Corneal Limbal Stem Cell Research Articles

Histopathology in the isolated chicken eye test and comparison of different stainings of the cornea

The isolated chicken eye (ICE) test, developed at our Institute, is accepted by the OECD for identification of severe eye irritants. The OECD ICE Guideline (No. 438) encourages preservation of the treated eyes for possible histopathology of the cornea, which is believed to strengthen evidence of absence or presence of irritation and to help clarify borderline effects by assessment of the corneal Depth-of-Injury. Histopathology of the cornea in addition to the normal slit-lamp microscope assessment of corneal effects has already been performed routinely in ICE tests at our Institute, using two standard stainings (H&E and PAS). In this study, three other stainings (AZAN, EVG and Trichrome), more specific for collagen-rich membranes such as basement- and Bowman’s membranes were examined with corneas exposed to four model compounds ranging from non- to severely irritating (corrosive). PAS appeared to be the superior staining method. Surprisingly, the well-known eye corrosive sodium hydroxide (NaOH, solid) did not visibly compromise the integrity of Bowman’s or the basement membrane. Based on our experience, histopathology of the treated cornea is confirmative in relation to the standard assessment of eye irritation by slit-lamp observation in the ICE and in certain cases can help to evaluate borderline effects. Besides establishing the depth of injury, additional investigation of corneal limbal stem cell damage after chemical exposure might be appropriate to determine reversibility or irreversibility of eye effects.

Pathologic epithelial and anterior corneal nerve morphology in congenital aniridic keratopathy

Abstract Purpose To document corneal morphology in Swedish families with congenital aniridia. Methods Detailed ophthalmic examinations were conducted in a number of affected and unaffected members. Digital slit lamp photography, anterior segment optical coherence tomography (ASOCT) and in‐vivo confocal microscopy (IVCM) examinations were performed bilaterally to document corneal morphology. Results Affected family members presented with different stages of aniridic keratopathy, with a corneal appearance varying from totally transparent to opaque and highly vascularised. Increased corneal thickness in affected members, particularly those with severe keratopathy, was noted by ASOCT. By IVCM, opaque corneas were characterized by active vessels and dense inflammatory cell infiltration. In corneas with milder keratopathy, pathologic epithelial findings included epithelial pleomorphism, focal epithelial opacities, and an absence of limbal epithelial crypts and focal stromal projections at the limbus. Nerves of the anterior cornea exhibited several distinct features, including an unusually close association of subbasal nerves with epithelial cells, an unusually high subbasal nerve density with highly branched nerves, and a prominent whorl region. Additionally, abnormally dense and tortuous anterior stromal nerves, attached to stromal keratocytes, were noted in unaffected members. Conclusion Altered epithelial morphology and a vigorous innervation of the anterior cornea were the most pronounced corneal findings in family members with milder forms of aniridic keratopathy. Further findings confirmed the known increase in corneal thickness and limbal stem cell abnormality in aniridia.

Management of ocular thermal and chemical injuries, including amniotic membrane therapy

To provide a concise review of the literature regarding potential management strategies of ocular thermal and chemical injuries. After experiencing a serious ocular surface burn, either thermal or chemical, the goal of therapy is to restore a normal ocular surface and corneal clarity. If extensive corneal scarring and/or limbal stem cell deficiency are present, techniques such as limbal stem cell grafting, amniotic membrane transplantation and possibly a keratoprosthesis can be employed to help restore vision. This article will review the literature available and discuss how these techniques have improved the prognosis of patients with serious thermal and chemical injuries. Ocular thermal and chemical injuries are a true ocular emergency and require immediate and intensive evaluation and treatment. The sequelae of an ocular burn can be severe and particularly challenging to manage. Improvements in the understanding of the pathophysiology of a radiant energy or chemical injury as well as advancements in ocular surface reconstruction have provided hope for patients in whom would otherwise have a dismal visual prognosis.

