Corneal Limbal Stem Cell Deficiency Research Articles

Duloxetine enhances PAX6 expression and suppresses innate immune responses in murine LPS-induced corneal inflammation

Background-aimPAX6 is a key regulator of eye development and epithelial homeostasis in the cornea. When deficient, chronic corneal inflammation, neovascularization and limbal stem cell deficiency can occur. Here we investigated the potential of duloxetine, a generic serotonin reuptake inhibitor that can upregulate PAX6 in vitro, for its in vivo activity in the context of corneal inflammation. MethodsDuloxetine tolerance was tested in a human limbal stem cell line and isogenic CRISPR-knockout PAX6+/− cells. C57BL/6-Wildtype mice were administered duloxetine eye drops at concentrations of 1 μM - 2 mM and tested for toxicity and corneal PAX6 expression. In LPS-induced corneal inflammation in mice, duloxetine's effect on PAX6 expression, corneal opacification and inflammatory responses were evaluated by in vivo corneal imaging, immunostaining, and whole-transcriptome microarray analysis. ResultsNo toxicity was observed in vitro for duloxetine concentrations up to 10μΜ. In vivo, duloxetine drops were well-tolerated up to 50 μM. Duloxetine drops at 10μΜ significantly upregulated PAX6 protein levels in the cornea by 30 % within 2 days. In the LPS model, duloxetine resulted in a sustained 33 % PAX6 protein upregulation in the cornea at 7 days, and in reduced opacity within 2 days, accompanied by a significant dampening of IL-17A signaling, neutrophil degranulation, microglial activation, macrophage markers, and MMP expression, despite non-significant changes in total inflammatory cell infiltration. ConclusionShort-term administration of a repurposed generic drug, duloxetine, upregulates PAX6 protein levels in the cornea of mice and exerts an anti-inflammatory activity by dampening innate immune responses.

Advances in Cell Transplantation Therapy for Limbal Stem Cell Deficiency.

Limbal stem cells (LSCs) are essential for maintaining corneal transparency and ocular surface integrity. Many external factors or genetic diseases can lead to corneal limbal stem cell deficiency (LSCD), resulting in the loss of barrier and corneal epithelial cell renewal functions. Stem cell transplantation is one of the primary treatments for LSCD, including limbal transplantation and cultivated limbal epithelial transplantation. In addition, a variety of non-limbal stem cell lines have been experimented with for LSCD treatment. Biological scaffolds are also used to support in vitro stem cell culture and transplantation. Here, we review the mechanisms of corneal maintenance by LSCs, the clinical stage and surgical treatment of LSCD, the source of stem cells, and the biological scaffolds required for in vitro culture. This study is a narrative retrospective study aimed at collecting available information on various aspects of surgical treatments for LSCD. Relevant literature was searched in a range of online databases, including Web of Science, Scopus, and PubMed from 2005 to March, 2023. A total of 397 relevant articles were found, and 49 articles with strong relevance to the studies in this paper were obtained and analyzed. Moreover, 11 of these articles were on the concept of LSCD and the mechanism of LESCs maintaining the corneal epithelium, 3 articles on the staging and grading of LSCD, 17 articles on cell transplantation methods and donor cell sources, and 18 articles on scaffolds for delivering stem cells. We also summarized the advantages and disadvantages of different cell transplantation methods and the benefits and limitations of scaffolds based on the above literature. The treatment of LSCD is determined by the clinical stage and whether it involves monocular or binocular eyes. Appropriate surgical techniques should be taken for LSCD patients in order to reconstruct the ocular surface, relieve symptoms, and restore visual function. Meanwhile, biological scaffolds assist in the ex vivo culture and implantation of stem cells.

Management of corneal neovascularization: Current and emerging therapeutic approaches.

Corneal neovascularization (CoNV) is a sight-threatening condition affecting an estimated 1.4 million people per year, and the incidence is expected to rise. It is a complication of corneal pathological diseases such as infective keratitis, chemical burn, corneal limbal stem cell deficiency, mechanical trauma, and immunological rejection after keratoplasties. CoNV occurs due to a disequilibrium in proangiogenic and antiangiogenic mediators, involving a complex system of molecular interactions. Treatment of CoNV is challenging, and no therapy thus far has been curative. Anti-inflammatory agents such as corticosteroids are the mainstay of treatment due to their accessibility and well-studied safety profile. However, they have limited effectiveness and are unable to regress more mature neovascularization. With the advent of advanced imaging modalities and an expanding understanding of its pathogenesis, contemporary treatments targeting a wide array of molecular mechanisms and surgical options are gaining traction. This review aims to summarize evidence regarding conventional and emerging therapeutic options for CoNV.

