Limbal graft transplantation: a rare implementation in pediatric limbal stem cell deficiency.

PURPOSE: To evaluate limbal graft transplantation success in pediatric patients with chemical injury-induced limbal stem cell deficiency (LSCD) using the 'LSCD Working Group' staging system. METHODS: Medical records of 11 eyes of 11 children who underwent limbal graft transplantation (limbal autograft/limbal allograft) were included. Surgical success was defined as improvement in the post-operative 1st year LSCD stage. RESULTS: The mean age was 12 ± 5 (4-17) years. Causative agent was alkaline in 4(36.4%) and acid in 3(27.2%) patients. Limbal autograft was performed in 9 (81.8%) eyes with unilateral LSCD, and allograft transplantation was performed in 2 (18.2%) eyes with bilateral LSCD. The mean follow-up time was 33.89 ± 30.73 (12-102.33) months. The overall limbal graft transplantation success rate was 72.7%. Among 9 patients who receive limbal autograft, 8 had improvement in post-operative LSCD stage, 1 had stable LSCD stage. Of the 2 patients who receive limbal allograft, post-operative LSCD stage remained the same in 1 and worsened in 1 patient. The mean time between injury and the surgery was 30.47 ± 30.08 (7-108.47) months. Penetrating keratoplasty was performed in 3 (27.2%) of 11 patients following limbal graft transplantation. CONCLUSION: Management of LSCD in children is challenging and appears to be somewhat different from that of adults. Limited data in the literature indicate that cultivated or simple limbal epithelial transplantations (CLET/SLET) are primarily preferred in children. Although the tendency to take small tissue from the healthy eye is noteworthy, conventional limbal allograft and autograft transplantations also show promising results without any further complications in at least 1 year follow-up period.

Three-Dimensional Reconstruction of Subbasal Nerve Density in Eyes With Limbal Stem Cell Deficiency: A Pilot Study.

PURPOSE: Corneal subbasal nerve parameters have been previously reported using 2-dimensional scans of in vivo laser scanning confocal microscopy (IVCM) in eyes with limbal stem cell deficiency (LSCD). This study aims to develop and validate a method to better quantify corneal subbasal nerve parameters and changes from reconstructed 3-dimensional (3D) images. METHODS: IVCM volume scans from 73 eyes with various degrees of LSCD (mild/moderate/severe) confirmed by multimodal anterior segment imaging including IVCM and 20 control subjects were included. Using ImageJ, the scans were manually aligned and compiled to generate a 3D reconstruction. Using filament-tracing semiautomated software (Imaris), subbasal nerve density (SND), corneal nerve fiber length, long nerves (>200 µm), and branch points were quantified and correlated with other biomarkers of LSCD. RESULTS: 3D SND decreased in eyes with LSCD when compared with control subjects. The decrease was significant for moderate and severe LSCD ( P < 0.01). 3D SND was reduced by 3.7% in mild LSCD, 32.4% in moderate LSCD, and 96.5% in severe LSCD. The number of long nerves and points of branching correlated with the severity of LSCD ( P < 0.0001) and with declining SND (R 2 = 0.66 and 0.67, respectively). When compared with 2-dimensional scans, 3D reconstructions yielded significant increases of SND and branch points in all conditions except severe LSCD. 3D analysis showed a 46% increase in long nerves only in mild LSCD ( P < 0.01). CONCLUSIONS: This proof-of-concept study validates the use of 3D reconstruction to better characterize the corneal subbasal nerve in eyes with LSCD. In the future, this concept could be used with machine learning to automate the measurements.

[Experimental evaluation of the efficacy of tissue-engineered constructs in the treatment of limbal stem cell deficiency]. / Eksperimental'naya otsenka effektivnosti primeneniya tkaneinzhenernoi konstruktsii v lechenii limbal'noi nedostatochnosti.

