Limbal Epithelial Transplantation Research Articles

Allogenic Simple Limbal Epithelial Transplantation Versus Amniotic Membrane Grafting in the Early Management of Severe-Grade Ocular Chemical Injuries—A Retrospective Comparative Study

To compare outcomes of management in the early stage of severe chemical injury (grade 4 and worse; Dua classification) with amniotic membrane grafting (AMG) alone vs allogenic simple limbal epithelial transplantation (alloSLET). Retrospective comparative interventional case series. Retrospective comparative interventional series. Records of patients with severe ocular chemical injury who underwent AMG alone (between 2009 and 2013) vs alloSLET (between 2013 and 2017) were analyzed for grade of injury, time of and interventions for epithelial healing, ocular surface status post healing (grade of symblepharon, and limbal stem cell deficiency [LSCD]), and type of and need for interventions in the chronic stage. Among patients presenting in early stage of severe chemical injury, 38 eyes (median age 11 years) managed with AMG alone were compared with 39 eyes (median age 8 years) managed with alloSLET. The mean time of presentation post injury was 33.85 ± 27.5 and 40.6 ± 23.5days in the AMG and alloSLET group, respectively. The rate of epithelial healing was faster in the alloSLET group and the difference was noted to be statistically significant (odds ratio [OR] 0.966, P= .001). Similarly, the lower occurrence of LSCD (OR 0.137, P= .004) and need for keratoplasty (OR 0.093, P= .003) favored alloSLET over AMG. Final best-corrected visual acuity of >20/200 was achieved in 39.4% and 53.8% in the AMG and alloSLET groups, respectively. AlloSLET helps in faster epithelialization of the surface, thus reducing the need for subsequent surgeries in the chronic stage and aiding faster visual rehabilitation. The outcomes of alloSLET appear superior to amniotic membrane grafting alone and should be considered in eyes with grade 4 and above (Dua classification) chemical injuries in the early stage.

Allogenic simple limbal epithelial transplantation (alloSLET) from cadaveric donor eyes in patients with persistent corneal epithelial defects

Background/aimTo describe the clinical outcome of allogenic simple limbal epithelial transplantation (alloSLET) utilising tissue from cadaveric donor eyes after failed re-epithelialisation of the corneal surface.MethodsMedical records of 14 eyes from...

Outcomes of Ipsilateral Simple Limbal Epithelial Transplantation, Tenonectomy, Mitomycin and Amniotic Membrane Transplantation for Treatment of Recurrent Pterygium.

To report on the outcomes of recurrent pterygium treated by ipsilateral simple limbal epithelial transplantation (SLET), mitomycin, tenonectomy, and amniotic membrane transplantation. A retrospective, interventional study was conducted including all patients with recurrent pterygium who underwent SLET surgery under a single surgeon using ipsilateral donor tissue with a minimum 6-month follow-up at Toronto Western Hospital, Canada. Outcome measures included the following: recurrence rates, best spectacle-corrected visual acuity, and postoperative complications. Ten eyes of 10 patients, aged 60.7 ± 18.5 years (range 23-79) with a mean follow-up time of 15.2 ± 10.0 months of which 50% (n = 5) were men, were included. Eight eyes (80%) had a history of 2 or less pterygium operations. Two patients had 3 and 5 previous pterygium operations, respectively. Concurrent limbal stem cell disease was noted in 6 eyes (60%). Average number of pterygium recurrences per eye was 1.9 ± 1.3 (range 1-5). Mean pre-op best-corrected visual acuity was 0.5 LogMAR (Snellen equivalent 20/60, range 20/20 to counting fingers). Best-corrected visual acuity remained the same or improved in 6 eyes (60%). Recurrence was noted in 1 eye (10%) with a history of 5 previous pterygium excisions and remained stable at the last follow-up. No patients required a second operation. Ipsilateral SLET with mitomycin, tenonectomy, and amniotic membrane transplantation is a novel technique to address recurrent pterygium. Concurrent limbal stem cell diseases are often present. Initial results demonstrate low recurrence. Visual improvement is modest. Stabilization of the ocular surface to improve vision is possible.

Optimization of Human Limbal Stem Cell Culture by Replating a Single Limbal Explant.

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Clinical observation of autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency

Objective: To discuss effect of autologous simple limbal epithelial transplantation (SLET) performed for unilateral limbal stem cell deficiency (LSCD). Methods: Retrospective case study. In this retrospective study, records of 7 patients (7 eyes) who had undergone autologous SLET for unilateral LSCD, with a minimum of 6 months of follow-up, were reviewed. Demographic details, etiology of LSCD, duration between ocular burn and SLET, prior surgery performed, presence or absence of symblepharon, pre-and post-operative visual acuity, and complications were noted. Results: Seven eyes of 7 patients underwent autologous SLET. With a follow-up of 6 months, a completely epithelialised and stable corneal surface was obtained in all recipient eyes. Visual acuity improved in all patients, while none of the eyes developed any complications. Conclusions: Autologous SLET is an effective and safe modality for treatment of unilateral LSCD. Clinical success rates and visual acuity improvement are equal to or better than those reported with earlier techniques. (Chin J Ophthalmol, 2019, 55:923-927).

