Limbal Stem Cell Grafts Research Articles

Clinical results of limbal stem cell graft of cadaveric donor in patients with bilateral limbal stem cell deficiency

Clinical results of limbal stem cell graft of cadaveric donor in patients with bilateral limbal stem cell deficiency

Outcomes of Human Leukocyte Antigen-Matched Allogeneic Cultivated Limbal Epithelial Transplantation in Aniridia-Associated Keratopathy-A Single-Center Retrospective Analysis.

Purpose:To assess the efficacy and safety of human leukocyte antigen–matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK).Methods:Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period.Results:The mean follow-up was 53.6 months (range 24–104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients.Conclusions:Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration.

Diagnoses and Outcomes of Prosthetic Replacement of the Ocular Surface Ecosystem Treatment-A Canadian Experience.

To investigate underlying diagnoses and outcomes of patients undergoing Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) treatment at the first Canadian PROSE center. A retrospective chart review was conducted on patients referred for PROSE treatment and fitted with PROSE devices from 2018 to 2020. Data were collected on diagnoses, presenting symptoms, previous lens modalities attempted, best-corrected visual acuities (BCVAs) pre-PROSE and post-PROSE, daily wear time, and failure rates. Best-corrected visual acuities pre-PROSE and post-PROSE were compared to evaluate visual improvement. In total, 78 patients (126 eyes) were analyzed. The most common diagnoses were keratoconus (n=39 eyes) and postcorneal graft (n=15) in the distorted cornea group, and limbal stem cell deficiency (n=17) and graft versus host disease (n=15) in the ocular surface disease (OSD) group. Most frequent symptoms included blur, photophobia, and pain. Most common lens modalities attempted pre-PROSE were conventional scleral lenses and glasses. The overall mean BCVA improvement was 0.40 logarithm of the minimal angle of resolution (logMAR) (4-lines Snellen) (P<0.0001). Best-corrected visual acuities improvement in the distorted cornea group (0.52 logMAR, 5-lines) was significantly greater than in the OSD group (0.29 logMAR, 3-lines) (P=0.004). Prosthetic replacement of the ocular surface ecosystem treatment can provide significant visual improvement for patients with distorted corneal surfaces and OSDs who failed other lens modalities.

Tectonic Keratoplasty in Patients with Non-traumatic, Non-infectious Corneal Perforations

Introduction:The study aims to report clinical results of tectonic keratoplasty for non-traumatic, non-infectious corneal perforations.Materials and Methods:The medical records of 12 patients who underwent tectonic penetrating keratoplasty between October 2014 and August 2018 at Ege University Ophthalmology Department were retrospectively reviewed.Results:The mean age of the patients was 52.92±30.34 (range, 2-82) years. The causes of corneal perforation were dry eye (neurotrophic keratopathy (n=4), limbal stem cell deficiency (n=2), exposure keratopathy (n=2) and graft versus host disease (n=1)) in 9 patients. In the remaining 3 patients, the etiology of perforation was not determined. The mean Visual Acuity (VA) was 2.98±0.39 (range, 1.8-3.1) LogMAR before the surgery. Despite conservative treatment, tectonic penetrating keratoplasty had to be performed in all patients in order to manage the perforation. Mean time in between initial examination and surgery was 10.75±12.04 (1-41) days. In 2 patients, allogenic limbal stem cell transplantation; in one patient, lateral tarsorrhaphy and in one patient symblepharon release with amniotic membrane transplantation were performed additional to tectonic keratoplasty. Mean follow-up time was 57.88±55.47 (4-141) weeks. Grafts were clear in 6 eyes and opaque in 5 eyes. The main causes of graft failure among opaque grafts were ocular surface disease (3), allograft rejection (1) and glaucoma-related endothelial failure (1). Phthisis bulbi was detected in one patient with congenital glaucoma due to vitreous loss at the time of perforation. The mean final VA in patients who had clear grafts was 1.83±1.03 (range, 0.8-3.1) LogMAR.Conclusion:To prevent serious complications in non-traumatic, non-infectious corneal perforations, providing anatomic integrity immediately is a must. If conservative treatment is inadequate or the perforation area is extensive, tectonic penetrating keratoplasty is indicated. Besides, it is important to manage the etiological risk factors in order to obtain successful clinical follow up.