Limbal stem cell deficiency (LSCD) is one of the leading factors negatively affecting the success of keratoplasty, and its treatment remains an urgent problem in ophthalmology. With the development of regenerative medicine, one of the promising approaches is the transplantation of tissue-engineered constructs from cultured limbal stem cells (LSCs) in biopolymer carriers. PURPOSE: This study was conducted to develop an experimental model of LSCD and evaluate the effectiveness of transplantation of a tissue-engineered construct consisting of cultured cells containing a population of LSCs and a collagen carrier. MATERIAL AND METHODS: The study was performed on 12 rabbits and included several stages. At the first stage, the physiological effects of collagen matrix implantation into the limbal zone were studied. At the second stage, tissue-engineered constructs consisting of LSCs on a collagen matrix were formed and their effect on the regeneration processes in the experimental LSCD model was analyzed. The animals were divided into 2 groups: surgical treatment (transplantation of the tissue-engineered construct) was used in the experimental group, and conservative treatment was used in the control group. Slit-lamp biomicroscopy with photo-registration, fluorescein corneal staining, optical coherence tomography of the anterior segment of the eye, and impression cytology were used to assess the results. RESULTS: No side reactions were observed after implantation of the collagen matrix into the limbal zone. One month after surgical treatment of the LSCD model in the experimental group, complete epithelization with minor manifestations of epitheliopathy was observed. In the control group, erosion of the corneal epithelium was noted. The time of corneal epithelization in the experimental and control groups was 9.2±2.95 and 46.20±12.07 days, respectively (p=0.139). According to the data of impression cytology, in the experimental group there were no goblet cells in the central part of the cornea, which indicates the restoration of corneal type epithelial cells, in contrast to the control group. CONCLUSION: Transplantation of a tissue-engineered construct from cultured limbal cells on a collagen membrane should be considered as a promising method for the treatment of limbal stem cell deficiency.

Clinical and pathologic characterization of a mouse model of graded limbal stem cell deficiency.

Limbal stem cell deficiency (LSCD) is a clinically challenging eye disease caused by damage to limbal stem cells (LSCs). Currently, the international consensus classifies LSCD into three clinical stages based on the disease severity. However, no existing animal models attempt to replicate the varying degrees of LSCD observed in clinical cases. The present study demonstrates an easy-to-create, reproducible, and reliable mouse model of graded LSCD. To achieve mild, moderate, or severe LSCD, filter paper rings with a variety of central angles (90°, 180°, or 270°) are utilized to deliver alkali burns to different sizes of the limbal area (1, 2, or 3 quarters). The animal model has successfully resulted in the development of clinical signs and pathological manifestations in escalating severity that are similarly observed in the three clinical stages of LSCD. Our study thus provides new insights into distinct pathological features underlying different grades of LSCD and serves as a new tool for further exploring the disease mechanisms and developing new effective therapeutics for repairing damaged LSCs.

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

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Long-term ocular surface stability in conjunctivolimbal autograft and ocular surface safety in the donor eyes.

PURPOSE: The current study is aimed to present the long-term results of the patients who underwent conjunctivolimbal autograft (CLAU) as the primary operation in unilateral limbal stem cell deficiency and the ocular surface safety of the donor eyes. The patients were followed up for five years or longer. METHODS: The records of all patients who underwent CLAU as the primary operation were retrospectively analyzed. Additional ocular surface operations, ocular surface stability, best-corrected visual acuity (BCVA), and ocular surface status of the donor eyes were investigated. RESULTS: The mean age of the patients at the time of transplantation was 35.07 ± 12.9 (12-60). Twenty-nine eyes of 29 patients were followed up for an average of 97.82 ± 34.45 (60-186) months. Additional ocular surface operation was required in 27.58% (8/29) of the eyes in order to achieve a stable ocular surface. Ocular surface stability was achieved in 82.75% (24/29) of the eyes at the end of the follow-up period. BCVA increased from 1.78 ± 0.82 to 0.91 ± 0.92 logMAR at the last visit (p < 0.001). Corneal ectasia and vascularization developed in one donor eye in the fifth postoperative year. CONCLUSIONS: CLAU tissues provide ocular surface stability with a successful vision result in the long term. CLAU theoretically carries risks including limbal stem cell deficiency in the donor eye. In the long-term follow-up of donor eyes after CLAU, ectasia and limbal stem cell deficiency were observed in one eye.

Severity Classification of Limbal Stem Cell Failure Due to Steven Johnson Syndrome in the Light of the Classification Consensus of Limbal Stem Cell Deficiency.

