Clinical outcome of autologous cultivated limbal epithelium transplantation

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.     

Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency

PURPOSE: To evaluate the survival of conjunctival limbal grafts and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency (LSCD) and the influence of several parameters as cause of LSCD, dry eye, keratinization, eyelid abnormalities, HLA compatibility, systemic immunosuppression, and keratoplasty (PKP) on surgical outcome. DESIGN: Prospective, noncomparative, interventional case series. METHODS: Thirty-three eyes of 31 patients with total LSCD that underwent conjunctival limbal grafts and AMT at the Department of Ophthalmology, Federal University of S?o Paulo were included in this study. Cumulative graft survival as well as the influence of several variables on surgical outcome was analyzed. RESULTS: Ten eyes (30%) underwent conjunctival limbal autograft and 23 (70%) underwent conjunctival limbal allograft from living HLA-matched donor. Graft survival was seen in 13 eyes (40%) at 1 year and in 11 eyes (33.3%) at 2 years, with a cumulative survival of 33% after a mean follow-up time of 33 months. Increase in postoperative visual acuity was observed in 20 eyes (60.6%) during this period. Marked impact on graft survival was observed for patients with Stevens-Johnson syndrome, dry eye, keratinization, eyelid abnormalities, and allogeneic conjunctival limbal transplantation (independently of HLA compatibility) (P < .05). Preoperative dry eye was the most important prognostic parameter for surgical outcome (P < .001). CONCLUSIONS: Conjunctival limbal grafts associated with AMT are useful for restoring corneal epithelium phenotype in eyes with total LSCD. However, the cumulative survival declines substantially over a 2-year period. Considering all investigated variables, dry eye was the most important prognostic parameter.     

Autologe Transplantation von kultiviertem Limbusepithel

Objective

In this study the clinical outcome of ex vivo expansion of autologous limbal epithelial cells on intact amniotic membranes (AM) for ocular surface reconstruction in limbal stem cell deficiency (LSCD) was investigated.

Patients and Methods

A total of 30 eyes in 28 patients (22 male and 6 female) with total (n=18) or partial (n=12) LSCD were treated by transplantation of autologous limbal epithelial cells after expansion on intact AM. The causes of LSCD in the patients were chemical and thermal burns (n=16), pterygium (n=9), tumor excision (n=2), perforating injury, mitomycin C-induced LSCD and epidermolysis bullosa (each n=1). Only eyes with a follow-up time of at least 9 months were included in the analysis. The main outcome criteria were restoration of ocular surface integrity and improvement of visual acuity (VA).

Results

The mean follow-up time was 28.9±15.5 months. An entirely stable corneal surface was reconstructed in 23 (76.7%) eyes. Visual acuity increased significantly in 21 (70%) eyes, was stable in 8 (26.7%) eyes and decreased in 1 (3.3%) eye. The mean visual acuity increased significantly (p<0.0001) from a preoperative value of 1.58±0.97 LogMAR to 0.6±0.49 LogMAR.

Conclusion

Transplantation of limbal epithelium cultivated on intact AM restores a stable corneal surface and results in a significant increase in visual acuity in most cases of LSCD. Autologous transplantation of cultivated limbal epithelium showed an excellent prognosis and outcome after long-term follow-up.     

Biomarkers of in vivo limbal stem cell function

PurposeTo evaluate in vivo parameters as biomarkers of limbal stem cell function and to establish an objective system that detects and stage limbal stem cell deficiency (LSCD).MethodsA total of 126 patients (172 eyes) with LSCD and 67 normal subjects (99 eyes) were included in this observational cross-sectional comparative study. Slit-lamp biomicroscopy, in vivo laser scanning confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) were performed to obtain the following: clinical score, cell morphology score, basal cell density (BCD), central corneal epithelial thickness (CET), limbal epithelial thickness (LET), total corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), and tortuosity coefficient. Their potential correlations with the severity of LSCD were investigated, and cutoff values were determined.ResultsAn increase clinical score correlated with a decrease in central cornea BCD, limbal BCD, CET, mean LET, maximum LET, CNFL, CNFD, CNBD, and tortuosity coefficient. Regression analyses showed that central cornea BCD, CET and CNFL were the best parameters to differentiate LSCD from normal eyes (Coef = 3.123, 3.379, and 2.223; all p < 0.05). The rank correlation analysis showed a similar outcome between the clinical scores and the central cornea BCD (ρ = 0.79), CET (ρ = 0.82), and CNFL (ρ = 0.71). A comprehensive LSCD grading formula based on a combination of these parameters was established.ConclusionsA comprehensive staging system combining clinical presentation, central cornea BCD, CET, and CNFL is established to accurately and objectively diagnose LSCD and stage its severity.     

