patients achieved an endothelial cell density of >500 cells, and the corrected distance visual acuity improved in 82% of the treated eyes. The results show that regenerative medicine for the cornea demonstrated a satisfactory efficacy and safety. Through translational research, we are expecting to establish a new treatment for waiting patients.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage. Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs), other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION: PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00273-5) contains supplementary material, which is available to authorized users.

Epidermal adult stem cells (EpiASCs) have the potential for unlimited proliferation and differentiation, however, the ability of these stem cells to activate corneal genetic programs in response to corneal stroma stimulation needs to be further validated. Herein, a feasible strategy was developed to reconstruct the damaged corneal surface in a goat model with total limbal stem cell deficiency (LSCD) by transplanting EpiASCs, which had been explanted and cultured from the skin of an adult ram goat and were then purified by selecting single cell-derived clones and cultivating them on a denuded human amniotic membrane (HAM). These artificial tissues were then successfully transplanted into ewe goats with total LSCD. Binding of EpiASCs to the base membrane of an EpiASCs-HAM-Sheet (EHS) indicated their proliferating status. After transplantation, the EpiASCs could survive in the host tissue and they reconstructed the damaged ocular surface of total LSCD. The crystal reconstructed corneal epithelium expressed CK3 and Pax-6 similar to normal corneal epithelium and expressed the Sry gene after transplantation. These results demonstrated that EpiASCs could be induced to differentiate into corneal epithelial cell types in a corneal microenvironment and had the ability to activate corneal genetic programs. This work offer a foundation for promoting tissue-engineered cornea into clinical application.

To determine the efficacy of subconjunctival bevacizumab injection after pterygium excision with limbal conjunctival autograft and limbal fixation suture. This retrospective study included a total of 150 eyes of 150 patients with primary pterygium who received three different procedures after pterygium excision, i.e., 49 eyes with limbal conjunctival graft (group A), 48 eyes with limbal conjunctival autograft with limbal fixation suture (group B), and 53 eyes with limbal conjunctival autograft with limbal fixation suture followed by bevacizumab injection (group C). Image analysis was performed using preoperative anterior segment photographs to measure parameters including relative length, relative width, relative area, and vascularity index of pterygium. Recurrence of pterygium was determined at 1 year after surgery, and outcomes were compared between the 3 groups. Risk factors related to recurrence were evaluated using univariate and multivariate analyses. Recurrence rates after 1 year were 18.4% (9/49), 8.3% (4/48), and 1.9% (1/53) in groups A, B, and C, respectively (P = 0.004). Multivariate analysis showed that patients in group C had significantly reduced risk of recurrence compared with those in group A (P = 0.009), whereas the risk of recurrence was not significantly different between groups A and B (P = 0.227) and groups B and C (P = 0.068), respectively. Among various parameters, higher vascularity index had significant correlation with increased risk of recurrence (P = 0.008). Bevacizumab injection after limbal conjunctival autograft and limbal fixation suture may effectively reduce recurrence after pterygium excision. The vascularity of pterygium was associated with a higher risk of recurrence.

Background Molluscum contagiosum, a pox virus infection, is likely to occur in the eyelid skin; however, corneal involvements by molluscum lesions are extremely rare. We report a case of molluscum contagiosum arising in the corneal limbus in an untreated AIDS patient, together with anterior segment optical coherence tomography (OCT) findings and histopathology of the excised tumor. Case presentation A 46-year-old man with AIDS was referred to our department for the management of an ocular lesion. Blood tests revealed an extremely low CD4+ T-cell count of 11 cells/μL, being strongly positive for anti-HIV antibody (591.36 S/CO) with a high copy number of HIV RNA (8070.0 × 100 copy/mL). Slit-lamp examination of his right eye showed a white nodule at the lower limbus. Anterior segment OCT findings of the nodule revealed a highly reflective elevated lesion, which was considered to involve the Bowman layer. The nodular lesion was excised from the limbus including the superficial corneal stroma, and then processed for histologic examination. Histopathology of the excised lesion showed acanthotic corneal epithelium containing swollen cells with eosinophilic inclusions known as molluscum bodies. He was diagnosed with molluscum contagiosum. Conclusions Anterior segment OCT findings provide useful information for morphological evaluations of and preoperative strategies against molluscum contagiosum.

Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of people for the cornea transplantation all over the world and the list of waiting patients is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and mitigates the adverse impact of induced inflammation.

Spectral-domain optical coherence tomography (SD-OCT) has been used to observe the morphology of the palisades of Vogt (POV) with satisfactory resolutions. In this study, we used SD-OCT to examine the microstructure of the POV in ocular surface disorders with limbal involvement. We detect subclinical limbal pathologies based on five parameters, including (1) decreased epithelial thickness, (2) loss of the sharp stromal tip, (3) loss of the smooth epithelial-stromal interface, (4) dilated stromal vessels, and (5) decreased POV density. Eighteen eyes of 10 patients with advancing wavelike epitheliopathy (AWE) and 15 eyes of 9 patients with phlyctenular keratitis/ocular rosacea were recruited. SD-OCT could detect abnormal changes in the POV in 100% of the lesion sites. In presumed-healthy areas of the diseased eyes diagnosed by slit-lamp biomicroscopy, SD-OCT detected abnormal changes in the POV in 100% of the eyes in both groups. In patients with unilateral disease, abnormal changes in the POV were detected by SD-OCT in 50% and 100% of presumed-healthy eyes diagnosed by slit-lamp biomicroscopy in the AWE group and phlyctenular keratitis/ocular rosacea group, respectively. SD-OCT is powerful in detecting POV changes in ocular surface disorders and can provide useful information that cannot be provided by slit-lamp biomicroscopy.

