Limbal Stem Cell Deficiency Patients Research Articles

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.

Prevalence of limbal stem cell deficiency at an academic referral center over a two-year period.

To evaluate the prevalence and clinical characteristics of limbal stem cell deficiency (LSCD) in the setting of a tertiary referral cornea practice at an academic center. A retrospective chart review was performed to identify all unique medical record numbers (MRNs) presenting to a single cornea specialist (JHH) at the University of Minnesota during calendar years 2019 and 2020. Records were queried and confirmed for a diagnosis of LSCD. Clinical characteristics of identified patients, including demographics, etiology of LSCD, severity of LSCD, treatment, and best corrected visual acuity (BCVA) at final follow-up, were documented. In total 1436 unique MRNs were identified over the study period. There were 61 individuals (91 eyes) diagnosed with LSCD, resulting in a prevalence of 4.25% (95% CI, 3.33-5.42). Of 91 eyes, 60 eyes were bilateral (65.9%). Among all eyes, ocular surface burns were the most common etiology (18.7%) followed by iatrogenic or medicamentosa (15.4%). There were 51 eyes (56.0%) that underwent some form of transplantation. The median BCVA at final follow-up was Snellen 20/80 (range 20/20 to no light perception). The prevalence of LSCD found at a cornea subspecialty tertiary referral center in our study was much higher than previously reported prevalence rates. This may reflect referral bias and potential underdiagnosis of LSCD in practices outside of subspecialty referral centers. The high prevalence rate in our study also suggests that LSCD patients are concentrated in subspecialty referral practices, with many having high morbidity disease. This constitutes a major health burden for these practices.

Evaluation of the factors that influence surgical outcome in conjunctival-limbal allograft transplantation.

To evaluate the surgical results and the variables affecting the outcomes in conjunctival-limbal allograft transplantation (CLAL). Patients who underwent CLAL for limbal stem cell deficiency (LSCD) between 2007 and 2019 were included in the study. LSCD staging was performed according to the staging system developed by the 'Limbal Stem Cell Working Group'. Stage 1C and higher stage LSCD patients were included in the study. 'Successful surgical outcome' was defined as improvement in LSCD stage at 1 year postoperatively. A total of 19 eyes of 19 LSCD patients were included. The mean age of the patients was 40.21 ± 14.65 (6-65) years, and the male/female ratio was 12/7. CLAL was performed in 9 (47.3%) patients with Stage 2B, 9 (47.3%) patients with Stage 3 and 1 (5.4%) with Stage 1C. LSCD aetiology; chemical injury (12), vernal keratoconjunctivitis (2), aniridia (1), corneal degeneration (1), and unknown (3). Surgery was successful in 52.6% of cases. Surgical success was associated with lower LSCD stage (p = 0.04). Lower grades of chemical injury at presentation and a longer time interval between injury and CLAL were associated with higher surgical success (p = 0.001; p = 0.001). The mean postoperative follow-up time was 50.77 ± 29.46 (6-98) months. Despite graft rejection and long-term use of immunosuppressants, CLAL is still one of the most preferred techniques in the treatment of bilateral LSCD. Preoperative LSCD stage and degree of chemical burn are important factors affecting the surgical outcome. Also, CLAL surgery should not be rushed and should be performed when inflammation has subsided.

Диагностика синдрома лимбальной недостаточности

Relevance. Limbal stem cell deficiency (LSCD) is a pathology that leads to substantial vision decrease, corneal neovascularization, corneal epithelial persistent erosions and have a negative impact on patient's the quality of life. It is known that the efficiency of corneal transplantation in LSCD patients is low and requires repeated interventions. One of the important problems is the lack of a clear diagnostic algorithm and a standardized set of diagnostic techniques for this condition. Purpose. Systematically analyze domestic and foreign literature on the currently existing methods for diagnosing LSCD. Material and methods. A systematic analysis of scientific publications from 1992 to 2021 was carried out. Its included domestic and foreign authors on the resources of PubMed, Medline, eLibrary, as well as dissertations and textbooks devoted to currently existing methods for diagnosing LSCD. Results. In addition to biomicroscopy, the main in vivomethods for diagnosing LSCD are staining of the ocular surface, impression cytology with the detection of specific cytokeratins, confocal microscopy, optical coherence tomography of the anterior segment (OST) and OST-angiography. Conclusion. Physician awareness and vigilance are required for the diagnosis of LSCD, especially in patients with conjunctivalization and corneal neovascularization. It is impossible to use only biomicroscopy and the patient's complaints, since LSCD is not always obvious. That is why diagnostics should include an integrated approach, including all available research methods, which are briefly described in Table No. 1 with their disadvantages and advantages. Key words: limbal stem cell deficiency, impression cytology, laser confocal scanning microscopy, goblet cells, cytokeratin, penetrating keratoplasty, cytokines

Cultured Autologous Corneal Epithelia for the Treatment of Unilateral Limbal Stem Cell Deficiency: A Case Series of 15 Patients.

Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27–79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60–98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.

Computerized Detection of Limbal Stem Cell Deficiency from Digital Cornea Images

Limbal Stem Cell Deficiency (LSCD) is an eye disease that can cause corneal opacity and vascularization. In its advanced stage it can lead to a degree of visual impairment. It involves the changing in the semispherical shape of the cornea to a drooping shape to downwards direction. LSCD is hard to be diagnosed at early stages. The color and texture of the cornea surface can provide significant information about the cornea affected by LSCD. Parameters such as shape and texture are very crucial to differentiate normal from LSCD cornea. Although several medical approaches exist, most of them requires complicated procedure and medical devices. Therefore, in this paper, we pursued the development of a LSCD detection technique (LDT) utilizing image processing methods. Early diagnosis of LSCD is very crucial for physicians to arrange for effective treatment. In the proposed technique, we developed a method for LSCD detection utilizing frontal eye images. A dataset of 280 eye images of frontal and lateral LSCD and normal patients were used in this research. First, the cornea region of both frontal and lateral images is segmented, and the geometric features are extracted through the automated active contour model and the spline curve. While the texture features are extracted using the feature selection algorithm. The experimental results exhibited that the combined features of the geometric and texture will exhibit accuracy of 95.95%, sensitivity of 97.91% and specificity of 94.05% with the random forest classifier of n = 40. As a result, this research developed a Limbal stem cell deficiency detection system utilizing features’ fusion using image processing techniques for frontal and lateral digital images of the eyes.

Cultivated autologous oral mucosal epithelial transplantation

According to the World Health Organization, corneal blindness is the fourth most common cause of blindness and visual impairment worldwide. In Russia, up to 18% of blindness is caused by corneal damage. Limbal stem cell deficiency (LSCD) is one of the causes of corneal blindness and visual impairment due to anterior epithelial replacement with fibrovascular pannus. Bilateral LSCD may develop in patients with aniridia, Steven-Jones syndrome, and severe corneal burns of both eyes, leading to severe decrease in visual acuity in both eyes and, as a consequence, physical disability associated with blindness. In such cases, cell therapy, based on autologous oral epithelial culture as an alternative to allogeneic limbus transplants, is proposed for reconstruction of the anterior corneal epithelium. This new treatment method promotes corneal reepithelization, better visual acuity, reduced nonspecific ocular complaints and improved quality of life of patients. The effectiveness and significant increase in the frequency of transparent engraftment of donor corneas after cell therapy drives huge interest in this topic all over the world. This review presents literature data on the features of histotopography and methods for obtaining a cultured autologous oral mucosal epithelium, on cell markers that are used to identify epithelial cells, and on methods for creating cell grafts for subsequent transplantation to the corneal surface in LSCD patients.

In vivo confocal microscopic characteristics of limbal stem cell deficiency

Objective: To investigate the cellular changes and quantitative analysis of basal cell density (BCD) and corneal epithelial thickness (CET) in limbal stem cell deficiency (LSCD) by in vivo confocal microscopy (IVCM). Methods: Prospective case-control study. A total of 35 eyes of 23 patients diagnosed with LSCD and 25 eyes from normal subjects were included in this study. Based on slit-lamp presentation and the global consensus on classification, the LSCD patients were classified into LSCD Ⅰ, LSCD Ⅱ and LSCD Ⅲ. Confocal images of the central cornea, and the superior, inferior, nasal and temporal limbus were inspected by IVCM. Morphologic characteristics of LSCD were summarized. The BCD and CET in all locations were measured. ANVOA or Kruskal-Wallis test was used for analysis when appropriate. A receiver operating characteristic was used to detect the diagnosis efficiency of BCD and CET. Results: The characteristics in the corneal epithelium of LSCD on IVCM included nested corneal epithelial cells, goblet cells with hyper-reflective spots, irregular basal cells and decreasing subbasal nerve density. The mean BCD of the LSCD group was (8 976±1 096) cells/mm2 in the central cornea. Compared to the control group, the BCD in the central cornea, superior, inferior, nasal and temporal limbus decreased by 30.2%, 26.0%, 28.7%, 29.3% and 30.2%, respectively (all P<0.007). The CET in the central cornea was (47.3±8.1) μm. The CET in the central cornea, superior, inferior, nasal and temporal limbus decreased by 27.9%, 23.7%, 20.6%, 26.9% and 23.1%, respectively, compared to the control group (all P<0.007). There was a decline of BCD and CET in more serious LSCD. Additionally, the decline of BCD and CET was shown in the unaffected region. The receiver operating characteristic showed the diagnosis efficiency of BCD in the corneal center and limbus (0.931 and 0.916) was superior to CET (0.853 and 0.817). Conclusions: There was a series of characteristic cellular changes in LSCD on IVCM. Both BCD and CET decreased significantly in LSCD. The BCD had higher diagnostic efficiency for LSCD.(Chin J Ophthalmol, 2020, 56:447-455).