Peeling-off in a reverse direction combined with corneal limbal stem cell transplantation for pterygium

Objective To investigate the the efficacy of peeling-off in a reverse direction com bined with corneal limbal stem cell transplantation for pterygium. Methods Conventional simple excision of pteiygium was performed on 34 eyes, so was peeling-off in a reverse direction plus corneal limbal stem cell transplantation on 42 eyes. Visual acuity, conjunctival graft healing, and surgical complications were observed postoperatively. The recurrence rate was statistically analyzed in the two procedures one year after surgery. Results Visual acuity was improved at different levels in all the patients. Conjunctival graft healed well in peeling-off plus corneal limbal stem cell transplantation with no complications. The recurrence rate was 14. 2% one year after the procedure. Conclusions Peeling-off in a reverse direction combined with comeal limbal stem cell transplantation for pterygium has fewer complications, a lower recurrence rate, and is worth popularizing. Key words: Peeling-off in a reverse direction; Stem cell transplantation; Pterygium

Ex VivoConstruction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin-coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane (AM). Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibers within the compressed gel were dense, homogeneous, and similar in structure to those within denuded AM. Limbal epithelial cells were successfully expanded upon the compressed collagen, resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology, and cell stratification. Similar protein expression profiles for cytokeratin 3 (CK3) and CK14 and no significant difference in CK12 mRNA expression levels by real-time polymerase chain reaction were also observed. This study provides the first line of evidence that a laminin-coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification, and differentiation to a degree that is comparable to the leading conventional scaffold, denuded AM.

Corneal surface reconstruction - a short review

Corneal surface reconstruction - a short review

The Boston Type I Keratoprosthesis: Improving Outcomes and Expanding Indications

To report the usefulness of the Boston type I keratoprosthesis (Massachusetts Eye and Ear Infirmary, Boston, MA) in the management of corneal opacification, corneal limbal stem cell failure, or both in a large single-surgeon series. Retrospective review of consecutive clinical case series. All patients undergoing keratoprosthesis implantation by a single surgeon (AJA) between May 1, 2004, and May 31, 2008. Data were collected regarding the preoperative characteristics of each eye undergoing keratoprosthesis implantation, the surgical procedure(s) performed, and the postoperative course. Statistical analysis was performed to identify factors influencing the incidence and severity of postoperative complications. Interval visual acuities, keratoprosthesis retention, and significant postoperative complications. Fifty-seven keratoprosthesis procedures were performed in 50 eyes of 49 patients. The primary indication for surgery was repeat corneal transplantation failure (68%), although no prior corneal surgery had been performed in 16% of eyes. Preoperative visual acuity was 20/200 or worse in all eyes, with 88% of eyes having preoperative vision of counting fingers, hand movements, or light perception. The percentage of eyes with postoperative visual acuity of 20/100 or better was 67% at 6 months (n = 45), 75% at 1 year (n = 28), 69% at 2 years (n = 13), and 100% at 3 years (n = 7). In the 31% of patients in whom preoperative vision in the contralateral eye was 20/50 or better, the postoperative vision in the operative eye improved to 20/50 or better in 47% at the last follow-up (average, 18 months; range, 4-49 months). The overall keratoprosthesis retention rate was 84% at an average follow-up of 17 months (79 person-years of follow-up), with 100% retention in 8 eyes with no history of prior corneal transplantation (14.8 person-years of follow-up). The most common postoperative complications were retroprosthetic membrane formation (22 eyes) and persistent epithelial defects (19 eyes). No cases of infectious endophthalmitis were encountered, and only 1 patient with a history of glaucoma required additional glaucoma surgery during the postoperative period. The Boston type I keratoprosthesis is an effective means of managing repeat corneal graft failure and corneal limbal stem cell failure with or without corneal opacification in patients with both unilateral and bilateral visual impairment.

The Effects of Wnt Protein on Proliferation and Stemness Maintenance of Corneal Limbal Stem Cells (CLSCs)

Purpose: To evaluate the effects of the Wnt protein on proliferation and stemness maintenance of cultured corneal limbal stem cells. Methods: We examined the expression of Wnt proteins by Western blot analysis. We then evaluated the effects of Wnt on cell proliferation by colony forming efficiency. -catenin activation using Wnt proteins was examined by immunocytochemistry. We also examined the effects of Wnt on proliferation and stemness maintenance by reverse transcriptase polymerase chain reaction of p63 and connexin43. Results: Wnt has a different effect on corneal epithelial stem cells. Colony forming efficiency was also significantly higher in treated Wnt2 and Wnt4 cells compared with controls. The Wnt2 and Wnt4 treated cells showed nuclear accumulation of -catenin. In addition, the limbal stem cell marker p63 was strongly expressed in Wnt2, Wnt4 Wnt5a, and Wnt5b. Connexin43 mRNA was also strongly expressed in Wnt5a, Wnt5b and Wnt7b cells. Conclusions: We suggest that Wnt2 and Wnt4 could lead to more effective proliferation and stemness maintenance for human corneal epithelial stem cells.

