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).     

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.     

Cultivated Limbal and Oral Mucosal Epithelial Transplantation

Stem cells located at the limbus are the ultimate source for regeneration of the corneal epithelium in normal and traumatized states. When limbal stem cells are dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. Its surgical management depends on laterality and severity of corneal-limbal involvement. Conventional methods of stem cell transplantation are conjunctival-limbal autograft (CLAU), conjunctival-limbal allograft (CLAL), and kerato-limbal allograft (KLAL) surgeries. Cultivated limbal epithelial transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET) on a carrier such as amniotic membrane are current surgical alternatives. These new surgical procedures are effective in stabilizing the ocular surface. The theoretical advantage of ex-vivo expansions over conventional methods is that only a small limbal or mucosal biopsy is needed, thus minimizing the risk to the donor eye; there is also a lower risk of rejection. They can be used in cases with unilateral or bilateral total stem cell deficiency. In the unilateral cases, the source for CLET is a healthy fellow eye and in bilateral cases the source can be living-related or cadaveric eyes. The oral explants do not have limbal stem cells, but they seem to be a source of limbal stem cell equivalents that are able to generate cornea-like epithelium under the proper culture conditions. The main advantage of COMET is that patients with bilateral LSCD can be treated with grafts derived from their own autologous oral mucosal cells. The long-term outcomes of COMET have to be elucidated.     

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.     

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells

PurposeTo construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation.MethodsA stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks.ResultsThe hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype.ConclusionThe tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.     

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.     

Perspektiven und aktueller Stand der Limbusstammzelltransplantation

The transplantation of limbal stem cells is one of the most challenging surgical approaches in ocular surface reconstruction. Partial and unilateral limbal stem cell insufficiency (LSCI) can be treated by fractionated abrasion or autologous limbal stem cell transplantation from the fellow eye. In cases of advanced bilateral and partial LSCI, ex vivo expansion of limbal stem cells on amniotic membranes or fibrin can be performed but all patients with complete bilateral LSCI must rely on allogenic limbal stem cell transplantation with high immunological risks. Attempts to combine allogenic limbal stem cell transplantation with mitomycin C and amniotic membrane transplantation are promising. In the laboratory, attempts to transdifferentiate bone marrow stem cells into corneal epithelial cells have been without success. Nonetheless, transdifferentiation of hair follicle stem cells into corneal epithelial cells looks promising. In parallel, research on the limbal stem cell niche is ongoing to elucidate the natural environment of limbal stem cells in order to improve ex vivo culture.     

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells

Corneal blindness caused by limbal stem cell deficiency (LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.     

Human limbal epithelial stem cell regulation,bioengineering and function

The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.     

Oberfl?chenrekonstruktion bei Limbusstammzellinsuffizienz

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.     

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.     

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.     

Limbal stem cell disease: Treatment and advances in technology

Anterior segment stem cell technology, due to its already well-defined corneal limbal stem cells with greater ease of evaluation, has been at the forefront of ophthalmic stem cell treatment and technology since 1997. This paper provides an overview of the current standard of care for treatment of limbal stem-cell deficient conditions and reviews recent treatment technologies using ex vivo expansion of cultivated limbal stem cells of the cornea.     

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.     

外伤性角膜缘干细胞缺陷症手术治疗的实验研究

目的 探讨兔不同类型的外伤性角膜缘干细胞缺陷的手术治疗方法及临床疗效。方法 建立兔完全性角膜缘干细胞缺陷和部分性角膜缘干细胞缺陷模型，7天后分别给予羊膜移植、自体角膜缘移植和羊膜 自体角膜缘移植，比较各组治疗后的临床表现。结果 兔完全性角膜缘干细胞缺陷组羊膜移植失败，而自体角膜缘移植和羊膜 自体角膜缘移植恢复了角膜上皮完整性和角膜缘屏障功能；部分性角膜缘干细胞缺陷组羊膜移植、自体角膜缘移植和羊膜 自体角膜缘移植均达到了重建眼表的目的。结论 对于角膜缘缺陷患者，必须了解其角膜缘干细胞缺乏程度，然后分别给予不同的治疗。     

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.     

Stage-related central corneal epithelial transformation in congenital aniridia-associated keratopathy

Purpose

To relate central corneal epithelial phenotype to degree of keratopathy in a limbal stem cell deficient population.

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.     

The role of amniotic membrane extract eye drop (AMEED) in in vivo cultivation of limbal stem cells

Background

Limbal stem cell transplantation (LSCT) is the definitive treatment for total limbal stem cell deficiency (LSCD). This study evaluates the anatomical and visual outcomes of a surgical technique supplemented by amniotic membrane extract eye drop (AMEED) for in vivo cultivation of limbal stem cells (LSCs).

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.