The utility of anterior segment optical coherence tomography angiography for the assessment of limbal stem cell deficiency

PurposeTo determine the utility of anterior segment optical coherence tomography angiography (AS-OCTA) in assessing limbal stem cell deficiency (LSCD). MethodsTwenty-six eyes of 24 LSCD patients, classified clinically into stage I, II and III, and 12 eyes of 12 healthy subjects were included. AS-OCTA images were analyzed by two masked observers, measuring the maximum corneal vascular extension (CoVE) from the limbus to the furthest vessel over the cornea, and corneal vascular thickness (CoVT) from the most superficial to the deepest corneal vessel. ResultsCoVE was 0.27 ± 0.10, 0.79 ± 0.21, 1.68 ± 0.89 and 2.53 ± 0.82 mm in controls, stage I, II and III LSCD, respectively (p < 0.001). The CoVT was 51.0 ± 19.4, 113.7 ± 36.6, 129.7 ± 39.3 and 336.0 ± 85.0 μm, respectively (p < 0.001). There was a significant difference in CoVE and CoVT between all stages compared to controls, and between stage I and III LSCD (p < 0.001). Further, CoVE showed a significant difference between stage I and II, whereas CoVT showed a significant difference between stage II and III LSCD (p < 0.001). BCVA showed strong correlation with CoVT (r = 0.765, p < 0.001) and moderate correlation with CoVE (r = 0.547, p = 0.001). AS-OCTA parameters showed excellent intra- and inter-class correlation coefficients (>0.900). ConclusionLSCD demonstrates significant changes in CoVE and CoVT as early as stage I LSCD in comparison to controls. CoVE and CoVT strongly correlate to both disease severity and BCVA. AS-OCTA may provide novel quantitative and non-invasive parameters to assess LSCD.

Keratograph 5M As A Useful And Objective Tool For Evaluating The Ocular Surface In Limbal Stem Cell Deficiency.

PurposeIn limbal stem cell deficiency, both the Ocular Surface Disease Index (OSDI) questionnaire and tear break-up time (BUT) are comparable between traditional methods and the Keratograph 5M. In this study, we aimed to correlate OSDI with Keratograph 5M interviewed OSDI, as well as slit-lamp tear BUT with Keratograph 5M noninvasive tear break-up time (NIKBUT) in limbal stem cell deficiency.Patients and methodsThirty-eight limbal stem cell-deficiency patients (76 eyes) from Virgen Macarena-Rocio Hospital (Seville, Spain) underwent this diagnostic test study. All patients completed the traditional OSDI. We measured the BUT, performed a Keratograph 5M analysis of NIKBUT first (employed for the analysis) followed by the average NIKBUT, the level of dryness, and conducted the OSDI questionnaire through an interview. For each pair of tests, we analyzed the means and applied an intraclass correlation coefficient (r), creating a Bland-Altman plot for data dispersion.ResultsAverage values were 47.5 points (±25.8), and 47.3 points (±27.5) for traditional OSDI and Keratograph OSDI, respectively (P =0.87); the r value indicates good agreement (0.72). The Bland-Altman plot followed a linear pattern, and the results were similarly distributed. The NIKBUT mean was shorter than the BUT mean (P = 0.007); the r value indicates moderate agreement (0.574). The Bland-Altman plot formed an almost horizontal line, with almost all values between the mean and two standard deviations.ConclusionKeratograph 5M is useful for the evaluation of the ocular surface in limbal stem cell deficiency. NIKBUT can substitute BUT based on its advantages of being noninvasive, objective, with intraobserver and interobserver repeatability and reliability. The Keratograph 5M OSDI is comparable to the traditional questionnaire.

Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus

Limbal Stem Cell Deficiency (LSCD) is a painful and debilitating disease that results from damage or loss of the Corneal Epithelial Stem Cells (CESCs). Therapies have been developed to treat LSCD by utilizing epithelial stem cell transplants. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients. A major hurdle has been the lack of vertebrate models to study CESCs. Here we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea stem cell deficiency, such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and can stimulate mitosis in the remaining cells in the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one half of the cornea. These cases initially develop localized stem cell deficiency characteristics on the treated side. The localized PUV treatment is also capable of stimulating some mitosis in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored. Our study provides insight into the response of cornea cells following stem cell depletion, and establishes Xenopus as a suitable model for studying CESCs, stem cell deficiency, and other cornea diseases. This model will also be valuable for understanding the nature of transplanted CESCs, which will lead to progress in the development of therapeutics for LSCD.

Comparison of the efficacy of different cell sources for transplantation in total limbal stem cell deficiency.

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 ± 9.9months in the ACLET group and 16.1 ± 5.8months 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 12months 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.

Ocular Surface Microbial Flora in Patients with Chronic Limbal Stem Cell Deficiency Undergoing Cultivated Oral Mucosal Epithelial Transplantation.

PURPOSE:The purpose of this study is to analyze the ocular surface microbial flora in patients with chronic limbal stem cell deficiency (LSCD) due to Stevens–Johnson Syndrome (SJS) and ocular chemical injury undergoing cultivated oral mucosal epithelial transplantation (COMET).METHODS:Patients of SJS and chemical injury who had bilateral total LSCD planned for COMET were studied. Conjunctival swab was taken before surgery. Parameters evaluated were organism cultured, sensitivity pattern, frequency of positive culture, and clinical impact on management strategy.RESULTS:Thirteen patients were included in which nine were males and four females. All patients had positive conjunctival swab culture. Most common organism isolated was Staphylococcus epidermidis, followed by Staphylococcus aureus and Pseudomonas aeruginosa. The staphylococcal species isolated were sensitive to all the conventional antibiotics while Pseudomonas cultured showed resistance to cefuroxime, ceftriaxone, and ceftazidime. Repeat conjunctival swab sent after a week of topical antibiotic therapy yielded positive culture of the same organism twice in 25% (3/12), thrice in 58.3% (7/12), and four times in 16.6% (2/12) of the patients. One patient had a polymicrobial flora with positive yield of S. aureus (thrice), S. epidermidis (twice), and P. aeruginosa (twice) in consecutive conjunctival swab culture in the absence of clinical infection. Two patients with persistent positive cultures had to undergo repeat oral mucosal harvesting as the transplantation of the cultivated explants had to be deferred.CONCLUSION:Ocular surface in LSCD patients yielded pathogenic organisms on culture. Poor ocular surface with absent tear film could be the contributing factors. It is important to perform the conjunctival swab culture before COMET surgery.

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells.

Corneal blindness caused by limbal stem cell deficiency (LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.

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.

Cincinnati Protocol for Preoperative Screening and Donor Selection for Ocular Surface Stem Cell Transplantation.

To describe our process for preoperative screening and donor selection for ocular surface stem cell transplantation (OSST). A 7-year retrospective chart review was performed on limbal stem cell deficiency patients. The inclusion criterion was all patients who underwent an OSST procedure. The exclusion criterion was eyes with unilateral disease in which an autograft was performed. Data for human leukocyte antigen (HLA) typing, virtual crossmatching, donor-specific antibody, and panel reactive antibody level were obtained. Of the included 142 eyes (104 patients), 19 patients had no recorded living donor availability data, and HLA typing was not performed on 16 patients. A total of 94 donors (mean 1.4 donors/patient, range 1-6) were tested for 67 recipients. For 2 patients with graft-versus-host disease, no further HLA typing was needed, as the donors were known HLA-identical donors. For 47 patients, only 1 donor was tested, whereas multiple donors underwent HLA typing for 20 patients. There were 73 ABO (blood group)-compatible matches for the 61 tested recipients, and only 1 recipient did not have any ABO-compatible donor. For the virtual crossmatch, there were 5 patients who did not have a compatible donor (positive virtual crossmatch). The best available donor match was a sibling for 41 recipients (65%), a parent for 19 recipients (30%), and an offspring for 3 recipients (5%). Our protocol for OSST preoperative screening and donor selection minimizes the antigenic burden for transplanted tissue by selecting the best available donor match.

A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells

Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro.Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded.Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC).Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.

Infectious Keratitis in Limbal Stem Cell Deficiency: Stevens-Johnson Syndrome Versus Chemical Burn.