Analysis of limbal stem cell deficiency by corneal impression cytology

PURPOSE: The impaired function of corneal epithelial stem cells, located in the limbus, is responsible for corneal surface damage and is clinically characterized by recurrent epithelial defects, conjunctivalization, neovascularization, and corneal opacity. The aim of this study was to investigate corneal limbal stem cell deficiency (LSCD) by means of the impression cytology (IC) technique, using antibodies against cytokeratin 19 (CK19) and cytokeratin 3 (CK3), and to evaluate the diagnostic potential of this approach. METHODS: Over a 3-year period (October 1998-June 2001), we collected 113 pairs of IC samples from the eyes of 85 patients with a range of ocular surface diseases and performed an immunocytochemical analysis of CK19 and CK3. Samples with more than 50% cellularity were considered suitable for diagnostic purposes, while samples with less than 50% cellularity were considered with caution. CK19-positive cells in corneal IC were considered an expression of LSCD. We arbitrarily scored LSCD as mild (<25% of CK19-positive cells), moderate (25-50%), and severe (>50%). RESULTS: One hundred thirteen pairs of IC specimens were obtained from 85 patients; 32 patients (37.6%) had alkaline burns, 18 (21.2%) had other chemical or physical corneal injuries, 13 (15.3%) had complications from wearing contact lenses, 8 (9.4%) had severe microbial keratitis, and 14 (16.5%) had suspicious limbal deficit due to other causes. Nine patients underwent bilateral sampling and 12 had to be resampled. Thirteen pairs of IC specimens were obtained during the follow-up of 8 patients who had undergone limbal stem cell transplantation. In 3 of these patients, IC confirmed reversion to corneal immunophenotype (CK3+/CK19-), whereas in 4, residual limbal damage was still evident; 1 patient relapsed. In the remaining 100 pairs of impressions, we found 77 cases of LSCD, whereas in 16 pairs, we did not find LSCD. Seven pairs were defined as "not valuable" because of the poor quality of both CK samples. Diffuse LSCD, moderate or severe in degree, was found in 26 of 32 patients (81.2%) with alkali burns, whereas mild diffuse LSCD or sectoral LSCD was found in 13 of 18 patients (72.2%) with other chemical-physical injuries, in 10 of 13 patients (76.9%) wearing contact lenses, in 7 of 8 patients (87.5%) with severe microbial keratitis, and in 12 of 14 patients (85.7%) with other corneal pathologies. The quality of impressions was assessed in 77 cases and found to be good or discrete for both CKs in 32 cases (41.5%) and poor in 45 (58.5%): in 46.7% of these cases, the IC was poor only for CK19 and in 45.4% only for CK3. CONCLUSIONS: Immunocytochemistry for seeking out CK19- and CK3-positive cells on corneal IC is a simple and practical method to investigate LSCD. We believe that this technique could have an important role in evaluating patients undergoing therapeutic penetrating keratoplasty to select those who would benefit from limbal stem cell transplantation. Since sampling has been shown to be a critical point, we believe that any improvement in this area will also help to improve the methodology and will contribute to its wider utilization.     