The purpose of this study was to compare the efficacy of allogeneic cultured limbal epithelial transplantation (ACLET) and cultivated oral mucosal epithelial transplantation (COMET) in treating total limbal stem cell deficiency (LSCD). In this retrospective cohort study, 73 patients (76 eyes) with total LSCD, including 41 patients (42 eyes) treated with ACLET and 32 patients (34 eyes) receiving COMET, were evaluated. The age, gender and injury cause of all patients were recorded. The mean follow-up was 23.3 ± 9.9 months in the ACLET group and 16.1 ± 5.8 months in the COMET group. A higher incidence of persistent epithelial defect was observed after COMET (P = 0.023). The overall ocular surface grading scores were all lower in the ACLET group than in the COMET group at 3, 6, and 12 months after surgery and the last follow-up. Kaplan-Meier survival curve analysis demonstrated a significantly higher success rate of ACLET (71.4%), compared with that of COMET (52.9%; P = 0.043). The risk of graft failure was higher in patients with entropion and trichiasis, incomplete eyelid closure and treated with COMET. The graft failure risk rate after COMET was 3.5 times higher than that of ACLET. For total LSCD patients, ACLET should be prioritized, since limbal epithelial cells have better ability to maintain corneal epithelial integrity and ocular surface stability and benefit the ocular surface when compared with oral mucosal epithelial cells. Preoperative and postoperative eyelid abnormalities should be corrected as early as possible.

Currently, regenerative medicine has attracted much attention among researchers investigating new methods to treat ocular surface diseases. Based on this new concept, cultivated limbal epithelial transplantation (CLET), whether in the form of autograft or allograft, has emerged as a promising surgical procedure for treating limbal stem cell deficiency (LSCD). Given that there is no updated comparison between autograft and allograft CLETs, the present review and meta-analysis aims to compare and determine the efficacy of two different CLET techniques, autologous versus allogeneic, based on a literature review of relevant studies. A comprehensive search of electronic databases, including PubMed, Web of Science, Cochrane Library, Embase and Scopus, for related articles was performed in March 2018 to obtain relevant articles and to conduct a meta-analysis investigating the success rate of ocular surface regeneration and two-line improvement in best-corrected visual acuity (BCVA) using autograft versus allograft transplantations. A total of 30 studies, including 1306 eyes from 1288 patients with LSCD, with a sample size ranging from 6 to 200 and follow-up period of 0.6–156 months, were reviewed. Of 1306 eyes, 982 (75.2%) underwent autograft and 324 (24.8%) received allografts from living or deceased donors. Meta-analysis revealed that there was no significant difference between autograft and allograft CLETs in terms of success rate and two-line BCVA improvement. The prospective studies showed a zero difference between the two groups; only two retrospective studies included in the analysis pulled the autografts up to 1.82 and 1.2 times more than allografts in terms of success rate and two-line BCVA improvement, respectively [pooled OR 1.82 (95% CI 0.80–4.11); pooled OR 1.2 (95% CI 0.54–2.65)]. There was no statistically significant evidence of bias in the meta-analysis in terms of success rates and two-line BCVA improvement. The present analysis revealed no significant differences in success rates or visual improvement between autograft and allograft surgical techniques.

Simple limbal epithelial transplantation (SLET) is a relatively new treatment for severe limbal stem cell deficiency. Outcomes of treatment are typically determined based on clinical manifestations. In this prospective-multicenter study, we aimed to analyze the epithelial phenotypes of the corneas after SLET using IVCM and IC, and correlated them with clinical findings. Ten eyes of nine patients, who underwent SLET (five autologous SLET and five living-related SLET) were recruited. A set of examinations included slit-lamp biomicroscopy, corneal in vivo confocal microscopy (IVCM), and impression cytology (IC) was performed in all eyes at least twice (≥ 3-month interval) postoperatively. Then, a correlation between findings of the three examinations was analyzed. There were seven eyes with clinical success (no central neovascularization) showed pure corneal epithelial phenotype or mixed corneal-conjunctival phenotypes (mostly cornea) in either IVCM or IC. Three eyes with clinical failure, presented with peripheral and central neovascularization, showed total or predominant conjunctival phenotype in IVCM and sole conjunctival phenotype in IC. From a total of 22 sets of examinations, there was a high correlation between clinical manifestation vs. IC (κ = 0.844, observed agreement = 81.82%) and a substantial correlation between clinical manifestation vs. IVCM (κ = 0.727, observed agreement = 76.19%) and between IVCM versus IC (κ = 0.729, observed agreement = 76.19%). In conclusion, IVCM and IC facilitate determination of epithelial phenotype of the cornea after SLET. There was a substantial to high correlation between IVCM, IC and clinical presentations. Findings observed by IVCM and IC may allow early detection of epithelial alterations in eyes underwent SLET before clinical recognition.

Background While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5+ LSCs derived from human cadaveric limbal tissue. Methods We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5+ LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells’ engraftment potential, biodistribution behavior, and safety. Results ABCB5+ LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD. Conclusion Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5+ LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient’s LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea.