Allogeneic limbo-deep anterior lamellar keratoplasty (Limbo-DALK)-A novel surgical technique in corneal stromal disease and limbal stem cell deficiency.

To describe a novel corneal surgical technique combining Deep Anterior Lamellar Keratoplasty (DALK) with grafting of allogeneic limbus (Limbo-DALK) for the treatment of eyes with corneal stromal pathology and limbal stem cell deficiency (LSCD). Clinical records of six Limbo-DALKs performed in five patients diagnosed with LSCD and corneal stromal pathology requiring keratoplasty were retrospectively reviewed. All patients were diagnosed with LSCD due to various pathologies including thermal and chemical burns, congenital aniridia or chronic inflammatory ocular surface disease. Parameters analysed included demographics, diagnoses, clinical history, thickness measurements using anterior segment OCT, visual acuity, and epithelial status. Regular follow-up visits were scheduled at 6 weeks as well as 3, 6, 9, and 12 and 18 months postoperatively. Main outcome measures were time to graft epithelialisation and the occurrence of corneal endothelial decompensation. Two grafts showed complete epithelial closure at 2 days, two at 14 days. In one eye, complete epithelial closure was not achieved after the first Limbo-DALK, but was achieved one month after the second Limbo-DALK. No endothelial decompensation occurred except in one patient with silicone oil associated keratopathy. Endothelial graft rejection was not observed in any of the grafts. Based on the data from this pilot series, limbo-DALK appears to be a viable surgical approach for eyes with severe LSCD and corneal stromal pathology, suitable for emergency situations (e.g. corneal ulceration with impending corneal perforation), while minimising the risk of corneal endothelial decompensation.

Blood‐derived trophic factors for the treatment of ocular surface diseases

The use of blood‐derived products (Eye Drop of Human Origin—EDHO; in Italian: Emocomponents for Not Transfusional Use, EUNT) for the treatment of various ocular surface diseases has become increasingly utilized in ophthalmic practice over the years. In particular, they are recommended as a third line option, when conventional tear substitutes, or active drugs, such as corticosteroids or Cyclosporine A, result unsuccessful in controlling either signs and, perhaps most importantly, subjective symptoms of discomfort and pain. The rationale for their use is based on the stimulation of cell proliferation and migration through the supply of metabolically active substances, in particular growth factors, and definitely mimicking the function of natural tears.EDHO have been used for the treatment of various ocular surface diseases, including dry eye, persistent corneal epithelial defect, corneal ulcer, neurotrophic keratitis, ocular surface burn, recurrent corneal erosion and limbal stem cell deficiency. However, the therapeutic potential of these products is still to be fully explored, as randomized clinical trials and internationally recognized harmonized guidelines are still needed to provide better evidence, improve quality of the final products and lead to a more widespread use of these therapies in daily clinical practice. The autologous source is still quite popular, although allogeneic sources appear to be a promising innovation. These might offer the solution of obtaining in advance products tailored for each patient and ocular surface disease, at scalable concentrations based on the severity of the disease and clinical parameters. There remain many unanswered questions regarding what might be considered optimal treatment, sketched as “The 5 Ws (and 2 H) for blood based eye drops”, to quote a review from our group (doi: 10.3390/jcm8091478). This presentation will discuss: 1. who is the patient to be treated, in terms of disease type, severity, and stage?; 2. Why is an EDHO needed, in terms of a target indication?; 3. When is it appropriate to prescribe an EDHO therapy?; 4. Where are EDHO be dispensed, and is a national/regional program a feasible solution to optimize resources?; 5. What is the EDHO of choice, which source and preparation are targeted for a given patient? And further: 1. how is EDHO standardized in terms of processing to ensure an optimal dilution, solvent, dispenser, storage time?; 2. How is EDHO delivered to the ocular surface, in terms of form, dose‐size modulation, length of treatment?An interdisciplinary work with transfusional medicine specialists is imperative to share information and competences, to achieve a better clinical management.