Idiopathic limbal stem cell deficiency

PURPOSE: To describe and to characterize the clinical findings and prognosis of patients with idiopathic limbal stem cell deficiency (LSCD). DESIGN: Retrospective case series. PARTICIPANTS/METHODS: We reviewed records from seven patients whose LSCD had been diagnosed clinically and confirmed by impression cytology and in whom the cause of LSCD was never identified. A detailed history, clinical records, and results of slit-lamp biomicroscopy, photography, vital staining, and impression cytology were evaluated. RESULTS: Six of seven patients (86%) were women, indicating a female predominance. Two patients were from the same family, whereas one other had a positive family history. Severe photophobia was noted in all patients and reduced vision in three patients. The main clinical findings included superficial vascularization, worse in the superior followed by the inferior and nasal cornea. The limbal regions showed a loss of limbal palisades of Vogt, and the adjacent peripheral cornea revealed an irregular and hazy epithelium with positive late fluorescein staining and the presence of conjunctival goblet cells by impression cytology. LSCD was bilateral in all patients but asymmetric in four. During a mean follow-up of 6.1+/- 3.8 years, the visual acuity decreased in both eyes of one patient after cataract extraction and in both eyes of two other patients without surgery. The process of conjunctivalization advanced in four patients (57%) and remained stable in three (43%) without surgical intervention. CONCLUSIONS: Idiopathic LSCD is a rare and as yet poorly recognized clinical entity, and the findings reported herein may help explore how progressive loss of limbal stem cells occurs. Correct diagnosis of idiopathic LSCD is important so that the patient will not be subjected to unnecessary surgeries, which may actually severely worsen the clinical course.     

Efficacy and outcome of simple limbal epithelial transplantation for limbal stem cell deficiency verified by epithelial phenotypes integrated with clinical evaluation

PurposeTo evaluate the efficacy and outcome of simple limbal epithelial transplantation (SLET) for limbal stem cell deficiency (LSCD) using epithelial phenotype detection integrated with clinical manifestation.MethodsThis prospective multicenter study included patients with LSCD who underwent autologous SLET (autoSLET) and living-related allogenic SLET (Lr-alloSLET). All patients were assessed by slit-lamp biomicroscopy, in vivo confocal microscopy (IVCM), and impression cytology with immunofluorescence staining (ICIF) before and after surgery. The criteria for success were the presence of a clinically non-conjunctivalized cornea and corneal epithelium detected by IVCM or ICIF. Otherwise, the case would be considered a failure. Visual improvement and risk factors for SLET failure were analyzed.ResultsA total of 28 eyes of 26 patients (11 autoSLET and 17 Lr-alloSLET) were included. The median age was 53 years (range, 35–63), and the follow-up time was 29.5 months (range, 17.5–39.8). The overall survival rate was 89.3% at 2 years and 75.6% at 3 years with no difference between autoSLET and Lr-alloSLET (p = 0.24). Seven eyes subsequently underwent penetrating keratoplasty. Immunohistochemistry analysis showed that all corneal buttons had corneal epithelium and limbal stem cell markers. Visual improvement was achieved in both SLET groups (p < 0.001). Failed SLET developed between 5 and 32 months postoperatively. However, absolute risk factors for SLET failure were unidentified.ConclusionThe efficacy of autoSLET and Lr-alloSLET for LSCD was excellent. Limbal explants can regenerate and restore the corneal surface while maintaining the characteristics of limbal stem cells as shown by epithelial phenotype detection and immunohistochemistry integrated with clinical evaluation.     

Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits

PURPOSE: To determine factors affecting the outcome of corneal surface reconstruction in rabbits with total limbal stem cell deficiency (LSCD), by using autologous limbal epithelial stem cells (LSC) ex vivo, expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered totally limbal stem cell deficient by n-heptanol debridement of the entire corneal epithelium followed by surgical removal of 360 degrees of limbal rim. After cytologic verification of LSCD, the fibrovascular pannus of each cornea was removed. Group I (n = 10) received a rabbit AM transplant, whereas groups II, III, and IV (n = 42) underwent transplantation of LSCs cultured on rabbit AM (LSC-AM graft) derived from a small limbal biopsy specimen from the right eye. Clinical outcome was graded as a success if a smooth, avascular corneal surface was restored, a partial success if more than two quadrants of corneal surface were smooth, or a failure if the corneal surface was revascularized and irregular. RESULTS: A long-term follow-up of more than 1 year was achieved. Compared with the 100% failure rate in group I, inclusion of expanded LSCs resulted in variable success rates in groups II, III, and IV (all P < 0.001). Kaplan-Meier survival analysis showed that different suturing techniques, subconjunctival injection of long-acting steroid, and tarsorrhaphy used in groups II (n = 17) and III (n = 13) did not significantly alter the outcome (P = 0.89). However, the use of a larger graft and human AM as a temporary patch with the explant retained for 1 week in group IV (n = 12) significantly improved the success rate to 83% (P = 0.002). Among eyes showing clinical failure, there was a significant correlation between the logarithm of the first day when an epithelial defect was noted and the time of graft failure (r(2) = 0.60, P < 0.001). Furthermore, the presence of severe lid deformity was borderline significant when correlated with failure cases in all four groups (P = 0.069). CONCLUSIONS: Ex vivo expansion of LSCs can be achieved by using rabbit AM culture. Such expanded LSCs can successfully reconstruct corneal surfaces affected by total LSCD. This animal model is useful to investigate culturing variables affecting epithelial stemness so that surgical reconstruction of corneas with total LSCD can be successfully performed. Furthermore, this model can be used to test the feasibility of gene therapies targeting LSCD in the future.     

Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series

Purpose: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal‐derived products and non‐human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. Methods: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Results: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11‐ to 28‐month follow‐up). Conclusions: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal‐derived products and non‐human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter‐species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.     

A systematic literature review of surgical interventions for limbal stem cell deficiency in humans

PURPOSE: To evaluate the relative benefits and to identify any adverse effects of surgical interventions for limbal stem cell deficiency (LSCD). DESIGN: Systematic literature review. METHODS: We searched the following electronic databases from January 1, 1989 through September 30, 2006: MEDLINE, EMBASE, Science citation index, BIOSIS, and the Cochrane Library. In addition, reference lists were scanned to identify any additional reports. The quality of published reports was assessed using standard methods. The main outcome measure was improvement in vision of at least two Snellen lines of best-corrected visual acuity (BCVA). Data on adverse outcomes also were collected. RESULTS: Twenty-six studies met the inclusion criteria. There were no randomized controlled studies. All 26 studies were either prospective or retrospective case series. For bilateral severe LSCD, keratolimbal allograft was the most common intervention with systemic immunosuppression. Other interventions included eccentric penetrating keratolimbal allografts and cultivated autologous oral mucosal epithelial grafts. An improvement in BCVA of two lines or more was reported in 31% to 67% of eyes. For unilateral severe LSCD, the most common surgical intervention was contralateral conjunctival limbal autograft, with 35% to 88% of eyes gaining an improvement in BCVA of two lines or more. The only study evaluating partial LSCD showed an improvement in BCVA of two lines or more in 39% of eyes. CONCLUSIONS: Studies to date have not provided strong evidence to guide clinical practice on which surgery is most beneficial to treat various types of LSCD. Standardized data collection in a multicenter LSCD register is suggested.     

Post-chemotherapy bilateral limbal stem cell deficiency

We report an extremely rare case of bilateral total limbal stem cell deficiency (LSCD) induced by long-term systemic chemotherapy for squamous cell carcinoma of the lung, and the outcomes of bilateral keratolimbal allograft transplantation (KLAT) in such a case. A 34-year-old male patient had underlying disease of squamous cell carcinoma in the right upper lung with extensive mediastinum and lymph node metastasis and received a series of systemic chemotherapy, including a combination of vinorelbine and cisplatin and a combination of gemcitabine, cisplatin, and docetaxel. According to clinical manifestations of ocular surface disorder, pathology findings, and immunostaining of cytokeratin 13 (CK13), LSCD secondary to systemic chemotherapy was diagnosed. KLAT was performed in both eyes. Although graft rejection developed in the right eye, complete re-epithelialization with favorable visual acuity occurred in the left eye 3 years after limbal transplantation.     

Molecular and cellular characterization of expanded and cryopreserved human limbal epithelial stem cells reveal unique immunological properties

Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin β1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.     