COMPARATIVE STUDY OF THE EFFICACY OF STEM CELLS IN CORNEAL REGENERATION IN A CHEMICAL BURN IN RABBITS

Objectives: This study compares the efficacy of stem cell transplantation in corneal regeneration and restoration of the limbic deficit in an experimental chemical burn in rabbits.&#x0D; Methods: Biopsy was performed of the limbus and the chemical burns for all rabbits, and we collected the amniotic membranes from a pregnant female rabbit. We kept a control group without transplantation, to study spontaneous and natural healing, and we transplanted the stem cells produced in vitro under the corneal epithelium burned. To compare the result, we tested a group for amniotic stem cell transplantation, a group for limbal stem cell graft, and another group for combined transplantation of both types of stem cells.&#x0D; Results: Transplanted rabbits develop permanent unilateral blindness due to a severe limbic deficit. The group receiving only amniotic stem cells shows temporary anatomical improvement without functional recovery. The two groups receiving limbal stem cells alone or combined with amniotic stem cells showed anatomical and functional satisfaction with quick recovery time for the combined transplantation.&#x0D; Conclusions: A simple chemical burn can establish permanent blindness. When the limbic deficit is important, spontaneous healing is not available. Transplantation of stem cell transplant is the only way to repair this deficit and regenerate the cornea. Only limbic stem cells can be sufficient. Amniotic stem cells can support and speed up the healing time when it combined to limbal stem cells graft. &#x0D; Peer Review History: &#x0D; Received 23 July 2020; Revised 14 August; Accepted 28 August, Available online 15 September 2020&#x0D; UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.&#x0D; Received file &#x0D; &#x0D; Average Peer review marks at initial stage: 5.0/10&#x0D; Average Peer review marks at publication stage: 7.5/10&#x0D; Reviewer(s) detail:&#x0D; Name: Dr. Mohamed Amin El-Emam&#x0D; Affiliation: Department of Pharmacology and Therapeutics, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria (PUA), Alexandria, Egypt.&#x0D; E-mail: mohamed.elemam@pua.edu.eg&#x0D; &#x0D; Name: Francesco Ferrara&#x0D; Affiliation: USL Umbria 1, Perugia, Italy&#x0D; E-mail: francesco.ferrara@uslumbria1.it&#x0D; &#x0D; Name: Maged Almezgagi&#x0D; Affiliation: Department of Immunology, Medical College of Qinghai University, Qinghai Xining 810001, China&#x0D; E-mail: 1902244017@qq.com&#x0D; &#x0D; Name: Dr. Asia Selman Abdullah&#x0D; Affiliation: University of Basrah, Iraq&#x0D; E-mail: asiaselman2016@gmail.com&#x0D; &#x0D; Comments of reviewer(s): &#x0D;

DISCRETION VALUES OF LIMBAL STEM CELL GRAFT WITH COMPARISON OF CONJUCTIVAL AUTOGRAFT IN PTERYGIUM EXCISION

Objective: To determine the recurrence rate of pterygium in cases treated with limbal stem stem cell graft versus connjuctival autograft. Materials and Methods: It was a randomized clinical trial conducted in Opthalmology department of Hayatabad Medical Complex Peshawar from January 2018 to February 2019 i.e one year study duration . A total of 60 diagnosed cases of pterygium of both sexes and all ages were included in the study . The cases were randomized into two groups i.e 30 cases were treated with stem cell graft while the other 30 cases were treated with conjunctival autograft .The recurrence rate of pterygium was determined in both the groups by following them for 6 months i.e at 1st , 3rd and 6th month of excision. Recurrence rate was calculated for both the procedures. Mean and standard deviations were used for quantitative variables while frequency and percentages were used for qualitative data. Results: A total of 60 diagnosed cases of pterygium were included in the study. Age of the study sample ranged from 22-55 years, with mean of 37±9.3 SD. About 36 (60%) cases were males and ,14 (40%) cases were females. Recurrence rate of pterygium was 3.3% in cases treated with limbal stem cell graft technique as compared to recurrence rate of 10% which was seen in cases treated with conjunctival autograft technique. Conclusion: Recurrence rate of pterygium was more in cases treated with conjunctival autograft as compared to those treated with limbal stem cell graft. So, limbal stem cell graft is more effective as compared to conjunctival autograft in treating pterygium and should be preferred treatment option for pterygium in order to decrease the recurrence rate and hence morbidity of the patient.