Background the sparsity of established tools for the grading of limbal stem cell deficiency hinder objective assessments of the clinical outcome of cultivated limbal epithelial cell transplantation. To advance towards the development of standards for the comparison of the outcomes of these bio-surgical protocols we have now applied a battery of recognized objective and patient-declared subjective outcome criteria to the autologous modality of cultivated limbal epithelial cell transplantation. Methods the prospective study involved ten patients (M/F = 9/1; mean age = 42.1 years) displaying overt unilateral limbal stem cell deficiency complying with the inclusion criteria described in Methods. Limbal biopsies were obtained from the contralateral eye and their outgrowths after 2-week cultures were transplanted on the affected eye after pannus resection. Outcomes were followed up for 12 months. the objective tests were scores for best-corrected visual acuity (BCVA); using the LogMAR scale, a multiparametric ocular surface score (OSS), and the Schirmer's test. Subjective scores were based on patient answers to a) perception of visual improvement/pain; b) the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25); and c) the 12-item Ocular Surface Disease Index Questionnaire (OSDI). All procedures were performed under good manufacture practices using solely xeno-free reagents. in all cases, a single biopsy was divided into two pieces and they were expanded in order to prevent outgrowth failure. in 5 patients, both biopsies generated healthy culture sheet. in those cases the lesser outgrowth were used for immune-histological characterization. Results the experimental parallel outgrowth samples showed a similar percent of p63 alpha(+)cells. PreOp and 12-month PostOp BCVAs and OSSs were, respectively, 1.15 +/- 0.70; 0.21 +/- 0.13 and 7.40 +/- 2.01; 2,30 +/- 1.30, (p < 0.05). Patient's responses to all three question sets except ocular pain were consistent with significant improvement (p < 0.05). Conclusion Objective clinical metrics demonstrate that in patients with limbal stem cell deficiency, cultivated limbal epithelial cell transplantation improves vision and ocular surface health and subjective visual perceptions., Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115S423]; USPHSUnited States Public Health Service [RO1-EY 029279, RO1-EY014878], OBS: the Scientific and Technological Research Council of Turkey (TUBITAK)(Project number:115S423); JMW: USPHS RO1-EY 029279 and RO1-EY014878.

Background It is rare for hepatolenticular degeneration [Wilson’s disease (WD)] to occur along with keratoconus (KC). In our report, a teenager was diagnosed with WD because of the discovery of Kayser–Fleischer (KF) ring in the cornea, and concomitant KC was found. Case presentation A 19-year-old male was diagnosed with KC due to a rapid decline in visual acuity within a short period of time. Ocular examination revealed the presence of ring-shaped, dense, brown sediment at the Descemet membrane of the bilateral limbus cornea, exhibiting characteristics similar to those of KF ring. Then, the patient was referred to the Department of Neurology and diagnosed with asymptomatic WD. During the next 5 years of follow-up, the patient has worn RGP lenses, routinely taken drugs that inhibit copper absorption and promote copper excretion, and maintained a low-copper diet. He has never exhibited obvious systemic symptoms associated with WD, such as neurological, mental, or hepatic dysfunction, and the color of the KF ring has grown obviously lighter. Moreover, the morphology of the cornea has stabilized. Conclusion Only one report of WD combined with KC was found in the literature. So far, there is no evidence of a correlation between the occurrence of the two diseases. However, a low-copper diet and active copper-reducing therapy may have played a role in stabilizing the patient’s condition in this case.

To determine the risk factors related to recurrence of pterygium after pterygium excision with limbal-conjunctival autograft.Retrospective, observational case series.The current study included 310 patients who underwent surgical removal of pterygium using limbal-conjunctival autografting. All surgery was performed by the same surgeon at a single Hospital from 2006 to 2011. Preoperative and postoperative patient data were collected. The patients were summoned to follow-up examinations. Procedures with at least 12 months of follow-up were included in the final analysis. Potential risk factors for pterygium recurrence were determined by Cox proportional-hazard linear analysis and recurrence-free survival time was evaluated using Kaplan-Meier method.The overall recurrence rate was 5.2% during an average follow-up time of 20.1±8.6 months (range, 12-37 months). According to Kaplan-Meier evaluation, the estimated time to recurrence was 34.2 ± 0.5 months (95% confidence interval 33.3-35.0 months) for the whole population. Multivariate analysis showed that age45 years old (RR = 3.582, 95% CI 1.984-13.044, P = 0.047), recurrent type of pterygia (RR = 4.856, 95% CI 1.794-13.142, P = 0.002), and vertical size of pterygium6.7 mm (RR = 2.529, 95% CI 1.053-6.075, P = 0.038) were significantly associated with increased risk of recurrence.Our findings suggest that young age, recurrent type of pterygia, and larger pterygial tissue are risk factors for pterygium recurrence after surgical excision. Hence, early excision of pterygium is recommended to decrease the recurrence rate.

Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC. Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay. In the DHL, the DNA content was reduced from 1.5 ± 0.3 μg/mg to 0.15 ± 0.01 μg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity. The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

To report the therapeutic efficacy and safety of topical 0.1% lodoxamide in the long-term treatment of superior limbic keratoconjunctivitis. Sixty-seven eyes of 34 patients with active SLK were studied. Therapeutic response was analyzed according to modified-Ohashi parameters. All eyes were treated with 0.1% lodoxamide twice daily, and those with moderate or severe inflammation received a short course (7–14 days) of 0.1% fluorometholone acetate at presentation and during a relapse. Patients were evaluated at regular intervals and followed up for ≥3 months on continuous therapy. Primary endpoints included inflammatory response; rates of inflammatory control and remission; relapses while on therapy or on remission; and therapeutic failure rate. The mean follow-up time on lodoxamide therapy was 15.3 months. The majority of eyes (82.0%) achieved control of inflammation in a mean time of 2.2 months. Of these, 42 (76.3%) eyes remained under control while on therapy for 13.8 months. There was a significant improvement of SLK-related signs by the third month on therapy (p

Corneal melting with perforation is a severe ophthalmic complication of autoimmune disorders such as rheumatoid arthritis. It requires urgent medical management in order to maintain the integrity of the globe and preserve vision. Treating this complication by penetrating keratoplasty is problematic due to the high rate of recurrence of corneal melting as well as other complications. We describe the use of a tectonic fresh-tissue corneolimbal covering graft. An interventional case series including three patients that presented to our tertiary center between 2000 and 2015 with corneal melting and perforation, secondary to rheumatoid arthritis. Emergency surgery included suturing of a 13.00- to 13.50-mm full-thickness fresh-tissue corneolimbal covering graft to the patient’s posterior limbal zone. The corneolimbal graft maintained the integrity of the cornea in all cases, by sealing the perforation and promoting the creation of a fibrovascular scar at the area of corneal melting. There were no complications, recurrences of host corneal melting, or perforation during the follow-up period. Fresh-tissue full-thickness corneolimbal grafts may be used to cover emergency corneal melting and perforations secondary to rheumatoid arthritis.