To investigate the incidence, clinical and microbiological characteristics, risk factors, and therapeutic outcome of infectious keratitis in patients with limbal stem cell deficiency (LSCD) related to Stevens-Johnson syndrome (SJS) and corneal chemical burn. Medical records of 90 eyes of 59 patients who were diagnosed with LSCD resulting from SJS (52 eyes of 29 patients) or corneal chemical burn (38 eyes of 30 patients) were reviewed. Infectious keratitis developed in 35% of LSCD patients with SJS (18 eyes, 14 patients) and in 18% of those with chemical burn (7 eyes, 7 patients). The development of infectious keratitis in SJS was significantly associated with the severity of chronic ocular surface complications in the cornea, conjunctiva, and eyelids and with the use of topical corticosteroids during the disease course. All cases of infectious keratitis following chemical burn occurred in patients with grade III or IV burn by Roper-Hall classification. Approximately 83% of culture-proven cases of infectious keratitis were bacterial infection, most of which (80%) were caused by Gram-positive bacteria. For resolution of infection, 17 eyes (68%) received surgery in addition to medical treatment, whereas 8 eyes (32%) received medical treatment alone. After infection resolution, the final visual acuity was decreased in 10 eyes (40%) compared with before infection. Infectious keratitis is a common complication of LSCD associated with SJS or severe chemical burn to the cornea. Despite medical and surgical treatments, the visual outcome is poor.

Epithelial Thinning in Limbal Stem Cell Deficiency

To investigate the epithelial thickness in the cornea and limbus in limbal stem cell deficiency (LSCD) by using in vivo laser scanning confocal microscopy. Cross-sectional comparative study. Confocal images of 48 eyes of 35 patients with LSCD collected by the Heidelberg Retina Tomograph III Rostock Corneal Module Confocal Microscope from 2010 to 2014 were analyzed. Volume Z-scans of the central cornea and the superior, nasal, inferior, and temporal limbus were included in the analysis. Eleven normal eyes served as control. Epithelial thickness in all locations was measured by 2 independent observers. The mean epithelial layer thickness was 48.6 ± 2.3 μm in the central cornea and 63.7 ± 11.3 μm in the limbus in the control. Compared with the epithelial thickness in normal control, the epithelial thickness in LSCD patients was reduced by an average of 20.2% in the central cornea and 38.5% in the limbus (all P < .05). The mean corneal epithelial thickness in patients with LSCD reduced 7.6%, 20.8%, and 61.3% in the early, intermediate, and late stage, respectively, compared to the control. In the limbus, the overall epithelial thickness decreased 30.0%, 39.7%, and 62.8% in the early, intermediate, and late stage of LSCD, respectively (all P < .05). Epithelial thinning correlated with the severity of LSCD in both cornea and limbus. In eyes with sectoral LSCD, a similar degree of epithelial thinning was also detected in the clinically unaffected limbal regions. Both corneal and limbal epithelia become progressively thinner in LSCD. Epithelial thickness could be used as a diagnostic measure of LSCD.

Culture optimisation of CD34‐positive corneal stromal stem cells and transdifferentiation into corneal epithelial cells

AbstractPurpose The integrity of the corneal epithelium is essential for clarity of vision. Under normal circumstances, superficial epithelial cells are shed into the tear film and regenerated by epithelial stem/progenitor cells located at the limbus. Damage or depletion of the limbal cells leads to conditions such as limbal stem cell deficiency (LSCD). Current treatment involves transplantation of limbal epithelial cells expanded ex‐vivo; however cell numbers are limited. In this study, we identify a source of multipotent stem cells located in the corneal stroma that express CD34 upon isolation. These cells demonstrate an ability to transdifferentiate into corneal epithelial cells.Methods CD34 expression diminishes during traditional tissue‐culture plastic propagation; in this study we optimised culture conditions for maintained and efficient expansion of CD34+ cells. We investigated the role of CD34 in epithelial transdifferentiation of stromal cells, using siRNA gene knockdown and analysis by immunocytochemistry, flow cytometry and qPCR.Results Early results suggest a three‐dimensional environment and semi‐solid medium intended for hematopoietic culture demonstrated extended CD34 expression. When cells were transferred to epithelial differentiation medium they showed an epithelial morphology, significantly increased cytokeratin 3 and 19 expression, and considerable upregulation of genes related to corneal epithelial cells (ABCG2, DeltaN63, LEF1, HES1, FRZB1, KRT19, DTC and CDH1).Conclusion This work will help produce methodologies to create cell banks for generation of corneal epithelium, from a corneal stromal stem cell source, leading to improved surgical and visual outcomes in LSCD patients.