Corneal epithelial restoration after penetrating keratoplasty in repeated failed cultivated limbal stem cell grafts

Abstract Purpose To report a case of corneal epithelial restoration after penetrating keratoplasty following multiple complications in repeated failed cultivated stem cell grafts. Methods This is a case report of a 62-year old patient who was treated for unilateral severe limbal stem cell deficiency after a chemical burn of the right eye. Results Two attempts to reconstruct the ocular surface using autologous cultivated limbal stem cell grafts (CLET) failed, and were complicated by perforations requiring two emergency penetrating keratoplasties. While the first donor cornea revascularised quickly, the corneal epithelium of the second remained transparent, avascular and stable until one year postoperatively. In vivo confocal microscopic examination confirmed presence of a healthy corneal epithelial phenotype. Conclusions Occurrence of serious adverse events are related to an inadequate epithelial homeostasis following CLET. The mechanisms of action of limbal stem cell transplantation are still poorly understood and the corneal epithelial integrity may be maintained without limbal input.

Treatment of total limbal stem cell deficiency with autologous ex vivo cultivated limbal epithelial stem cell graft

Treatment of total limbal stem cell deficiency with autologous ex vivo cultivated limbal epithelial stem cell graft

The Differences Between Tumor Necrosis Factor-α (TNF-α) Expression in Fleshy and Atrophy Pterygium

Background: Pterygium is a conjungtival fibrovascular degenerative process which fleshy type was different to atrophy type, in which post operative recurrencies was high especially fleshy type. The exact pathogenesis were not clearly understood, it was reported that many inflammation mediators had a play role, including Tumor Necrosis Factor-α (TNF-α). Objective of the study was to revealed a different expression of TNF-α in fleshy and atrophy type. Method: Cross-sectional study design have been done with independent variable was TNF-α and dependant vaiable was pterygium type. Samples consist of 20 patients fleshy type and 20 patients atrophy type, both groups were given fluorometholone eye drop for 1 week prior surgery to suppress the degree of inflammation. The baseline data was recorded e.g. demography, the size of pterygium, Tear Break Up Time (TBUT), Schirmer test and Ferning test. Pterygium excission followed by limbal stem cell graft, and examination of TNF-α expression by immunohistochemistry. TNF-α expression was analyzed using 2 x 2 table. Result: Baseline data of both groups were not significantly different, except TBUT (p=0.000) and the clinical characteristic of pterygium (p=0.000). TNF-α expression in fleshy type tend to be higher (total Allred score mean 4.70) but not significantly different than atrophic type (total Allred score mean 4.54 and p=0.61). Previous fluorometholone treatment prior to surgery might be suppressed the inflammatory cytokine. Conclusion: No significant difference of the TNF-α expressions between fleshy and atrophy type of pterygium. Keywords: pterygium, fleshy type, atrophy type, TNF-α, immunohistochemistry

In Vivo Confocal Microscopy 1 Year after Autologous Cultured Limbal Stem Cell Grafts

To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation. Prospective, interventional, noncomparative, masked case series. Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye). Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patient's injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohen's κ coefficient. Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface). We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea. Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft.

English

Amniotic Membrane Transplantation is currently being used for a continuously widening spectrum of ophthalmic indications. It has gained widespread attention as an effective method of reconstruction of the ocular surface. Amniotic membrane has a unique combination of properties, including the facilitation of migration of epithelial cells, the reinforcement of basal cellular adhesion and the encouragement of epithelial differentiation. Its ability to modulate stromal scarring and its anti-inflammatory activity has led to its use in the treatment of ocular surface pathology as well as an adjunct to limbal stem cell grafts. Amniotic membrane transplantation has been used for reconstruction of the corneal surface in the setting of persistent epithelial defects, partial limbal stem cell deficiency, bullous keratopathy and corneoscleral ulcers. It has also been used in conjunction with limbal stem cell transplantation for total limbal stem cell deficiency. Amniotic membrane grafts have been effectively used as a conjunctival substitute for reconstruction of conjunctival defects following removal of pterygia, conjunctival lesions and symblephara. More recently, amniotic membrane has been used as a substrate for ex vivo cultivation of limbal, corneal and conjunctival epithelial cells. This article reviews the current literature on the applications of amniotic membrane

Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation

BackgroundTo determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency.MethodsIn vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique.ResultsThe majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity.ConclusionWe conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

Results of a phase I/II clinical trial: standardized, non‐xenogenic, cultivated limbal stem cell transplantation

AbstractWe describe the results of a phase I/II clinical trial for standardized, non‐xenogenic, cultivation and “no touch” surgical transplantation of limbal stem cell grafts. 18 eyes of 18 patients were transplanted with either autologous (n=15) or allogenic (n=3) limbo‐amnion composite grafts that were generated using a standardized culture protocol free of xenogenic culture products and transplanted using a standardized “no touch” surgical technique. In vitro cellular outgrowth and phenotype of the cultivated graft was assessed prior to transplantation. Corneal neovascularization, central corneal opacity, pain, photophobia and visual acuity were investigated pre and post transplantation. The limbal epithelial cells showed an average outgrowth of 14.2mm by day 14. The majority of the cells displayed a progenitor phenotype: p63, CK14, desmoglein, ABCG2 bright and CK3/12 dim protein expression. The transplant recipients were followed up for a mean of 22 months. 12 out of the 18 transplant recipients were graded successful, giving an overall success rate of 67%. The ocular surface photographs for pre‐ and post stem cell transplantation, showed a significant (p=0.007) reduction in the percentage area of corneal neovascularization.

Ocular Surface Rreconstruction with Allogeneic Limbal Stem Cell and Autologous Oral Mucosal Graft: Two Cases

Ocular Surface Rreconstruction with Allogeneic Limbal Stem Cell and Autologous Oral Mucosal Graft: Two Cases

Boston type 1 keratoprosthesis outcomes in ocular burns

To report the outcomes of Boston type I keratoprosthesis (BKPro) in the management of ocular burn injuries. This was a prospective study including all cases of BKPro implantation for ocular burns at the External Diseases and Cornea Service of the Federal University of São Paulo, between February 2008 and February 2010. Ten patients (10 eyes) were enrolled. Procedures performed to manage ocular injury were identified, and data were collected regarding patients' ocular history, surgical procedure(s) performed, and postoperative outcomes, including visual acuity, retention, complications and required surgical procedures. A total of 11 Type 1 BKPro were implanted in 10 eyes of 10 patients. The mean follow-up period was 25.7 ± 10.8 months. Preoperative best-corrected visual acuity (BCVA) ranged from count fingers to light perception. Postoperative BCVA was better than 20/200 in 90% of the patients and better than 20/60 in 60% of the patients. The overall BKPro retention rate was 90%. The most common complications were retroprosthetic membrane formation (50%) and persistent corneal epithelial defect evolving to corneal melting (40%). Patients who underwent ocular surface procedures such as limbal transplantation prior to BKPRo implantation had a lower incidence of corneal melting/thinning (p = 0.07), although this was not statistically significant. The anatomical and functional results identified in this study support the use of BKPro in managing bilateral limbal stem cell deficiency secondary to ocular burns.

