Treatment Of Limbal Stem Cell Deficiency Research Articles

Advances in Cell Transplantation Therapy for Limbal Stem Cell Deficiency.

Limbal stem cells (LSCs) are essential for maintaining corneal transparency and ocular surface integrity. Many external factors or genetic diseases can lead to corneal limbal stem cell deficiency (LSCD), resulting in the loss of barrier and corneal epithelial cell renewal functions. Stem cell transplantation is one of the primary treatments for LSCD, including limbal transplantation and cultivated limbal epithelial transplantation. In addition, a variety of non-limbal stem cell lines have been experimented with for LSCD treatment. Biological scaffolds are also used to support in vitro stem cell culture and transplantation. Here, we review the mechanisms of corneal maintenance by LSCs, the clinical stage and surgical treatment of LSCD, the source of stem cells, and the biological scaffolds required for in vitro culture. This study is a narrative retrospective study aimed at collecting available information on various aspects of surgical treatments for LSCD. Relevant literature was searched in a range of online databases, including Web of Science, Scopus, and PubMed from 2005 to March, 2023. A total of 397 relevant articles were found, and 49 articles with strong relevance to the studies in this paper were obtained and analyzed. Moreover, 11 of these articles were on the concept of LSCD and the mechanism of LESCs maintaining the corneal epithelium, 3 articles on the staging and grading of LSCD, 17 articles on cell transplantation methods and donor cell sources, and 18 articles on scaffolds for delivering stem cells. We also summarized the advantages and disadvantages of different cell transplantation methods and the benefits and limitations of scaffolds based on the above literature. The treatment of LSCD is determined by the clinical stage and whether it involves monocular or binocular eyes. Appropriate surgical techniques should be taken for LSCD patients in order to reconstruct the ocular surface, relieve symptoms, and restore visual function. Meanwhile, biological scaffolds assist in the ex vivo culture and implantation of stem cells.

Limbal stem cell therapy.

To highlight the progress and future direction of limbal stem cell (LSC) therapies for the treatment of limbal stem cell deficiency (LSCD). Direct LSC transplantation have demonstrated good long-term outcomes. Cultivated limbal epithelial transplantation (CLET) has been an alternative to treat severe to total LSCD aiming to improve the safety and efficacy of the LSC transplant. A prospective early-stage uncontrolled clinical trial shows the feasibility and safety of CLET manufactured under xenobiotic free conditions. Other cell sources for repopulating of the corneal epithelium such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells are being investigated. The first clinical trials of using MSCs showed short-term results, but long-term efficacy seems to be disappointing. A better understanding of the niche function and regulation of LSC survival and proliferation will lead to the development of medical therapies to rejuvenate the residual LSCs found in a majority of eyes with LSCD in vivo. Prior efforts have been largely focused on improving LSC transplantation. Additional effort should be placed on improving the accuracy of diagnosis and staging of LSCD, and implementing standardized outcome measures which enable comparison of efficacy of different LSCD treatments for different severity of LSCD. The choice of LSCD treatment will be customized based on the severity of LSCD in the future. New approaches for managing different stages of LSCD are being developed. This concise review summarizes the progresses in LSC therapies for LSCD, underlying mechanisms, limitations, and future areas of development.

A Challenging Case of Simple Limbal Epithelial Transplantation (SLET) in Limbal Stem Cell Deficiency Following Chemical Injury: A Case Report

Introduction : Limbal stem cell deficiency (LSCD) often arises following trauma or inflammation that hinders corneal epithelium healing, commonly caused by chemical or thermal injuries. Despite significant advancements, treating LSCD remains challenging. This case highlights a challenging case of simple limbal epithelial transplantation (SLET) in LSCD following chemical injury.
 Case Illustration : A 20-year-old male presented with reduced vision in left eye following an alkaline chemical injury (grade III) was diagnosed with unilateral LSCD. Upon examination, conjunctivalization of the peripheral cornea, a central corneal cicatrix, and stromal neovascularization present after previous necrotomy, tenoplasty, and amniotic membrane transplantation (AMT). The patient then underwent SLET, with limbal biopsies from the collateral eye. Four months after surgery the visual acuity improved from 3/60 to 6/15 with spectacles. However, the cornea conjunctivalization still developed despite improved visual acuity.
 Discussion : To effectively manage the recurrence of stem cell deficiency, other stem cell transplantation techniques are needed to manage the problem. Combining different limbal stem cell transplantation approaches can be customised to address the problem and achieve lasting results.
 Conclusion : Prompt diagnosis and treatment of LSCD is crucial to prevent complications and blindness. SLET is a surgical option for LSCD after chemical injury, but this report highlights a challenging case and the need for the alternatives approaches.

Matrix-Assisted Cell Transplantation for the Treatment of Limbal Stem Cell Deficiency in a Rabbit Model.