To compare the outcome of allogenic ex vivo cultivated limbal stem cell transplantation (LSCT) versus cadaveric keratolimbal allograft in patients with limbal stem cell deficiency (LSCD). In this prospective interventional study from Army Hospital Research and Referral in New Delhi, 50 eyes of 40 patients with ocular surface disorder having LSCD underwent either cultivated LSCT (group 1: 25 eyes of 20 patients) or keratolimbal allograft transplantation (group 2: 25 eyes of 20 patients). The outcome measures were gain of two or more lines of best-corrected visual acuity (BCVA), absence of conjunctival vascularization over cornea, corneal neovascularization regression, regression of visually significant corneal opacity (iris details poorly visible), Schirmer’s 1 ≥10 s, tear break-up time (TBUT) ≥10 s, and absence of fluorescein staining. At 1 year of follow-up, both groups experienced significant improvement in all outcome measures, but there was no significant difference between both groups in improvement of two or more lines of BCVA (P = 0.841), absence of conjunctival vascularization over cornea (P = 0.812), corneal neovascularization (P = 0.074), visually significant corneal opacity (P = 0.075), Schirmer’s 1 ≥10 s (P = 0.814), TBUT ≥10 s (P = 0.382), or absence of fluorescein stain (P = 1.00). Both allogenic ex vivo cultivated LSCT and cadaveric keratolimbal allograft transplantation are comparable in terms of visual gain and ocular surface restoration.

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3–5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.

Purpose To report a technique to prevent pterygium recurrence using the principle of contact inhibition. Methods Two hundred and fifteen patients (232 eyes; average age, 64.1 years) with primary pterygia who underwent pterygium surgery at the Hara Eye Hospital between 1999 and 2012. We retrospectively evaluated the patients who underwent the following procedure to prevent pterygium recurrence. The surface conjunctiva on the pterygium body was not removed. After removing the pterygium body, by placing a narrow pedicle autoconjunctival flap along the corneal limbus and tying it tightly to the front area of the residual conjunctiva, there is no room for the active residual tissue to proliferate, thus preventing a recurrence by contact inhibition. The key factor is conjunctival suturing, which establishes face-to-face contact of both areas of cut conjunctival tissue. Mitomycin C is applied locally for 3 min intraoperatively and 5 days postoperatively. The main outcome measure was the prevention of pterygium recurrence using this technique. Results By the end of the average follow-up of 5 years 4 months, three eyes (1.3%) had a recurrence. Among the 232 eyes, 23 eyes had large pterygia extending to the pupillary area. Using the surgical technique, there were no recurrences. No specific characteristic of the recurrence was found in association with the eye, sex, and preoperative grade. Conclusions This surgery has three relevant features: (1) reconfirmation of the effect of contact inhibition, (2) the anatomic structure of the conjunctival sac scarcely changes postoperatively, because the surface conjunctiva of the pterygium body is not removed, and (3) a low recurrence rate. Electronic supplementary material The online version of this article (doi:10.1007/s00417-016-3558-9) contains supplementary material, which is available to authorized users.

To conduct a long-term follow-up study evaluating the efficacy and safety of transplantation of preserved limbal allograft and amniotic membrane for recurrent pterygium. This was a retrospective, non-comparative, interventional case series conducted at a private eye hospital. Eighty-four eyes of 80 patients with recurrent pterygium were included in the study. The mean number of previous surgeries for pterygium was 1.36 ± 0.98 (range, 1–8). All subjects received transplantation of preserved limbal allograft and amniotic membrane. The mean follow-up period was 73.0 ± 38.1 months (range, 12–154 months). Pterygium recurred in 10 eyes (11.9 %). The mean period to recurrence was 16.3 ± 11.3 months (range, 5–33 months). Symblepharon was cured in 21 eyes, persisted in 2 eyes, and newly occurred in 3 eyes. Diplopia was cured in eight eyes, persisted in five eyes, and newly occurred in one eye. As for complications, intraocular pressure elevations over 21 mmHg were recognized in ten eyes of nine cases, in which the intraocular pressure was controlled by reduction of topical steroid in four eyes and by addition of topical prostaglandin derivatives in six eyes. Twenty-four eyes (28.6 %) gained two lines or more of Landolt best spectacle-corrected visual acuity (BSCVA), 56 eyes (66.7 %) stayed within one line from preoperation, and four eyes (4.8 %) lost two lines or more. There were no major complications and no graft rejection. Transplantation of preserved limbal allograft and amniotic membrane is a safe and effective procedure for recurrent pterygium.