Evaluation of Limbal Stem Cells in Patients With Type 2 Diabetes: An In Vivo Confocal Microscopy Study.

This study aimed to investigate corneal limbus changes in patients with type 2 diabetes mellitus (DM) using in vivo confocal microscopy (IVCM) and explore the correlation between their ocular manifestations and systemic status. Fifty-five patients with type 2 DM and 20 age-matched controls were included. The following IVCM parameters were compared between the 2 groups: palisades of Vogt (POV), corneal epithelial thickness (CET), basal cell density (BCD), subbasal nerve plexus, and dendritic cell density. All subjects underwent blood and urine sampling for laboratory analysis, including fasting blood glucose, glycated hemoglobin, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C-reactive protein, urinary albumin-to-creatinine ratio, urine albumin, and urine creatinine. The correlations between IVCM parameters and blood biomarkers were detected. Receiver operating characteristic curve was used for selecting the cutoff value of risk factors for corneal stem cell injury in patients with DM. Compared with controls, patients with DM displayed a significant reduction of POV (superior region, P = 0.033; inferior region, P = 0.003; nasal region, P < 0.001; temporal region, P < 0.001), central CET (44.8 ± 3.6 μm vs. 51.9 ± 3.6 μm, P < 0.001), central corneal BCD (7415.5 ± 563.2 cells/mm 2 vs. 9177.9 ± 977.8 cells/mm 2 , P < 0.001), and peripheral corneal BCD (6181.3 ± 416.5 cells/mm 2 vs. 8576.3 ± 933.2 cells/mm 2 , P < 0.001). Dendritic cell density (41.0 ± 33.7 cells/mm 2 vs. 24.6 ± 7.8 cells/mm 2 , P = 0.001) was significantly higher in the DM group. The following weak correlations were shown between IVCM parameters and blood biomarkers: central corneal BCD was negatively correlated with DM duration (r = -0.3, P = 0.024), TC (r = -0.36, P = 0.007), and LDL (r = -0.39, P = 0.004). The presence of POV in the superior region was negatively correlated with TC (r = -0.34, P = 0.011) and LDL (r = -0.31, P = 0.022). Cutoff values of 1.215 mmol/L for HDL, 1.59 mmol/L for TG, or 4.75 mmol/L for TC were established to distinguish patients with a high risk from a low risk for stem cell damage. Patients with type 2 DM displayed a lower positive rate of typical POV and a decrease in BCD, CET, and subbasal nerve density. The most relevant indicators for stem cell phenotypes were DM duration, TC, and LDL. Lipid status in diabetic patients could be a predictor of risk for developing corneal limbal stem cell deficiency. Further studies with larger sample sizes or basic research are needed to verify the results.

Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

BackgroundMesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium.MethodsLimbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs.ResultsThe serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90+, CD73+, CD105+, and ≤ 6% were positive for CD45−, CD34−andHLA-DR−. Immunofluorescence analysis confirmed similar expression of Pax6+, COLIV+, ABCG2+, ABCB5+, VIM+, CD90+, CD105+, CD73+, HLA-DR−andCD45−, αSMA− in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium.ConclusionThe findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.

3D bioprinting as a prospective therapeutic strategy for corneal limbal epithelial stem cell deficiency.

Limbal epithelial stem cells (LESCs) are responsible for the maintenance and repair of the corneal surface. Injuries and diseases of the eye may result in a vision condition called limbal stem cell deficiency (LSCD). Without limbal stem cells, the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy as a treatment method was first described in 1997, and LESCs cultured from either patients or donors have been used to treat LSCD successfully. However, the main source of cornea for LSCD treatment is from donors, which are too few to meet the demand (less than 1:70 of cases). The global shortage of donor cornea promotes the need for studies exploring corneal limbus alternatives. Many problems still remain unresolved, such as original geometry reconstruction, corneal epithelial regeneration, and ocular optical function restoration. 3D bioprinting has garnered tremendous attention in recent years, and significant advances have been made in fabricating cell-laden scaffolds. These advancements could lead to a promising treatment for LSCD. It is possible that alternative limbus stem cells can be constructed using 3D printing, which, in corneal limbus regeneration, enables personalized corneal implants and fabrication of single- or multilayer corneal limbus equivalents with corneal limbal stem cells. In this review, the progress, applications, and limitations of the most influential works regarding current treatments of LESC deficiency are discussed. The advantages of 3D bioprinting are illustrated, and some first promising steps toward the creation of a functional cornea limbus with 3D bioprinting are discussed. Finally, insights into the prospects and technical challenges facing the future research of 3D bioprinting of corneal limbus alternatives in vivo and in vitro are provided.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Corneal Epithelial Repair In Vitro.