Curative effect comparison of throe different operation methods combined with mitomycin-c for pterygium

Objective To compare the curative effect of three different operation methods for pterygium, Methods 120 eyes of 90 patients with pterygium were randomly divided into three groups:Group A, B and C. 35 eyes in Group A received normal pterygium incision with sclera exposed;40 eyes in Group B received pterygium incision with plantation of amniotic membrane;45 eyes in Group C received pterygium incision with transplantation of corneal limbal stem cells, and mitomycin-c combined with three methods, Follow-up after operation lasted from 12 months to 2 years, and the reoccurrence in each group was compared. Results 10 eyes out of 35 eyes in Group A had reoccurrence (28.57%),3 eyes out of 40 eyes in Group B (7.5%) and 2 eyes out of 45 eyes in Group C (4.44 %),The comparison between Group A and Group B or Group C (P<0.05) was of significant difference. But there is no difference between Group B and Group C. Conclusion Aterygium incision with plantation of amniotic membrane and autologous corneal limbal stem cell transplantation can decrease the recurrent rate. Especially pterygium incision with transplantation of corneal limbal stem cells is an ideal method of pterygium surgery. Key words: Pteryium; Limbal stem cell; Amniotic membrane; Transplantation; Mitomycin-C

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins: the outer, epithelial layer develops from the ectoderm overlying the lens vesicle, whereas the stroma and the endothelium have mesenchymal origin. In the adult organism, the outermost corneal epithelium is the most exposed to environmental damage, and its constant renewal is assured by the epithelial stem cells that reside in the limbus, the circular border of the cornea. Cell turnover in the stromal layer is very slow and the endothelial cells probably do not reproduce in the adult organism. However, recent experimental evidence indicates that stem cells may be found in these layers. Damage to any of the corneal layers leads to loss of transparency and low vision. Corneal limbal stem cell deficiency results in severe ocular surface disease and its treatment by transplantating ex vivo expanded limbal epithelial cells is becoming widely accepted today. Stromal and endothelial stem cells are potential tools of tissue engineering and regenerative therapies of corneal ulcers and endothelial cell loss. In the past few years, intensive research has focused on corneal stem cells aiming to improve the outcomes of the current corneal stem cell transplantation techniques. This review summarizes the current state of knowledge on corneal epithelial, stromal and endothelial stem cells. Special emphasis is placed on the molecular markers that may help to identify these cells, and the recently revealed mechanisms that could maintain their "stemness" or drive their differentiation. The techniques for isolating and culturing/expanding these cells are also described.

Harvesting keratolimbal allografts from corneoscleral buttons: a novel application of cyanoacrylate adhesive

Aim: To describe an alternative and novel technique using cyanoacrylate glue to achieve successful limbal tissue dissection, from an organ culture media stored corneoscleral button, without an artificial anterior chamber....

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

The treatment effect of two transplantation of pteryginm combine mitomycin C

Objective To compare the treatment effect of amniotie membrane transplantation(AMT) combine mitomycin C(MMC) and corneal limbal stem cell autograft transplantation combine mitomycin C surgery.Methods 234 pterygium patients(239eye) were randomly divided into two groups: routine resection and AMT combine mitomycin C,limbal epithelial autograft transplantation combine mitomycin C. Results The recrudescnce (3.25 % ) of AMT combine mitomycin C is lower than routine resection and limbal epithelial autograft transplantation combine mitomycin C(4.31% ). But there was no significant difference between these two groups. Conclusion The treatment of AMT combine mitomycin C is as good as Iimbal epithelial autograft transplantation combine mitomyein C and the recrudescnce is lower. Key words: Pterygiurn; Corneal transplantation; Mitomycin C; Amnion