Correlation of long term phenotypic and clinical outcomes following limbal epithelial transplantation cultivated on amniotic membrane in rabbits

AIM: To determine the epithelial phenotype in rabbits with total limbal stem cell deficiency (LSCD) after reconstruction with autologous limbal epithelial stem cells ex vivo expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered total LSCD, verified by impression cytology. The fibrovascular pannus of each cornea was removed. Group I (n = 10) received rabbit AM transplantation alone, while groups II-IV (n = 42) underwent transplantation of LSC cultured on rabbit AM (LSC-AM) from a small limbal biopsy taken from the right eye. Clinical outcome was graded as "success," "partial success," or "failure" depending on the corneal smoothness and avascularity. Epithelial phenotype was determined by immunostaining and graded as "corneal (K)," "conjunctival (J)," or "mixed (M)" depending on expression of K3 and Muc5AC. RESULTS: After 1 year follow up, group I showed 100% failure and groups II-IV showed 26% success (p<0.001). Clinical failure correlated with J phenotype p = 0.001), while clinical success correlated with K phenotype p = 0.01). When the phenotypic outcome was used for comparison, J phenotype was significantly high in group I (p = 0.003), while K phenotype was significantly high in groups II-IV (p<0.05). CONCLUSION: There is a strong correlation between clinical success and resultant corneal epithelial phenotype. Ex vivo expanded LSC can successfully reconstruct corneal surfaces with unilateral total LSCD.     

组织工程角膜上皮移植重建眼表面的临床研究

目的 观察组织工程角膜上皮移植重建眼表面治疗完全性角膜上皮干细胞缺乏的短期临床效果.方法 系列病例研究.6例(6眼)单眼全角膜缘干细胞缺乏患者,包括碱烧伤3例、爆炸伤2例、热烧伤1例.采用去上皮羊膜组织作为载体,体外扩增患者自体健眼角膜上皮干细胞,构建组织工程角膜上皮.然后进行组织工程角膜上皮移植重建眼表面.术前及术后检查指标包括裸眼视力、裂隙灯显微镜、超声生物显微镜、泪液分泌试验.结果 患者自体来源的角膜上皮干细胞在去上皮羊膜上培养3周后均可形成直径15 mm的复层上皮片.组织工程角膜上皮均成功移植于所有受体眼表面.移植术后3个月,所有患者角膜上皮完整光滑,角膜瘢痕及纤维血管翳明显减轻,6例患者视力均有不同程度的提高.结论 患者自体来源的角膜上皮干细胞构建的组织工程角膜上皮移植可以成功重建全角膜缘干细胞缺乏患者的眼表面,为这类患者的复明带来希望.     

Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency

OBJECTIVE: To minimize the risk to the donor eye when a conjunctival limbal autograft is performed for unilateral total limbal stem cell deficiency (LSCD), a new approach has been reported of expanding limbal epithelial progenitor cells from a small limbal biopsy cultured on amniotic membrane (AM). Herein, we present for the first time the morphologic and phenotypic outcome of one such patient. DESIGN: Interventional case report. METHODS: A 31-year-old male with a severe acid burn to his left eye received AM transplantation at the acute stage and a keratolimbal allograft (KLAL) at the chronic stage for total LSCD. As an alternative to combat the failed KLAL, the above-mentioned new surgical procedure was performed. The corneal button, obtained after a penetrating keratoplasty performed 5.5 months later, and a normal corneal button as a control were submitted to hematoxylin-eosin and immunofluorescence staining for keratin K3, connexin 43, goblet-cell mucin MUC 5AC, laminin 5, and integrins alpha3beta1 and alpha6beta4. MAIN OUTCOME MEASURES: Clinical and immunohistologic features. RESULTS: The resultant epithelium was stratified with five to six cell layers and anchored to laminin 5 of the amniotic basement membrane via integrins alpha3beta1 and alpha6beta4 in a manner similar to the normal corneal epithelium. Intriguingly, the epithelial phenotype was limbal and not corneal, based on the negative expression of keratin K3 and connexin 43 of the basal epithelium. CONCLUSIONS: The technique described ensures the preservation of amniotic basement membrane, which allows formation of adhesion complexes and maintains normal corneal architecture. The preservation of a limbal epithelial phenotype on the reconstructed corneal surface indicates that AM provides a unique stromal environment conducive to the preservation and expansion of limbal epithelial progenitor cells.     