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.

Therapeutic effect of secretome from TNF-α stimulated mesenchymal stem cells in an experimental model of corneal limbal stem cell deficiency.

To explore the secretome efficacy in tumor necrosis factor (TNF)-α stimulated mouse mesenchymal stem cells (MSCs) in a murine model of corneal limbal alkali injury. Corneal limbal stem cell deficiency (LSCD) was created in the eyes of male C57 mice. Concentrated conditioned medium from TNF-α stimulated MSCs (MSC-CMT) was applied topically for 4wk, with basal medium and conditioned medium from MSCs as controls. Corneal opacification, corneal inflammatory response, and corneal neovascularization (NV) were evaluated. Corneal epithelial cell apoptosis, corneal conjunctivation, and inflammatory cell infiltration were assessed with TUNEL staining, CK3 and Muc-5AC immunostaining, and CD11b immunofluorescence staining, respectively. The effect of TSG-6 was further evaluated by knockdown with short hairpin RNA (shRNA). Compared to the controls, topical administration of MSC-CMT significantly ameliorated the clinical symptoms of alkali-induced LSCD, with restrained corneal NV, reduced corneal epithelial cell apoptosis, and inhibition of corneal conjunctivation. In addition, MSC-CMT treatment significantly reduced CD11b+ inflammatory cell infiltration, and inhibited the expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6). Furthermore, the promotion of corneal epithelial reconstruction by MSC-CMT was largely abolished by TSG-6 knockdown. Our study provides evidence that MSC-CMT enhances the alleviation of corneal alkali injuries, partially through TSG-6-mediated anti-inflammatory protective mechanisms. MSC-CMT may serve as a potential strategy for treating corneal disorders.

Клинические результаты трансплантации аутологичного культивированного эпителия полости рта при дисфункции стволовых клеток лимба роговицы

The review presents an analysis of clinical trials results for autologous cultured oral mucosal epithelium transplantation (COMET) in patients with bilateral corneal limbal stem cell deficiency (LSCD) over the past 15 years. Detailed characteristics and evaluation are given for anatomical outcomes, visual acuity changes, and complication rates. The results obtained during the analysis confirm the consistency of the concept of corneal re-epithelization by means of COMET. COMET promoted persistent corneal re-epithelization in 81.5% of cases, and visual acuity improvement in 78.8% of patients with LSCD. COMET does not require systemic immunosuppression, and it is accompanied by much smaller numbers and significantly lower grades of complications compared with keratoprosthesis. About 15% of patients experienced developing superficial peripheral corneal neovascularization regressed spontaneously by 12 months of observation. Based on the COMET clinical trials results, the management of patients with bilateral LSCD is under optimization by reference to the pathogenesis of the underlying disease. Thus, autologous cultured oral mucosal epithelium transplantation seems promising for further studies and introduction into routine clinical practice. Key words: limbal stem cell deficiency, ocular surface reconstruction, corneal epithelium, oral mucosal epithelium, transplantation.

Ocular Features in a Large Cohort of Indians With Xeroderma Pigmentosum.

Xeroderma pigmentosum (XP) is an extreme hypersensitivity to sunlight causing skin freckling and pigmentary changes because of defective DNA repair mechanisms. The purpose of this article is to evaluate the spectrum of ocular and systemic features in XP at a tertiary eye care center in India over 32 years. Data from 418 eyes of 209 patients diagnosed with XP from 1987 to 2018 were reviewed retrospectively for demographics, complaints, ocular features, systemic associations, and their management. Median age at diagnosis was 2 years (interquartile range, 0.5-5 years). A total of 124 patients (59.3%) were men. There was parental consanguinity in 74.4% cases. Common ocular complaints were photophobia (47.1%), ocular discomfort (45%), defective vision (36.6%), redness (13.4%), tissue growth (12%), white spot (11.2%), and pain (10.5%). At presentation, 43.5% had corneal scars (45.5% were located inferiorly and 70.9% covered visual axis). Corneal vascularization and limbal stem cell deficiency were noted in 37.4%. A total of 56% of patients had at least 1 tumor, and 6.7% had neurological abnormalities. At least 1 ocular surgery was performed in 37.8% of patients. Ophthalmic surgical interventions included tumor excision (23%), keratoplasty (13.4%), and nontumor ocular surface surgery (3.4%). XP is a disorder that has high ocular morbidity in Indian patients. The recognition of common signs and symptoms and relative frequency of various ocular complications with time trends will help in managing and reducing the sequelae of this otherwise untreatable and progressive disease.