Adult-onset xanthogranuloma (AOX) of the corneoscleral limbus is a rare inflammatory condition of unknown aetiology. Similar to limbal juvenile xanthogranuloma (JXG), it presents as a growing mass at the corneoscleral junction. Limbal AOX and JXG can lead to sight-threatening complications if not managed in a timely manner. This systematic review summarises the main clinical and histopathological features of limbal AOX/JXG and discusses the management of this uncommon disease. We performed a literature search in the MEDLINE database for all historical entries, using the search terms “limbus”, “limbal” and “xanthogranuloma”, and retrieved all articles reporting on limbal xanthogranuloma. After refining the search to articles relevant to limbal AOX, we were able to identify ten adult cases of limbal AOX and compare those with all reported cases of limbal JXG. Clinically, AOX usually presents as an isolated smooth, yellowish, dome-shaped nodule at the corneoscleral junction, similar to an ocular presentation of JXG, with which it also shares similar histopathological features. Limbal JXG and AOX may represent the same disease entity. Diagnosis relies on the clinical presentation, pathology and immunohistochemical profile. Spontaneous regression is unlikely, and thus prompt surgical intervention should be considered to prevent sight-threatening complications. Xanthogranuloma should be included in the differential diagnosis of corneoscleral limbal masses in patients of all age groups.

In this article, human limbal niche cells (LNC) or bone marrow derived mesenchymal stem cells (BMMSC) were used to prevent limbal stem cell deficiency (LSCD) in an alkali burn rabbit model and their results were compared. The epithelial cell defect area, corneal neovascularization, and the print cell cytometry were quantified to grade the severity of LSCD. Three months after the alkali burn, a partial LSCD was observed in the control group (no treatment) indicated by chronic corneal epithelial defects, positive corneal fluorescein staining, neovascularization and goblet cell migration. In contrast, the severity of LSCD in both the LNC and BMMSC transplantation groups was dramatically reduced as shown by smaller epithelial cell defects, decreased fluorescein sodium staining, decreased neovascularization and decreased goblet cell density. Interestingly, the LNC group was shown to more effectively prevent LSCD than the BMMSC group. Further analysis indicated subconjunctivally transplanted LNCs were more powerful than BMMSCs to prevent LSCD, at least partially, due to increased activation of SCF-c-Kit signal. We conclude that LNCs are a more powerful resource than BMMSCs to prevent LSCD in an alkali burn rabbit model, at least partially due to increased activation of SCF signaling.

To investigate the safety and efficacy of intraoperative conversion of unsuccessful canaloplasty technique into 180-degree metal or 360-degree suture trabeculotomy. In this retrospective observational case series, 35 eyes of 35 patients with open-angle glaucoma (OAG) who had undergone unsuccessful canaloplasty technique and underwent intraoperative conversion were studied. In 17 eyes (group A), the procedure was converted into 180-degree metal trabeculotomy because of unsuccessful circumferential Schlemm’s canal (SC) catheterisation. Group B included 18 eyes that were converted into 360-degree trabeculotomy because of cheese wiring of tension suture through the trabecular meshwork (TM) after successful complete catheterisation of SC. Median preoperative IOP was 22.0 mmHg (range 16–34) in group A, and 22.0 mmHg (range 15–48) in group B. Median IOP was significantly reduced in both groups postoperatively, at the 12-month examination to 15.0 mmHg (range 9–21) representing a reduction of 36 % in group A, and to 14.5 mmHg (range 8-21) representing a reduction of 34 % in group B. Median glaucoma medication number decreased significantly from 4 (range 2–4) and 3 (range 1–4) preoperatively to 2 (range 0–4) and 1 (range 0–3) at 12 months in groups A and B respectively. Hyphema was the commonest complication, with an incidence of 76 % in group A and 78 % in group B. 180-degree metal or 360-degree suture/catheter trabeculotomy are safe and effective surgical backup methods to control IOP in cases of unsuccessful intracanalicular placement of tension suture during canaloplasty surgery.

Current imaging techniques for the characterization of differentiated corneal limbal stem cells are destructive and cannot be used in eye bank for monitoring the regenerated epithelium in culture. We presented a minimally invasive, multimodal, marker-free imaging method for the investigation of epithelia regenerated with cultured human donor corneal limbal epithelial stem cells. Two-photon fluorescence and harmonic generation signals were collected from specimens in culture and used for evaluating the structure and morphology of epithelia cultured on two different bio-scaffolds; in addition, donor human corneal tissues were used as controls. The method provided reliable information on the organization of cellular and extracellular components of biomaterial substrates and was highly sensitive to determine differences between the density packing arrangement of epithelial cells of different biomaterials without relying on inferences from exogenous labels. The present minimally invasive standardized quality control methodology can be reliably translated to eye banks and used for monitoring harvested corneal limbal stem cells growth and differentiation in bioengineered materials.

Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

To compare the role of fresh and dry preserved amniotic membrane transplantation (AMT) in partial limbal stem cell deficiency (LSCD). 30 Patients of partial LSCD involving

We retrospectively studied the efficacy and safety of different surgical approaches to treating pediatric limbal dermoids with regard to intra and postoperative complications.The data of 12 consecutive patients (14 eyes), who underwent monocentric surgery of a limbal demoid in the past 9 years, were retrospectively analyzed for intra and postoperative complications. Group one consists of eleven eyes: seven eyes with a bare-sclera deep lamellar excision of the dermoid and four eyes with an additional amniotic membrane transplantation subsequent to excision. Group two consists of three eyes: two eyes treated with Mitomycin C 0.02% over 2 min following the excision, and one eye treated with Mitomycin C and amniotic membrane transplantation after removal of the dermoid. Follow-up ranged from 2 to 53 months (median 17 months).Intraoperative complications did not occur in any of the patients. Postoperatively, all patients showed corneal reepithelialization within a week. Limbal stem cell deficiency with a pseudopterygium developed in four eyes, one treated with bare-sclera excision and the others with amniotic membrane transplantation. One pseudopterygium had to be removed surgically because of visual acuity deterioration. Not a single eye treated with Mitomycin C developed a pseudopterygium.The transplantation of amniotic membrane following removal of a limbal dermoid cannot prevent the occurrence of a pseudopterygium. However, the use of Mitomycin C seems to have a protective effect.