OBJECTIVES: To examine and to understand the limbal stem-cell deficiency (LSCD) because of Steven-Johnson syndrome (SJS) in line with the new classification system for the first time in the literature. METHODS: Medical records of patients with LSCD because of SJS were reviewed retrospectively. In addition to demographic data and ophthalmologic or systemic findings, anterior segment photographs of the patients were reviewed retrospectively. Limbal stem-cell deficiency severity was graded according to the classification published by the Limbal Stem Cell Working Group. RESULTS: Twenty-four eyes of 14 patients with eye involvement secondary to SJS were included in the study. The mean age of the patients was 36.09±16.70 (9-58) years and the female-to-male ratio was 11:3. The anterior segment photographs of the patients were evaluated by two independent masked observers. Limbal stem-cell deficiency severity was graded according to the classification published by Deng et al. Corneal opacity was divided into three stages according to the area of involvement. Corneal opacity was classified as Stage I if the central 5 mm region of the cornea was not affected, as Stage II if the central 5 mm region of the cornea was affected, and as Stage III if the entire corneal surface was affected. Limbal involvement was classified as Stage A if it was below 50%, as Stage B if it was between 50% and 100%, and as Stage C if it was 100%. CONCLUSION: This is the first study in the literature to describe and classify LSCD because of SJS, according to the new LSCD classification. Consistent with the results, LSCD follows a bimodal distribution. Most patients demonstrated severe (Stage III-32.14%) or mild (Stage IA-21.42%) LSCD.

Correlation Between Anterior Chamber Angle Status and Limbal Stem Cell Deficiency in Primary Angle-Closure Glaucoma.

PURPOSE: To investigate the correlation between the opening and closing states of anterior chamber angle (ACA) and the density of limbal epithelial basal cells (LEBCs) in subjects with primary angle-closure glaucoma (PACG). DESIGN: Cross-sectional observational study. METHODS: A total of 54 eyes of 29 patients diagnosed with PACG were included in the study. Fifty-four eyes from normal subjects were included as control. Automatic evaluation system for ultrasound biomicroscopy images of anterior chamber angle was used to assist ophthalmologists in identifying the opening or closing state of ACA, and the in vivo confocal microscopy (IVCM) was used to evaluate the density of LEBCs in different directions. RESULTS: (1) The average density of LEBCs in the superior, inferior, nasal, and temporal limbus of the eyes in the PACG group was lower than that in the control group, and this pattern did not align with the density distribution observed in the control group. (2) In the early, moderate and advanced PACG, the density of LEBCs corresponding to the closed angle was lower than that in the control group (P < .05). Compared with the density of LEBCs corresponding to the closed angle and the open angle, the closed angle of PACG in the early, moderate and advanced stages was less than that in the open angle (P < .05 in the early and moderate stages; advanced stage P > .05). (3) The basal cell density was processed by dimensionless analysis. In the data calculated by averaging and minimizing, both closed angle dimensionless values were smaller than the open angle (P < .05). (4) Comparative analysis was conducted among the normal, open-angle, and closed-angle conditions in the superior, inferior, nasal, and temporal limbus. In the early stage of PACG, significant differences were observed in 4 limbal regions (P < .05), while in the moderate PACG stage, this difference was noted in 3 limbal regions (P < .05). In advanced PACG, 2 limbal regions exhibited significant differences (P < .05). These findings suggest that during the early PACG stage, angle closure is the predominant influencing factor on LEBCs density, while in the advanced stage, the decrease in density is attributed to a combination of angle closure and the natural progression of the disease. CONCLUSIONS: There is a significant correlation between anterior chamber angle status and LEBCs. Advanced PACG and angle closure should be highly suspected of the occurrence of limbal stem cell deficiency (LSCD).

Comparative analysis of long-term results of three epithelial cell transplantation procedures for treating limbal stem cell deficiency.