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells

PurposeTo construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation. MethodsA stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. ResultsThe hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. ConclusionThe tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

Ethical issues in living-related corneal tissue transplantation

The cornea was the first human solid tissue to be transplanted successfully, and is now a common procedure in ophthalmic surgery. The grafts come from deceased donors. Corneal therapies are...

Long-term outcome of using Prosthetic Replacement of Ocular Surface Ecosystem (PROSE) as a drug delivery system for bevacizumab in the treatment of corneal neovascularization

PurposeTo report the long-term outcome of Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) for delivery of bevacizumab in the treatment of corneal neovascularization (KNV). MethodsRetrospective, non-comparative, interventional case series of 13 sequential patients treated for KNV at the BostonSight between 2006 and 2017. In all cases, PROSE treatment was initiated for management of ocular surface disease and patients wore PROSE consistently on a daily wear basis prior to bevacizumab treatment. Patients applied a drop of 1% preservative free bevacizumab to the reservoir of PROSE device twice daily. Patients continued with daily wear of the device during treatment and afterwards. Results13 patients (8 female and mean age of 45 years) are included with a mean follow-up of 5.1 years (range 6 months–11 years). Underlying ocular diagnoses included Stevens-Johnson syndrome (7), ocular chronic graft-versus-host disease (2), corneal transplant (2), contact lens-related corneal ulcer and limbal stem cell deficiency (1), and familial dysautonomia (1). Median duration of bevacizumab use was 6 months (range 3 months–10 years). Twelve cases (92%) had regression of KNV and 10 cases (77%) had improved best-corrected visual acuity (BCVA) with treatment. Median BCVA improved from −1.1 (LogMAR) at baseline, to −0.66 at end of bevacizumab treatment, and remained −0.63 at last follow-up (P = 0.047). KNV progressed in one eye after discontinuation of bevacizumab. There were no ophthalmic or systemic complications. ConclusionsTopical bevacizumab used in PROSE is effective in treating KNV and improving vision. Long-term follow-up reveals durable response and no complications.

Corneal Limbal Stem Cell Deficiency in Children with Stevens–Johnson Syndrome

To determine the incidence of corneal limbal stem cell deficiency (LSCD) as chronic ocular sequelae in children with Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and analyze the factors associated with LSCD development. Retrospective case series. Medical records were reviewed of pediatric patients who had been admitted to Seoul National University Hospital with SJS/TEN and who were diagnosed as having acute ocular involvement. For each record, the following data were collected: demographic information, underlying diseases, causative agents, acute systemic and ocular manifestations, systemic and ocular treatments, chronic ocular complications including LSCD, and visual acuities. Of 19 children with SJS/TEN, LSCD developed in 6 (32%) patients at a mean of 12.3 ± 21.3months after the onset of SJS/TEN (median 3.5months). Severity of acute systemic involvement including elevation of liver enzyme levels and serum C-reactive protein levels was significantly correlated with the development of LSCD (P= .0038) and chronic ocular complications (P= .0044). The presence of corneal epithelial defect necessitating the use of therapeutic contact lenses in the acute phase was also associated significantly with LSCD development. Combined penetrating keratoplasty and limbal allograft were performed in 3 of 6 LSCD patients, and grafts failed in 2 (67%) patients because of infection. At the last follow-up, visual acuities were significantly poorer in patients with LSCD compared with those without LSCD (P= .0055). Corneal LSCD occurred in 32% of pediatric patients with SJS/TEN, leading to poor visual outcome. Severity of acute systemic involvement was significantly associated with the development of LSCD and chronic ocular complications.