Simple limbal epithelial transplantation: Current status and future perspectives.

Damage to limbal stem cells as a result of injury or disease can lead to limbal stem cell deficiency (LSCD). This disease is characterized by decreased vision that is often painful and may progress to blindness. Clinical features include inflammation, neovascularization, and persistent cornea epithelial defects. Successful strategies for treatment involve transplantation of grafts harvested from the limbus of the alternate healthy eye, called conjunctival‐limbal autograft (CLAU) and transplantation of limbal cell sheets cultured from limbal biopsies, termed cultured limbal epithelial transplantation (CLET). In 2012, Sangwan and colleagues presented simple limbal epithelial transplantation (SLET), a novel transplantation technique that combines the benefits of CLAU and CLET and avoids the challenges associated with both. In SLET a small biopsy from the limbus of the healthy eye is divided and distributed over human amniotic membrane, which is placed on the affected cornea. Outgrowth occurs from each small explant and a complete corneal epithelium is typically formed within 2 weeks. Advantages of SLET include reduced risk of iatrogenic LSCD occurring in the healthy cornea at harvest; direct transfer circumventing the need for cell culture; and the opportunity to perform biopsy harvest and transplantation in one operation. Success so far using SLET is comparable with CLAU and CLET. Of note, 336 of 404 (83%) operations using SLET resulted in restoration of the corneal epithelium, whereas visual acuity improved in 258 of the 373 (69%) reported cases. This review summarizes the results of 31 studies published on SLET since 2012. Progress, advantages, challenges, and suggestions for future studies are presented.

Limbal stem cell transplantation and keratoprosthesis

AbstractLimbal stem deficiency (LSCD) is a severe ocular disease which may lead to vision impairment or corneal blindness. Beside conservative options like topical and systemical medical therapy, chronic stage treatment options include conjunctival replacement procedures and limbal autografting. If limbal cell transplanation in form of either cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET) or conjunctival limbal autograft (CLAU) and corneal transplantation are not successful a Keratoprosthesis in form of a Boston Keratoprosthesis or a Osteo‐odonto‐ Keratoprosthesis might be the last chance for these patients. In the following presentation indication and surgical options in LSCD will be discussed.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy

BackgroundLimbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives.MethodsWe compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA.ResultsXSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells.ConclusionsThe combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.Graphical abstract

Xeno‐free Cultivated Limbal Epithelial Stem Cell Transplantation: result of an ongoingphase I/II clinical study

AbstractLimbal stem cell deficiency (LSCD) is a condition that results from a loss of limbal stem cells or a dysfunctional limbal microenvironment. The term refers to a highly heterogeneous group of pathologies ranging from acquired diseases such as chemical burns to severe autoimmune conditions, iatrogenic trauma and genetic abnormalities such as aniridia. Here we report the clinical results of an ongoing clinical trial (2015–2020) using a cultivated limbal epithelial transplant (CLET) for a variety of LSCD causes including chemical burns, thermal burns and aniridia, both unilateral and bilateral. Currently 28 CLET surgeries have been performed on 23 eyes, with a median follow up of 19 months (range 2‐42 months). Seventeen patients were treated with autologous CLET, 4 patients with allogenic CLET from a living‐related HLA‐matched donor and 2 patients with HLA‐matched cadaveric donor CLET. Five patients required a second CLET and an elective penetrating keratoplasty (PK) was offered least one year after the CLET. Eleven patients are currently graded as a total anatomical success (47.8%), two patients are grade as partial success (8.7%), eight cases were deemed total failures (34.8%) and two patients were excluded due to a complication (8.7%). BCVA, pain and photophobia outcomes denoting functional success are also presented.

Evolution of techniques of limbal stem cell transplantation for restoration of ocular surface in ocular surface disorder with limbal stem cell deficiency

Purpose: Evolution of techniques of limbal stem cell transplantation for restoration of ocular surface in ocular surface disorder with limbal stem cell deficiency. Materials and Methods: It is an observational study in which 32 eyes of LSCD were observed with different surgical techniques. Outcome parameters were evaluated in terms of visual acuity, corneal clarity, symblepharon score, vascularisation. Statistical analysis was done using Annova t-test with significance level P ( Result: The mean preoperative logMAR best-corrected visual acuity (BCVA) in CLAU, SLET, KLAU, and KLAL were 2.150.70, 2.700.20, 2.750.22 and 2.800.11 respectively which improved to 1.250.50, 2.000.42, 2.010.15 and 2.300.18 respectively after 6 months of follow up. Improvement in structural parameter were also seen in different surgical techniques. Conclusion: Patients in which CLAU and SLET technique were used, had shown better improvement in Visual and structural outcome as compared to those in which KLAU and KLAL technique were used. Keywords: Limbal stem cell deficiency (LSCD), Limbal stem cell transplantation (LSCT) Auto conjunctival limbal graft (CLAU), Simple limbal epithelial transplantation (SLET), Keratolimbal autograft (KLAU), Keratolimbal allograft (KLAU), Amniotic membrane transplant (AMT).