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases.     

Limbal Stem Cell Transplantation and Complications

ABSTRACT

Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).     

Modified Allogenic Simple Limbal Epithelial Transplantation Followed by Keratoplasty as Treatment for Total Limbal Stem Cell Deficiency

Purpose: To describe a modified allogenic simple limbal epithelial transplant (SLET) technique with large donor tissue explants followed by keratoplasty.

Methods: A 69-year-old with conjunctival melanoma on her left eye developed total limbal stem cell deficiency (LSCD) after multiple cycles of topical mitomycin. She also had herpes stromal keratitis while on treatment with mitomycin. She underwent modified allogenic SLET with large donor limbal tissue explants glued on the cornea directly and the amniotic membrane placed over the limbal explants.

Results: Unfortunately she had recurrence of herpes simplex keratitis that caused worsening of stromal scarring and neovascularization. She underwent penetrating keratoplasty to improve vision. There were no postoperative complications and the corneal graft remains transparent 11 months after penetrating keratoplasty with 6/12 best-corrected visual acuity.

Conclusions: Modified SLET with large donor limbal explants followed by keratoplasty is an effective approach to restore corneal transparency in cases of total LSCD.     

A systematic review of cellular therapies for the treatment of limbal stem cell deficiency affecting one or both eyes

PurposeThis systematic review (SR) assessed the efficacy, safety and cost-effectiveness of cell-based therapy to manage limbal stem cell deficiency (LSCD), a sight-threatening orphan condition most frequently associated with severe chemical or thermal burns. LSCD has historically been treated by transplanting limbal tissue. In 1997, a new treatment, cultured limbal epithelial autografts, was described for unilateral LSCD. In cases of bilateral disease cultured autologous oral mucosa stem cells have been used. The relative efficacy of different cultured tissue procedures is unknown.MethodsA protocol was registered with PROSPERO (CRD42017081117). Searches were conducted in 14 databases and 6 conference websites. Two reviewers independently selected studies, conducted data extraction and assessed risk of bias. One reviewer extracted individual patient data (IPD); a second checked extracted data. Data were assessed to determine the feasibility of statistical analysis, with Bayesian synthesis used to estimate improvement achieved by different treatments.ResultsFifty-two studies were eligible for inclusion (1113 eyes); 41 studies (716 eyes) reported IPD. No evidence was identified on cost-effectiveness. This SR was unable to confirm that any of the types of ex vivo cultured stem cell transplants identified for LSCD treatment were statistically superior when assessed against the outcomes of interest.ConclusionsWe believe this SR is the first to include IPD analysis of LSCD data. There is no evidence for the superiority of any method of limbal stem cell transplant. Confirmation of the safety and efficacy of this treatment modality is challenging due to heterogeneity within and between the studies identified. Therefore, recommendations for future research are proposed.     

Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies

The self renewal capability of limbal epithelial stem (LEST) cells is fundamental to the maintenance and healing of corneal epithelium. Limbal stem cell deficiency (LSCD), due to dysfunction or loss of LEST cells, therefore presents as persistent epithelial defects, corneal vascularization, conjunctivalization etc. Stem cell-based therapy, in its simplest form – limbal autograft, has been used successfully for more than a decade. For bilateral LSCD, similar approaches with limbal allografts have been unsuccessful largely due to strong immune rejection. Therefore, as an alternate strategy for treating bilateral LSCD, ex vivo expansion of the remaining LEST cells or autologous stem cells sourced from other potential sites is being explored. Different culture systems (with and without xenobiotic supplements) using substrates like amniotic membrane or fibrin gels have been used successfully for ex vivo LEST cell maintenance and reproduction by imitating the stem cell niche. This paper is organized into sections reviewing the LEST cells, LSCD and various stem cell-based approaches for treating LSCD and discussing future direction and challenges.