Ocular Surface Reconstruction with the Autologous Conjunctival Epithelium and Establishment of a Feeder-Free and Serum-Free Culture System

Transplantation of the autologous cultured corneal limbal epithelium and oral mucosal epithelium is a standard technique for ocular surface reconstruction under corneal limbal stem cell deficiency. As an option for bilateral cases, we recommend utilization of autologous conjunctivae for ocular surface reconstruction. Autologous conjunctival epithelium sheet transplantation was effective for bilateral corneal limbal stem cell deficiency without symblepharon or severe keratinization. Moreover, we established a feeder-free and serum-free culture system of the limbal epithelium. This system can be applied for culturing conjunctival epithelia. Autologous cultured conjunctival epithelium transplantation is a practical option for treating bilateral corneal limbal stem cell deficiency.

Amniotic membrane use for management of corneal limbal stem cell deficiency

The current article reviews the most recent surgical techniques for management of corneal limbal stem cell deficiency (LSCD) using amniotic membrane tissue. Early successes with amniotic membrane transplantation (AMT) for the treatment of ocular surface disorders have encouraged clinicians to investigate new applications. The use of AMT as a temporary patch in emergency cases in which LSCD may develop has considerably improved the prognosis of these patients. Amniotic membrane does not have stem cells of its own, but it supports regeneration of limbal epithelial stem cells (LESCs). Similarly, the combination of AMT with classic surgical techniques has enhanced the surgical success rates in most case series. Furthermore, based on its advantageous properties as a cell carrier, new applications to support in-vivo and ex-vivo cell expansion have been reported recently. LSCD constitutes a general indication for AMT. Based on the clinical scenario, AMT may be performed alone to support regeneration of LESCs, in combination with other surgical techniques, or even supporting the in-vivo or ex-vivo expansion of LESCs.

Diagnosis of corneal limbal stem cell deficiency.

A state of limbal stem cell deficiency (LSCD) can be secondary to a number of etiologies, resulting in either a reduction in the total number of limbal stem cells or an abnormality in stem cell function. Initially, the epithelium becomes irregular and hazy; however, this condition may progress to persistent corneal epithelial defects, stromal scarring, ulceration, and even perforation. Since LSCD secondary to a variety of etiologies may be reversible, and various factors are prognostic of disease progression, timely diagnosis is important. This review will describe current knowledge of diagnostic techniques for LSCD and understanding of epithelial stem cell function. Conjunctivalization, regarded as the most reliable clinical finding diagnostically, can be identified as late staining of epithelium with fluorescein. While identifying loss of the palisades of Vogt by slit-lamp examination, can provide a high suspicion of LSCD, but this is not diagnostic. Impression cytology is a simple, noninvasive technique that aids in the diagnosis of LSCD, but a negative result also cannot rule out the diagnosis. Recent findings have also shown that imaging techniques including in-vivo confocal microscope and optical coherent tomography can also aid in diagnosing LSCD; however, several challenges remain before these techniques become standard diagnostic methods in clinical practice. Meanwhile, determination of the absence of limbal epithelial crypts and focal stromal projections using image reconstruction techniques may assist in the diagnosis of LSCD. Furthermore, histologic markers may help not only to improve sensitivity and specificity of conventional techniques in diagnosis of LSCD, but also to identify human limbal stem cells and determine their number and function in LSCD. Efforts to develop and improve techniques for diagnosing LSCD are ongoing. Increased knowledge of limbal stem cells and components of their niches may not only help in understanding the pathogenesis of LSCD but may improve its diagnosis, thereby ameliorating the prognosis of patients with this devastating disease.

Stem cells in regenerative medicine – from laboratory to clinical application – the eye

Stem cells are currently one of the most researched and explored subject in science. They consstitue a very promising part of regenerative medicine and have many potential clinical applications. Harnessing their ability to replicate and differentiate into many cell types can enable successful treatment of diseases that were incurable until now. There are numerous types of stem cells (e.g. ESCs, FSCs, ASCs, iPSCs) and many different methods of deriving and cultivating them in order to obtain viable material. The eye is one of the most interesting targets for stem cell therapies. In this article we summarise different aspects of stem cells, discussing their characteristics, sources and methods of culture. We also demonstrate the most recent clinical applications in ophthalmology based on an extensive current literature review. Tissue engineering techniques developed for corneal limbal stem cell deficiency, age-related macular degeneration (AMD) and glaucoma are among those presented. Both laboratory and clinical aspects of stem cells are discussed.