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a rabbit model

The integrity and transparency of the cornea plays a key role in preserving vision. This paper reports a procedure to create an artificial sheet of corneal epithelium from cryopreserved limbal stem cells (LSCs) and to use this for corneal transplantation. Corneal LSCs were isolated from biopsy specimens of rabbit limbal lamellar and cryopreserved in liquid nitrogen at 2–4 passages. The cells were grown in culture medium for 12–14 days on top of a cell-free human amniotic membrane framed on a nitrocellulose sheet. The corneal epithelium generated was transplanted into the right eyes of 14 LSC deficient (LSCD) rabbits (seven experimental animals, seven controls) with corneal damage. The seven LSCD rabbits in the experimental group were transplanted with a corneal epithelial sheet generated from the cryopreserved corneal LSCs. Four LSCD rabbits were used as the vehicle control and were transplanted with a cell-free amniotic membrane, and the remaining three LSCD rabbits were negative controls without transplantation. Over a 2-month recovery period, 2/7 animals in the experimental group recovered completely, four recovered partially and one did not respond. In the control groups, three negative controls and three vehicle controls lost their vision completely, and one of the vehicle controls partially recovered transparency of the cornea Following treatment, corneal transparency of the experimental rabbits was significantly improved compared to controls ( P < 0.05). The results indicated that cryopreserved corneal LSCs can repair damaged rabbit cornea, suggesting a possible new clinical approach to reconstruction of corneal epithelium.

Transplantation of Ex Vivo Cultured Limbal Epithelial Stem Cells: A Review of Techniques and Clinical Results

Ex vivo cultured limbal epithelial stem cells have been used successfully to treat corneal limbal stem cell deficiency. We identified 17 reports of the application of this novel cell-based therapy in humans. In addition we identified four reports of the use of culture oral mucosal epithelial cells to treat limbal stem cell deficiency. We examined these reports to discern the success rate, complication rate, visual outcome, whether there is an optimal technique and which patients are the most likely to benefit. We also discuss the different culture methods employed and the regulations governing cell banks that are providing this service. We found that the techniques used to cultivate and transplant cells varied, but that no individual method was clearly superior. The reported success rate is similar across all studies for both allografts and autografts. The clinical indications for this treatment are not clearly defined as indicated by the variety of disorders treated. Follow-up is limited and the long-term success rate is yet to be established. Nonetheless, we conclude that there is sufficient evidence to support the continued use and refinement of this procedure as a treatment for corneal stem cell deficiency.

In vivo fibreoptic confocal imaging (FOCI) of the human ocular surface

Recent developments in the miniaturization of confocal imaging technology have resulted in the development of a hand-held confocal microscope probe. There are many structures of interest in the human eye that are within reach of a fluorescence-mode confocal microscope; this study assessed the feasibility of in vivo human ocular imaging. Safety analysis was undertaken to ensure that the laser light applied to the ocular surface structures constituted no threat to patient safety. A fibreoptic confocal imaging (FOCI) probe using an illumination wavelength of 488 nm was applied to the ocular surface of four volunteers after topical administration of sodium fluorescein. Stabilization of the probe on the ocular surface was difficult, but movement artefacts could be minimized to a satisfactory level in most subjects by a variety of procedures. High-quality images of conjunctival epithelial and goblet cells, lamina propria structures, accessory lacrimal glands, lacrimal ducts and superficial sclera were obtained. Lateral resolution was 1-1.5 microm and axial resolution was approximately 30 microm; individual erythrocytes could be seen in conjunctival vessels. The rete ridges and intervening epithelial components, including the probable location of corneal limbal stem cells, could be viewed, although it was not possible to distinguish cell subgroups. The study showed that fluorescence-mode imaging of the ocular surface is a viable and promising tool for assessment of diseases and processes involving superficial ocular structures. Refinement of equipment and techniques, particularly probe stabilization, is necessary to realize fully the potential of FOCI for ocular use.