With the development of regenerative medicine in ophthalmology, the identification of cells with high proliferative potential in the limbal area has attracted the attention of ophthalmologists and offered a new option for treatment in clinical practice. Limbal stem cell deficiency (LSCD) is an identified eye disease with a difficult and negative outcome, for which the traditional treatment is keratoplasty. This study sought to evaluate the efficacy of matrix-assisted cell transplantation consisting of in vitro-cultured autologous limbal stem cells (LSCs) and type I collagen for the treatment of LSCD in rabbits. LSCD was induced in 10 rabbits by a combination of mechanical limbectomy and alkali burns. Cells were cultured on a plate for 14 days before being transferred to a collagen-based matrix for another 7 days. Rabbits were divided into two groups as follows: the experimental group (five rabbits) received matrix-assisted cell transplantation, while the control group (five rabbits) received only conservative therapy with anti-inflammatory eye drops. During the postoperative period, all rabbits were examined using slit-lamp biomicroscopy with photo-registration and fluorescent staining, impression cytology and anterior segment optical coherence tomography (AS-OCT). Rabbits were euthanized at 30 and 120 days, and their corneas were processed for histology and immunohistochemistry. As a consequence, rabbits in the experimental group demonstrated the restoration of the corneal epithelium and transparency without epithelial defects. Moreover, goblet cells were absent in the central zone of the corneal epithelium. In conclusion, our new method of treatment enhanced the corneal surface and is an effective method of treatment for LSCD in rabbits.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell–matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

3D bioprinting as a prospective therapeutic strategy for corneal limbal epithelial stem cell deficiency.

Limbal epithelial stem cells (LESCs) are responsible for the maintenance and repair of the corneal surface. Injuries and diseases of the eye may result in a vision condition called limbal stem cell deficiency (LSCD). Without limbal stem cells, the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy as a treatment method was first described in 1997, and LESCs cultured from either patients or donors have been used to treat LSCD successfully. However, the main source of cornea for LSCD treatment is from donors, which are too few to meet the demand (less than 1:70 of cases). The global shortage of donor cornea promotes the need for studies exploring corneal limbus alternatives. Many problems still remain unresolved, such as original geometry reconstruction, corneal epithelial regeneration, and ocular optical function restoration. 3D bioprinting has garnered tremendous attention in recent years, and significant advances have been made in fabricating cell-laden scaffolds. These advancements could lead to a promising treatment for LSCD. It is possible that alternative limbus stem cells can be constructed using 3D printing, which, in corneal limbus regeneration, enables personalized corneal implants and fabrication of single- or multilayer corneal limbus equivalents with corneal limbal stem cells. In this review, the progress, applications, and limitations of the most influential works regarding current treatments of LESC deficiency are discussed. The advantages of 3D bioprinting are illustrated, and some first promising steps toward the creation of a functional cornea limbus with 3D bioprinting are discussed. Finally, insights into the prospects and technical challenges facing the future research of 3D bioprinting of corneal limbus alternatives in vivo and in vitro are provided.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells

Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.

Prosthetic Replacement of the Ocular Surface Ecosystem for Limbal Stem Cell Deficiency: A Case Series.

To assess outcomes of limbal stem cell deficiency (LSCD) in patients treated with Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE). Retrospective case series. Patients with LSCD who received PROSE treatment were included. Data including best-corrected visual acuity (BCVA) and LSCD staging before and after PROSE dispensing were collected to characterize each case. Five eyes of four patients were included. All patients were female, with an age range of 21 to 80 years. Each patient received a PROSE device with diameters ranging from 16 to 18.5 mm. Follow-up ranged from 11 to 29 months. Tolerated wear times ranged from 3.5 to 10 hr daily. Four eyes showed improved BCVA and unchanged LSCD staging as per the global consensus after PROSE treatment. Three of these eyes had stage 3 and one had stage 1C LSCD at diagnosis. The fifth eye had worse BCVA and recurrence of stage 3 LSCD post-living-related conjunctival limbal allograft transplant despite PROSE treatment. Prosthetic Replacement of the Ocular Surface Ecosystem may be a viable treatment for LSCD, including severe cases, because it can provide symptom relief and improve vision. Its customizability, as demonstrated in this study, is beneficial for troubleshooting issues with fitting. Future studies are needed to further assess PROSE as treatment for LSCD.

Long-term survival in non-human primates of stem cell-derived, MHC-unmatched corneal epithelial cell sheets.

SummaryWhen corneal epithelial stem cells residing in the corneal limbus become dysfunctional, called a limbal stem cell deficiency (LSCD), corneal transparency is decreased, causing severe vision loss. Transplantation of corneal epithelial cell sheets (CEPS) derived from stem cells, including induced pluripotent stem cells, is a promising treatment for LSCD. However, the potential effect of human leukocyte antigen (HLA) concordance on CEPS transplantation has not been addressed. Here, we show that there is no difference in the immune response to CEPS between HLA-matched and -unmatched peripheral blood mononuclear cells in mixed lymphocyte reactions. CEPS transplantation in cynomolgus monkeys revealed that the immune response to major histocompatibility-unmatched CEPS was not strong and could be controlled by local steroid administration. Furthermore, programmed death ligand 1 was identified as an immunosuppressive molecule in CEPS under inflammatory conditions in vitro. Our results indicate that corneal epithelium has low immunogenicity and allogeneic CEPS transplantation requires mild immunosuppression.