To evaluate the feasibility of partial lamellar keratoplasty (LK) for treatment of peripheral corneal disease (PCD) using a graft from the corneoscleral rim preserved in glycerin.Patients who underwent LK for PCD at Shandong Eye Hospital from January 2006 to December 2012 were included. Corneoscleral rims of donor grafts, out of which the corneal buttons had been punched and used for penetrating keratoplasty, were preserved in glycerin and used for LK procedures. A trephine of 7.5-8.0 mm was used to mark the central cornea. An excision was made according to the size of the lesions, after which a partial ring-shaped corneoscleral graft was transplanted to repair the peripheral cornea. Visual acuity, refractive astigmatism, and complications were monitored.There were 26 patients (28 eyes), including 15 patients with Terrien's marginal degeneration, eight with Mooren's ulcer, two with Wegener's granulomatosis, and one with peripheral corneal ulcer. The mean follow-up was 15 months (range, 6-36 months). Uncorrected visual acuity was improved by a mean of 3.17 Snellen chart lines (P = 0.010), and best spectacle-corrected visual acuity was improved by a mean of 0.85 Snellen chart lines (P = 0.045) at 6 months after surgery. The refractive astigmatism decreased by 5.1 ± 8.6 diopters (P = 0.003). Two patients suffering graft ulcers (recurrence) were successfully treated by a second partial LK. No graft rejection occurred.Partial LK using a graft from the corneoscleral rim appears to be effective in the treatment of patients with PCD. This approach saves the donor graft, which is important in the countries or regions with scarcity of donor tissue.

The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63--a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).

Background To compare corneal epithelial thickness (CET) and limbal epithelial thickness (LET) measured by anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscope (IVCM) in normal subjects, and evaluate the consistency between them. Methods Thirty-eight normal subjects (17 men and 21 women) were enrolled in this study. AS-OCT was performed at central cornea and the superior, inferior, nasal and temporal limbus. Then followed by IVCM examination performed at the same location. Agreement was analyzed by mean difference (AS-OCT minus IVCM), 95 % limits of agreement (LoA) (1.96 standard deviation of the difference), and Bland-Altman analysis. Results The average CET measured by AS-OCT and IVCM was 55.6 ± 4.0 μm and 51.9 ± 4.9 μm respectively. The value measured by IVCM was significantly lower than that measured by AS-OCT (P = 0.015). The average LET values tested by AS-OCT were 10.3 and 10.9 % less at nasal and temporal quadrant (nasal: P = 0.019, temporal: P = 0.003), and were similar as those measured by IVCM at superior and inferior quadrant. In subjects older than 40 years, CET and LET values measured by AS-OCT were significantly higher than those by IVCM. Such differences were not found in subjects ≤ 40 years old. Conclusions CET values measured by IVCM are lower than those by AS-OCT, while LET values measured by two devices have good agreement. These two techniques have their own advantages in measuring epithelial thickness and are mutually complementary.

Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts. Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry. The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone. Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.

The aim of this study is to present the limbal stem cell deficiency (LSCD) cases with features resembling dyskeratosis congenita (DC), a heritable disease of stem cells principally caused by telomerase deficiency. The clinical, laboratory and molecular findings of four cases are presented. A complete systemic examination was performed in a standardized manner for each patient. Laboratory measurements included investigations of the tests used for screening DC. All eight known disease-causing genes in DC (DKC1, TERC, TERT, NOP10, NHP2, TINF2, C16orf57, and TCAB1) were screened for mutations. The family members of the cases were also assessed, when possible. In all four patients, multisystem involvement was present, along with the disorder affecting corneal LSCs. The affected tissues were mainly the skin and its adnexa, the oral cavity and the hematopoietic system, which are rapidly renewing tissues, consistent with the presence of a stem cell disorder. Similarly affected cases were seen in different generations in families, suggesting an underlying inherited disorder. No mutation was detected in any of the known disease-causing genes in these patients. Based on the presented cases and with the contribution of the review of previously reported DC cases available, we suggest that DC is one of the inherited causes of LSCD and that those cases presenting with LSCD might represent a subgroup of DC caused by mutations in an as yet undefined gene.

Limbal stem cells (LSC) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell (HLECs) transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. A major challenge is the ability to identify LSC in vitro and in situ, and one of the major controversies in the field relates to reliable LSC markers. This study was carried out to evaluate the culture of a limbal biopsy on human amniotic membrane (HAM): directly on the chorionic side and on intact epithelium, and the expression of the stem cell associated markers: ABCG2, p63. HAM has been extensively used for ocular surface reconstruction and has properties which facilitate the growth of epithelial cells controlling inflammation and scarring.