This study compared the long-term outcome of different epithelial transplantation techniques to treat limbal stem cell deficiency (LSCD). We conducted a retrospective 15-year comparative systematic cohort study of patients with LSCD who underwent either cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET), or cultivated oral mucosal epithelial transplantation (COMET). We reviewed the demographic data, etiology, LSCD severity, best-corrected visual acuity, surgical outcomes, and complications. A total of 103 eyes of 94 patients (mean age, 45.0 ± 16.4 years) with LSCD were enrolled. The most common cause of LSCD was chemical injury (42.7 %). The median follow-up time was 75 months. The success rates of CLET, SLET, and COMET were 45.5 %, 77.8 %, and 57.8 %, respectively. The 7-year survival rates after CLET, SLET, and COMET were 50.0 %, 72.2 %, and 53.2 %, respectively. Steven-Johnson syndrome (SJS) had a significantly lower survival rate than other causes (p < 0.001), but SLET had a significantly higher survival rate than CLET (p = 0.018) and COMET (p = 0.047). Visual improvement of more than four Snellen lines was achieved in 53.1 % of successful cases and 28.2 % of failed cases. SJS, Schirmer I test <5 mm, and the presence of postoperative recurrent epithelial defects were significant risk factors for a failed surgery. All epithelial transplantation techniques had favorable long-term surgical outcomes. More than half of the patients achieved a stable ocular surface and visual acuity improvement up to 7 years postoperatively. SLET tends to have a better surgical outcome than CLET and COMET, especially in patients with SJS.

Corneal Irregularity Induced by Transepithelial Photorefractive Keratectomy in a Patient With Partial Limbal Stem Cell Deficiency.

PURPOSE: The aim of this study was to present a case of transepithelial photorefractive keratectomy (trans-PRK) laser ablation in a patient with partial limbal stem cell deficiency (LSCD) that resulted in corneal irregularity. METHODS: A 23-year-old man with bilateral partial LSCD underwent trans-PRK for myopia correction 2 months before presentation to our department. Trans-PRK ablation was performed with a phototherapeutic keratectomy ablation profile set at 60 µm in the OD and 57 µm in the OS and 8 mm zone. At the time of presentation, the patient complained of decreased visual acuity. Corrected distant visual acuity was 20/20 (-3.25, -0.75 × 180 degrees) and 20/50 (-3.00, -3.00 × 180 degrees) in the OD and the OS, respectively. No further ocular history was reported other than prolonged soft contact lens use for myopia. Pre-trans-PRK tomography (WaveLight Oculyzer II diagnostic system, WaveLight GmbH, Germany) revealed bilateral superior steepening with corresponding epithelial thinning as obtained by the epithelial map using Optovue optical coherence tomography (Visionix Luneau technology). Slit-lamp examination showed bilateral superficial neovascularization, suggestive of early-stage LSCD. RESULTS: A nonuniform laser stromal ablation resulting in corneal irregularity and decreased visual acuity was observed 2 months postoperatively. During the 6-month follow-up, corneal tomography was stable with only minimal improvement. At that time, corrected distant visual acuity remained 20/20 (-2.75, -0.75 × 160 degrees) in the OD and 20/50 (-3.00, -3.00 × 180 degrees) in the OS. CONCLUSIONS: Trans-PRK seems to be a contraindication in cases with corneal epithelial irregularities and could lead to abnormal and nonuniform stromal ablation .

Quintuple Procedure for Ocular Surface Reconstruction and Visual Rehabilitation.

PURPOSE: The aim of this study was to describe a combined surgical approach for ocular surface and visual rehabilitation in patients with limbal stem cell deficiency, corneal scarring, and cataract. We aimed to introduce this combined approach as an alternative to a staged approach and to describe the intraoperative use of a diamond ophthalmic burr (DOB) and optical coherence tomography (OCT) to achieve and confirm a smooth graft-host interface during the anterior lamellar keratoplasty (ALK) portion of the procedure. METHODS: The quintuple procedure described herein consists of a modified ALK, cataract extraction, intraocular lens implantation, simple limbal epithelial transplantation, and temporal tarsorrhaphy. Intraoperative OCT and DOB were used to guide the creation of the stromal bed during the ALK. The procedure was performed in a patient with limbal stem cell deficiency and corneal scarring after a chemical ocular burn. RESULTS: The tarsorrhaphy was removed at 3 months postoperatively once complete corneal epithelialization was observed. The best-corrected visual acuity improved from light perception preoperatively to 20/30 at 16 months postoperatively with the use of scleral contact lenses. At this postoperative time point, the cornea was clear and compact, and the ocular surface was stable. CONCLUSIONS: The quintuple procedure allowed for visual and ocular surface rehabilitation in cases in which follow-up for a staged approach was not possible. Intraoperative OCT allowed for real-time visualization and dissection of the stromal bed, whereas the use of the DOB helped achieve a smooth stromal surface for an optimal graft-host interface.

Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency.

PURPOSE: To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). METHODS: A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. RESULTS: No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). CONCLUSION: ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

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

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

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

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their niche involving numerous signaling pathways, such as Wnt. Wnt proteins are secreted morphogens that play critical roles in embryonic development, stem cell proliferation, self-renewal, tissue regeneration, and remodeling in adults. It has been shown that a small molecule Wnt mimic could improve LSCs expansion ex vivo. Damage to the LSCs and/or their niche can lead to limbal stem cell deficiency (LSCD), a condition that can cause corneal blindness and is difficult to treat. This study explored if repopulating residual LSCs in partial LSCD through Wnt activation could be a novel therapeutic approach. To mimic LSCD due to a chemical injury, single cultured LSCs were exposed to various concentrations of sodium hydroxide. A progressive loss of the LSCs phenotype was observed: the percentage of p63bright cells and cytokeratin (K)14+ cells decreased while the percentage of K12+ increased. Wnt activation was attained by treating the LSCs with lithium chloride (LiCl) and a small-molecule Wnt mimic, respectively. After 18 h of treatment, LSCs proliferation was increased, and the LSCs phenotype was recovered, while the untreated cells did not proliferate and lost their phenotype. The percentage of p63bright cells was significantly higher in the Wnt mimic-treated cells compared with untreated cells, while the percentage of K12+ cells was significantly lower. These findings suggest that local Wnt activation may rescue LSCs upon alkaline injury.

Opposing Roles of Blood-Borne Monocytes and Tissue-Resident Macrophages in Limbal Stem Cell Damage after Ocular Injury.

Limbal stem cell (LSC) deficiency is a frequent and severe complication after chemical injury to the eye. Previous studies have assumed this is mediated directly by the caustic agent. Here we show that LSC damage occurs through immune cell mediators, even without direct injury to LSCs. In particular, pH elevation in the anterior chamber (AC) causes acute uveal stress, the release of inflammatory cytokines at the basal limbal tissue, and subsequent LSC damage and death. Peripheral C-C chemokine receptor type 2 positive/CX3C motif chemokine receptor 1 negative (CCR2+ CX3CR1-) monocytes are the key mediators of LSC damage through the upregulation of tumor necrosis factor-alpha (TNF-α) at the limbus. In contrast to peripherally derived monocytes, CX3CR1+ CCR2- tissue-resident macrophages have a protective role, and their depletion prior to injury exacerbates LSC loss and increases LSC vulnerability to TNF-α-mediated apoptosis independently of CCR2+ cell infiltration into the tissue. Consistently, repopulation of the tissue by new resident macrophages not only restores the protective M2-like phenotype of macrophages but also suppresses LSC loss after exposure to inflammatory signals. These findings may have clinical implications in patients with LSC loss after chemical burns or due to other inflammatory conditions.

P49-A103†Allogeneic limbo-DALK: a novel surgical technique for patients with corneal stromal disease and limbal stem cell deficiency.

PURPOSE: To describe a novel corneal surgical technique combining Deep Anterior Lamellar Keratoplasty (DALK) with grafting of allogeneic limbal stem cells (limbo-DALK) as treatment for eyes with corneal stromal pathology and limbal stem cell deficiency (LSCD). METHODS: This is a series of six Limbo-DALKs in five eyes of five patients. One patient received a second limbo-DALK after graft failure following the first procedure. Two of the donor corneae were HLA matched. Clinical records of included patients were reviewed retrospectively. All patients had been diagnosed with LSCD due to various pathologies. Analysed data included demographic data, diagnoses and clinical history, graft visualization and thickness measurements by anterior segment OCT, visual acuity and epithelial status. Follow-up visits were 6 weeks and 3, 6, 9, 12 and 18 months postoperatively with final suture removal at 18 months and further follow-up examinations twice yearly thereafter. RESULTS: Two grafts showed total epithelial closure after 2 days, two after 14 days. In one eye, full closure of corneal epithelium did not occur after the first limbo-DALK, but could be achieved one month after second limbo-DALK. No endothelial graft rejection was seen. CONCLUSION: Based on data from this pilot series, limbo-DALK seems to be a novel viable surgical approach for eyes with severe LSCD and stromal corneal pathology.