Ophthalmic transplantology: Anterior segment of the eye – Part I

SummaryBackgroundTransplantology is a quickly developing field of ophthalmology. It currently is able to treat many inherited, degenerative, inflammatory, traumatic, and cancerous diseases. This review outlines recent concepts and methods of treating ocular diseases with tissue and cell grafts. Ocular transplants related to the anterior part of the eye, including the conjunctiva and the cornea, are reviewed in Part 1.Material/MethodsThe scientific literature dated from January 2005 to July 2011 was thoroughly searched using Medline and PubMed. Publications dated 2009, 2010, and 2011 were analyzed in detail. Search terms were as follows: auto-, homo-, heterologous transplantation, eyeball, ocular adnexa, anterior segment of the eye, cornea, lamellar keratoplasty, stem cells, cultured cells. Further data were found at the website of the Eye Bank Association of America.ResultsNearly all tissues of the anterior segment of the eye (the conjunctiva, sclera, eye muscles, and cornea) are transplanted. Because of the recent significant progress in the field, cornea transplantation was analyzed in more detail, specifically procedures such as limbus grafts and anterior and posterior lamellar keratoplasty. Indications, advantages, and drawbacks of the transplant techniques were also reviewed.ConclusionsRecent progress in the field of cornea transplants allows treatment at the level of the endothelium and the use of cultured limbal epithelial stem cell grafts. However, compared with previous techniques, modern and multilayered transplant techniques of the cornea require much more expertise and longer training of the surgeon, as well as expensive and technologically advanced equipment. The availability of donor tissue is still the main limitation affecting all transplants. Therefore, cell culturing techniques such as stem cells, as well as artificial cornea projects, seem to be very promising.

Long‐term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns

Chemical burns cause depletion of limbal stem cells and eventually lead to corneal opacity and visual loss. We investigated the long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency. Prospective, non-comparative interventional case series. Sixteen eyes from 16 patients with severe, unilateral limbal stem cell deficiency caused by chemical burns. Autologous ex vivo cultured limbal stem cells were grafted onto the recipient eye after superficial keratectomy. Clinical parameters of limbal stem cell deficiency (stability/transparency of the corneal epithelium, superficial corneal vascularization and pain/photophobia), visual acuity, cytokeratin expression on impression cytology specimens and histology on excised corneal buttons. At 12 months post-surgery, evaluation of the 16 patients showed that 10 (62.6%) experienced complete restoration of a stable and clear epithelium and 3 (18.7%) had partially successful outcomes (re-appearance of conjunctiva in some sectors of the cornea and instable corneal surface). Graft failure (no change in corneal surface conditions) was seen in three (18.7%) patients. Penetrating keratoplasty was performed in seven patients, with visual acuity improving up to 0.8 (best result). For two patients, regeneration of the corneal epithelium was confirmed by molecular marker (p63, cytokeratin 3, 12 and 19, mucin 1) analysis. Follow-up times ranged from 12 to 50 months. Grafts of autologous limbal stem cells cultured onto fibrin glue discs can successfully regenerate the corneal epithelium in patients with limbal stem cell deficiency, allowing to perform successful cornea transplantation and restore vision.

Standardized Limbal Epithelial Stem Cell Graft Generation and Transplantation

To describe a standardized, xenogenic-free protocol for the manufacture of limbal epithelial stem cell grafts and a "no touch" surgical technique for its standardized transplantation. Antwerp University Hospital, Antwerp, Belgium. The limbo-amnion composite graft is generated by cultivating limbal epithelial stem cells on a standardized (thermolysin treated and spongy layer removed) amniotic membrane, stretched within an interlockable amnion ring. The cells are cultured in CnT-20 medium with the addition of 1% human AB serum for a period of 2 weeks. Fibrin glue is applied to the surgically prepared recipient's cornea and in one fluid motion, the composite graft within the amnion ring construct is transferred from culture and positioned onto the graft bed. The required size is cut out at the level of the limbus by means of a trephine and/or microsurgical scissors. The lightweight, plastic interlockable ring offered stability to the graft during culture, transport, and transplantation. The use of the standardized amniotic membrane, within the amnion ring construct, improves reproducibility of the results and therefore heralds elective surgery. Rapid transplantation of a wrinkle-free graft, using a sutureless, “no touch" technique was achieved and this allowed precise tailoring of the graft to the recipient bed. This is the first time a standardized, clinical grade protocol has been described for manufacturing limbal epithelial grafts with an efficient surgical technique that prevents postsurgical graft shrinkage and improves corneal integration. The quick, sutureless, and manipulation-free technique ensured transplantation of viable, proliferating limbal epithelial stem cells.

Evaluation of Molecular Markers in Corneal Regeneration by Means of Autologous Cultures of Limbal Cells and Keratoplasty

To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome. Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success" or a "failure," depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers. After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (DeltaNp63alpha) after transplantation. Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.

Techniques for Culture and Assessment of Limbal stem Cell Grafts

The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSC D) has been reported in many studies. A ccording to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as “medicinal products” and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and ΔNp63α, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.