Medium term results in keratoprostheses with biocompatible and biological haptic

Corneal grafts or limbal stem cell transplantation are often unsuccessful in patients with severe ocular surface disorders such as severe dry eye syndrome, symblepharon or diffuse vascularisation. In those patients, a keratoprosthesis (KPro) may be an alternative for the recovery of vision. Various KPro differ from each other in the material of the haptic that supports the optic cylinder. The haptic may be made of biocompatible or biological material such as tibia bone (TKPro) or dentine and alveolar bone (osteo-odonto-keratoprosthesis, OOKP). On the basis of our experience, we wanted to comment on the value of different KPro. Over the last 10 years we have implanted a total of 35 KPro, 29 with biological haptic (25 OOKP and four TKPro), and six KPro with biocompatible haptic (one Legais KPro, five Pintucci KPro). A follow up examination was carried out approximately every6 months. The patients gained a visual acuity of > or =0.9 in 20.6%, of > or =0.5 in 52.9%, of > or =0.2 in 61.8% and a significant improvement in visual acuity in 76.5%, respectively. There was no significant difference between the various types of KPro concerning the best postoperative visual acuity. All patients showing poor improvement had a pre-existing end stage secondary glaucoma or other retinal damage. The median follow-up was 2.9 years (maximum 8) for OOKP, 1 year for TKPro, 1 year (maximum 2) for Pintucci Kpro and 6 month for Legeais KPro. During this period, only one of the KPro with biological haptic was lost (one TKPro after 1 year), compared with four out of six of the KPro with biocompatible haptic (P<0.0001). Fixation of the KPro by a root of the patient s own tooth (OOKP) leads to the best results in the long-term follow up, as our results as well as the literature demonstrate. As long as a KPro is in place, the visual acuity is as good as the retinal function. For the ranking of different types of KPro, the percentage and the duration of the anatomic success are most important. The comparability of the various KPro results may be limited, since the patients were not randomised and the four groups differ in number.

Mucous membrane pemphigoid: an update

To review articles on mucous membrane pemphigoid, published between June 2004-May 2005. Decreased glycosylation of mucin was found in patients with ocular cicatricial pemphigoid. A unique antigen in oral mucous membrane pemphigoid has not yet been identified. Increased vascular cell adhesion molecule and intercellular adhesion molecule 1 expression was found in skin of patients affected by mucous membrane pemphigoid. Autoreactive T cells to an epitope of bullous pemphigoid antigen 180 kilodaltons were identified in the blood of some patients with mucous membrane pemphigoid. Circulating IgA against an antigen in mucous membrane pemphigoid was found in about 20% of patients, without prognostic significance. Enhanced sensitivity for direct immunofluorescence was reported if skin biopsy specimens were stored for 24 hours in saline. An enzyme-linked immunosorbent assay for detection of circulating autoantibodies against laminin-5 was developed. Sensitivity was higher than indirect immunofluorescence on salt-split skin and immunoblotting. Patients with younger onset (<60 years) of ocular cicatricial pemphigoid were found to have disease evolution similar to that of an older group (>70 years) but were visually impaired earlier in life. Intravenous immunoglobulin as treatment of ocular cicatricial pemphigoid was found to be superior to conventional immunosuppressants, with fewer side effects and better long-term outcome for halting disease activity. Intraoperative adjunction of mitomycin C during fornix reconstruction with amniotic membrane resulted in achieving a deeper fornix in 83% of patients with various cicatrizing conjunctivitis. Transplantation of cultured epithelial cells of oral mucosa in corneal limbal stem cell deficiency was successful in improving visual acuity and reestablishing corneal transparency in mid- to advanced ocular cicatricial pemphigoid. Further advances have been achieved in the field of mucous membrane pemphigoid.

Autografts of cultured stem cells transplantstion in treatment of pterygium

目的 观察自体角膜缘干细胞移植术治疗翼状胬肉的临床效果.方法翼状胬肉11例12只眼,采用手术显微镜下切除局部病灶,然后行自体角膜缘干细胞移植术,术后随访6～15个月(平均11个月).结果术后角膜上皮愈合良好,无排斥反应或其他并发症发生.随访期间1只眼复发,复发率为8.3%.结论应用自体角膜缘干细胞移植术为受损的角膜缘提供新的健康干细胞,可有效地防止翼状胬肉复发,起到美容的效果。