The limbal niche in the corneoscleral junction of the eye, habitat of the limbal epithelial stem cells (LESC), facilitates corneal epithelial regeneration by providing physical support and chemical signalling. Anatomical structures within the limbus, namely, limbal epithelial crypts and focal stromal projections, are believed to function as a putative niche for LESCs. In this study, the impact of age on the topography of this niche was investigated. Also, the relationship between niche topography and limbal epithelial cell phenotype was assessed. Ex vivo imaging of the limbus in cadaveric tissue of donors aged from infancy to 90 years was carried out using electron and confocal microscopy. The data suggested that the area occupied by the crypts was sharply reduced after the age of 60 years. The niche microstructures also became smoother with donor age. The phenotypic assessment of cultured limbal epithelial cells harvested from donors of different ages showed that the levels of putative stem cell markers as well as telomerase activity and telomere length remained unchanged, regardless of niche topography. However, the colony forming efficiency of the cultures was significantly reduced with age (p

Keratolimbal allograft (KLAL) transplantation has been proved to be a useful surgical procedure for limbal stem cell deficiency patients. However, information about in vivo ocular surface changes in those patients is limited, due to the lack of a reliable and non-invasive technique for closely monitoring the changes of KLAL grafts. The aim of this study is to characterize the cellular changes in the limbus and central cornea after KLAL in patients with severe ocular chemical injury, using in vivo laser scanning confocal microscopy (LSCM). This is a prospective, noncomparative, observational case series. Twenty-three patients (23 eyes) with total limbal stem cell deficiency due to ocular chemical injury were recruited. KLAL with or without other concurrent surgery were performed. LSCM and slit-lamp examination were performed on the limbus and the central cornea before surgery and at 6 and 12 months postoperatively. Presence of palisades of Vogt, limbal basal epithelial cell density within the palisades of Vogt (LEC), limbal dendritic cells (DC) density, and central corneal basal epithelial cell (CEC) density were assessed by LSCM. All patients completed 12 months of follow-up. Twenty-one patients were male and two were female, with a mean age of 39.5 ± 12.5 years. Six cases were due to acid burns, and the others were alkali burns. Palisades of Vogt were observed in all surviving grafts but were absent in graft failure. The epithelial cells in the central cornea of the failed graft had lost the classic polygonal morphology of the normal corneal basal epithelial cells. The cell density of LEC and CEC decreased significantly, whereas DC density increased in the failed grafts over time. In vivo LSCM is a useful tool for monitoring the cellular changes in KLAL grafts, and has the potential to diagnose the failure of KLAL grafts at the cellular level.

Pterygium is a chronic fibrovascular overgrowth on the corneal surface and is often associated with inflammation, astigmatism and obstructed vision. The common treatment is surgical removal but post-operative recurrences with more aggressive behavior are common. However, there is a controversy in the pathogenesis of primary pterygium between limbal stem cell failure versus proliferation. In this study, we explore the proliferative and migratory aptitude in pterygium by characterizing the growth and migration pattern of pterygial cells in the head (on the cornea), the neck (over the focal limbus), and the body (on the conjunctiva) epithelia of 12 full-length primary pterygia. Immunofluorescence and quantification analyses showed a spatial expression pattern of markers for stem cells, cell growth, and matrix metalloproteinases. Beside the basal epithelia in all three regions, p63α(strong) cells were located in suprabasal layers in head, weak in the body and absent in neck. Pertinent cell proliferation in head than body epithelia was revealed by its higher colony-forming efficiency. ATP-binding cassette transporter glycoprotein family member-2 and cytokeratin-15 were found mainly in the body basal epithelia, similar to that in normal conjunctiva. Much fewer proliferating stem-like cells in the neck region supported the limbal failure as a cause of pterygium formation. Pax6, matrix metalloproteinase-2 and -9 were more expressed in the head than in the other two regions. Our results indicate the importance of pterygium head in tissue growth and invasion and its likely involvement in post-operation recurrence.

To evaluate the short- and long-term in vivo safety and efficacy of topical bevacizumab (Avastin) application for treatment of corneal neovascularization secondary to a variety of corneal diseases. Thirty eyes of 27 patients with progressive corneal neovascularisation (not responding to conventional anti-inflammatory treatment) due to different underlying corneal diseases received topical bevacizumab (Avastin) eye drops (5 mg/ml Bevacizumab) for 0.5–12 months (five times/day on average). At each visit, a routine Snellen visual acuity assessment was performed, followed by ophthalmic examination including fluorescein staining. Changes of corneal neovascularization and vessel diameter were assessed using morphometry of standardized digital corneal photographs. Five patients (five eyes) developed new corneal epithelial defects during topical bevacizumab treatment. In 22 patients, no new epithelial defects were observed. None of the 27 patients complained about any drug-related ocular or systemic adverse events during follow-up. No allergic reactions were observed. Corneal photographs of 21 eyes (19 patients) could be assessed. The mean reduction in vascularized area during treatment was 61%. The mean reduction in vessel diameter under topical Avastin therapy was 24%. Off-label topical bevacizumab therapy against corneal neovascularization secondary to different corneal diseases was generally well-tolerated for up to 12 months. Bevacizumab (Avastin) eye drops inhibit corneal neovascularization, and lead to a reduction of the vessel diameter. Our results suggest that off-label use of Bevacizumab eye drops is a relatively safe and well-tolerated option for the treatment of corneal neovascularization. Care should be taken in patients with epithelial defects and neurotrophic keratopathy.

Nicotinamide adenine dinucleotide (NAD(+)), a precursor of molecules involved in cell regulatory processes, is released in extra-cellular compartments after stress or inflammation.This study investigates the expression in the human cornea of CD38 and CD157, two NAD(+)-consuming ectoenzymes and surface receptors. The analysis in corneal epithelial and stromal cells was performed by means of multiple approaches, which included immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and confocal microscopy. The presence of enzymatically active NAD(+)-consumers in intact corneal cells was analyzed by high performance liquid chromatography (HPLC)-based assays. The results obtained show that CD38 and CD157 are expressed constitutively by corneal cells: CD38 appears as a 45-kDa monomer, while CD157 is a 42- to 45-kDa doublet. The molecules are enzymatically active, with features reminiscent of those observed in human leukocytes. CD38 is expressed by cells of the suprabasal limbal epithelium, whereas it is not detectable in central corneal epithelium and stroma. CD157 is expressed by basal limbal clusters, a p63(+)/cytokeratin 19(+) cell subset reported to contain corneal stem cells, and by stromal cells. The results of the work indicates that the human cornea is equipped with molecular tools capable of consuming extracellular NAD(+), and that CD157 is a potential marker of corneal limbal cells in the stem cell niche. The presence and characteristics of these ectoenzymes may be exploited to design drugs for wound repair or for applications in tissue transplantation.