Bevacizumab Induces Upregulation of Keratin 3 and VEGFA in Human Limbal Epithelial Cells in Vitro.

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.

331.3: Simple limbal epithelial transplantation (SLET) in a patient with limbal stem cell deficiency.

331.3: Simple limbal epithelial transplantation (SLET) in a patient with limbal stem cell deficiency.

Short- and Long-Term Results of Xenogeneic-Free Cultivated Autologous and Allogeneic Limbal Epithelial Stem Cell Transplantations.

Purpose:To evaluate the short- and long-term success rates of xenogeneic-free cultivated limbal epithelial stem cell transplantation (CLET) for the treatment of limbal stem cell deficiency (LSCD).Methods:Thirteen patients with LSCD underwent an autologous (n = 9) or allogeneic (n = 4) CLET. The primary end point was to assess the long-term anatomical success rate of transplanted grafts at a follow-up of at least 3 years, in comparison with the short-term outcomes. Secondary end points involved reviewing functional improvement, patient-reported symptoms, and change in percentage area of corneal vascularization in both short-term and long-term.Results:The mean short- and long-term follow-up periods were 2.1 ± 0.38 years and 6.7 ± 1.81 years, respectively. The total anatomical success rate was 46.1% in the short-term, but it decreased to 23.1% in the long-term. A partial success rate of 30.8% was observed in both short- and long-term, and the failure rate increased from 23.1% to 46.1%. The mean percentage of vessel area decreased from 12.11% ± 5.29% preoperatively to 7.82% ± 6.70% in the short-term and increased to 8.70% ± 6.32% in the long-term. There was a significant improvement in best-corrected visual acuity (P = 0.044) in the short-term although not in the long-term (P = 0.865).Conclusions:This study shows that anatomical and functional success rates of CLET decrease over time. We believe that the decline of success is related to the extent of disease, cell origin, and lack of niche protection because subtotal LSCD and autologous donor cells confer a higher chance of success in the long-term.

Mini-Simple Limbal Epithelial Transplantation Versus Conjunctival Autograft Fixation With Fibrin Glue After Pterygium Excision: A Randomized Controlled Trial.

To compare the outcomes between mini-simple limbal epithelial transplantation (mini-SLET) and conjunctival autograft (CAG) fixation with fibrin glue after excision of pterygium. Ninety-two eyes with primary nasal pterygium were prospectively randomized into a CAG control arm and a mini-SLET study arm. Follow-up examinations were set at the first and third day, at weeks 1, 2, and 4, and at the third and sixth month after surgery. The primary outcome measure was the recurrence rate at 1, 3, and 6 months after surgery, whereas the secondary outcome measures were the intraoperative time, postoperative symptoms, and other complications. Eighty-two eyes completed the 6-month follow-up interval. Of the 42 eyes that underwent CAG, 4 (9.5%) exhibited recurrences, whereas only 1 of the 40 eyes (2.5%) treated with mini-SLET had recurrence (P = 0.358). The time taken for surgery in the study group (21.4 minutes) was greater as compared to the control group (15.1 minutes) (P < 0.001). The postoperative median symptom (foreign body sensation, lacrimation, pain, and irritation) score in the CAG group was significantly more for all symptoms on day 1 and day 3; however, on day 7, it was significantly more for pain and irritation only. Except dellen formation (1 in each group), both groups exhibited different other complications, although less in the study group. The study group exhibited a positive trend of less recurrence with reduced postoperative symptoms and other complications. More similar studies are required in future to validate the results.

Autologous limbal stem cell transplantation: a systematic review of clinical outcomes with different surgical techniques

PurposeTo conduct a systematic review on outcomes of three different techniques of autologous limbal stem cell transplantation (LSCT): conjunctival-limbal autografting (CLAu), cultivated limbal epithelial transplantation (CLET) and simple limbal epithelial...

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.

Systematic review and meta-analysis investigating autograft versus allograft cultivated limbal epithelial transplantation in limbal stem cell deficiency.

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

Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells.

Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.

Epithelial analysis of simple limbal epithelial transplantation in limbal stem cell deficiency by in vivo confocal microscopy and impression cytology.

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

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.