Background To evaluate the effect of lamellar keratoplasty combined with limbal stem cells, using amniotic membrane, autologous conjunctiva, and pseudopterygium to reconstruct external eyes for severe ocular burns with symblepharon. Methods Thirty eyes of 29 patients had severe symblephara resulting from eye burns. According to the range of the symblepharon and the loss of limbal stem cells, partial lamellar keratoplasty combined with partial limbal stem cell treatment was performed in 19 eyes, and total lamellar keratoplasty with total limbus was performed in 11 eyes. All patients had amniotic membrane and autologous conjunctival transplantation, and the pseudopterygium was preserved to reconstruct the fornix. Results Symblephara were completely relieved in 19 eyes. They remained partially in ten eyes in strip-like form, but seven of these were completely relieved after further autologous conjunctival transplantation. One eye was treated with tarsorrhaphy for eyelid malformation. The remaining pseudopterygium became thinner after the operation and showed no symblepharon. Immune rejection occurred in eight corneal grafts; clarity was restoredin fourof these,while there was graft neovascularization in the remaining four. Conclusions Depending on the area of symblepharon and the loss of limbal stem cells, partial or total lamellar keratoplasty combined with limbal stem cells, using amniotic membrane, autologous conjunctiva and pseudopterygium to reconstruct external eyes appears to be effective in treating severe ocular burns with symblepharon. Pseudopterygium can partly substitute autologous conjunctiva in ocular surface reconstruction.

Risk factors for corneal transplantation failure include both immunologic factors, such as graft rejection, corneal neovascularization, and peripheral anterior synechiae, as well as non-immunologic factors, such as ocular surface disorders (OSD) and glaucoma. This review highlights the necessity of having healthy ocular surface epithelia, tears, and eyelids. It presents different types of OSD, their underlying pathology, and their impact on native cornea and corneal grafts. In addition, a range of proposed donor and surgical factors influencing surface integrity following corneal transplant are addressed. Current medical and surgical research, both pre- and post-operative that promise to further improve the outcome of corneal grafts in the context of OSD are discussed.

To report histopathologic changes of the ocular surface pannus in patients with severe limbal stem cell deficiency (LSCD). Corneal and conjunctival pannus tissues from 29 patients undergoing ocular reconstruction with cultured limbal cell transplantation were included. The medical records of these patients were reviewed for demographics, aetiologic diagnosis, type of injury, interval between the initial insult and excision of pannus, and medical history involving human amniotic membrane (HAM) or limbal transplantation. The paraffin-embedded tissues were reviewed for epithelial changes, type–degree of fibrosis, degenerative changes, vascular changes, conjunctivalization of corneal surface, and evidence of residual HAM. We attempted a clinicopathologic correlation to understand the pathogenesis of pannus formation in LSCD. The 29 tissues were from 29 eyes of patients with primary aetiology of chemical burn in 89.6% (undetermined in 10.4%) of cases. The pannus showed epithelial hyperplasia in 62%, active fibrosis in 66%, severe inflammation in 21%, giant cell reaction in 28%, and stromal calcification in 14% cases. Goblet cells were seen over the cornea in 64% cases; their absence was associated with squamous metaplasia of the conjunctiva and with long duration of insult. Evidence of residual HAM was noted in 42% cases. The commonest cause of severe LSCD is alkali-induced injury. Goblet cells over the cornea were seen in 60% of cases. HAM used for ocular surface reconstruction could persist for long periods within the corneal pannus, thus raising the need for further studies with long-term follow-up.

Among all adult somatic stem cells, those of the corneal epithelium are unique in their exclusive location in a defined limbal structure termed Palisades of Vogt. As a result, surgical engraftment of limbal epithelial stem cells with or without ex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency. Nevertheless, compared to other stem cell examples, relatively little is known about the limbal niche, which is believed to play a pivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells. This review summarizes relevant literature and formulates several key questions to guide future research into better understanding of the pathogenesis of limbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing on the limbal niche.

Little information is currently available on the long-term safety of eyes following limbal tissue donation. We have therefore evaluated long-term refractive changes, corneal surface stability, and visual outcome following limbal tissue donation of 180 degrees with permanent amniotic membrane transplantation (AMT). Four healthy eyes of four patients underwent limbal keratectomy involving half of the limbal circumference (half limbal) for the purpose of limbal donation with permanent AMT. The best corrected visual acuity (BCVA), astigmatic changes by manifest refraction, and corneal surface changes as assessed by slit-lamp examination and by impression cytology were evaluated. The mean follow-up period was 20.8 months (range: 19–24 months). BCVA remained unchanged in three eyes (75%), but eye one showed a BCVA decrease of two lines due to newly developed filamentary keratitis caused by Sjogren’s syndrome. The average astigmatism change was an increase of 0.31 diopters. Conjunctival invasion toward the center beyond the edge of the keratectomy did not occur in any of the eyes, as assessed by impression cytology. Partial conjunctivalization within 2 h on keratectomy sites covered with amniotic membrane developed in three eyes (75%). Limbal donation of 180 degrees with permanent AMT seems to be a safe procedure, demonstrating stable astigmatism and an intact corneal surface for at least 1.5 years following the initial donation.