A Review of the Diagnosis and Treatment of Limbal Stem Cell Deficiency.

Limbal stem cell deficiency (LSCD) can cause significant corneal vascularization and scarring and often results in serious visual morbidity. An early and accurate diagnosis can help prevent the same with a timely and appropriate intervention. This review aims to provide an understanding of the different diagnostic tools and presents an algorithmic approach to the management based on a comprehensive clinical examination. Although the diagnosis of LSCD usually relies on the clinical findings, they can be subjective and non-specific. In such cases, using an investigative modality offers an objective method of confirming the diagnosis. Several diagnostic tools have been described in literature, each having its own advantages and limitations. Impression cytology and in vivo confocal microscopy (IVCM) aid in the diagnosis of LSCD by detecting the presence of goblet cells. With immunohistochemistry, impression cytology can help in confirming the corneal or conjunctival source of epithelium. Both IVCM and anterior segment optical coherence tomography can help supplement the diagnosis of LSCD by characterizing the corneal and limbal epithelial changes. Once the diagnosis is established, one of various surgical techniques can be adopted for the treatment of LSCD. These surgeries aim to provide a new source of corneal epithelial stem cells and help in restoring the stability of the ocular surface. The choice of procedure depends on several factors including the involvement of the ocular adnexa, presence of systemic co-morbidities, status of the fellow eye and the comfort level of the surgeon. In LSCD with wet ocular surfaces, autologous and allogeneic limbal stem cell transplantation is preferred in unilateral and bilateral cases, respectively. Another approach in bilateral LSCD with wet ocular surfaces is the use of an autologous stem cell source of a different epithelial lineage, like oral or nasal mucosa. In eyes with bilateral LSCD with significant adnexal issues, a keratoprosthesis is the only viable option. This review provides an overview on the diagnosis and treatment of LSCD, which will help the clinician choose the best option amongst all the therapeutic modalities currently available and gives a clinical perspective on customizing the treatment for each individual case.

Managing Limbal Stem Cell Deficiency with Simple Limbal Epithelial Transplantation – Perspective and Practical Guide

Limbal stem cell deficiency (LSCD) is an important cause of blindness. Various etiological factors can cause this challenging disease and several ocular and systemic comorbidities often make the management challenging in these cases. Limbal stem cell transplantation (LSCT) is the gold standard treatment for LSCD. Various techniques of LSCT such as conjunctival limbal autograft and cultured limbal epithelial transplantation have been described to treat this condition. Simple limbal epithelial transplantation (SLET) is an innovative technique of LSCT that has established itself as a successful modality for treating LSCD while addressing limitations of other procedures such as cost, reach and replicability. This review attempts to present a detailed account of SLET comprising of case selection criteria, surgical procedure, post operative care and complications and comparative details of the various techniques of LSCT. We also discuss pertinent tips related to the procedure which we hope will be beneficial for both beginner surgeons and those already practicing SLET.

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

Observational study of cenegermin for the treatment of limbal stem cell deficiency associated with neurotrophic keratopathy.

Neurotrophic keratopathy (NK) and limbal stem cell deficiency (LSCD) have high morbidity and require aggressive management to prevent permanent vision loss. Cenegermin, a recombinant human nerve growth factor, was approved by the Federal Drug Administration in 2018 for the treatment of NK. To determine the efficacy and safety of cenegermin in the treatment of LSCD associated with NK. Prospective cohort study. Patients diagnosed with LSCD and NK who had failed conventional treatment were enrolled in this prospective open-label study. Patients were treated with cenegermin for 8 weeks. The primary objective was to determine whether the area of abnormal epithelium decreased following treatment. Corneal sensation, visual acuity (VA), and LSCD severity were also evaluated. Six eyes of 5 patients were included in the study. Cenegermin significantly improved the area of abnormal corneal epithelium in 5 of 6 eyes, measuring 73% of total corneal area at the initial visit and 48% at the final visit (P = .036). Corneal sensation improved in all patients, Cochet-Bonnet aesthesiometry measured 14.7 and 26.7 mm at the initial and final visit, respectively (P = .009). VA improved in 4 out of 6 eyes, with mean initial logMAR VA of 1.67 and final logMAR VA of 1.19 (P = .045). Finally, LSCD grading improved using the Aravena scoring system; however, this difference was not statistically significant (P = .14). One patient presented with an epithelial defect at baseline, which resolved following treatment. No patient withdrew from the study due to adverse effects. Cenegermin effectively improved the cornea epithelium, VA, and corneal sensation in patients with LSCD and NK who had failed prior treatment. Further studies are necessary to better understand the anatomical changes and to confirm our results with a larger randomized control trial. The study was registered at ClinicalTrials.gov with identifier NCT04552730 (https://clinicaltrials.gov/ct2/show/NCT04552730).

Regulation of Limbal Epithelial Stem Cells: Importance of the Niche.

Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFβ. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.

Proof-of-concept study of electrospun PLGA membrane in the treatment of limbal stem cell deficiency

ObjectiveThe aim of this study was to assess the safety of poly-lactic co-glycolic acid (PLGA) electrospun membranes as carriers for limbal tissue explants for treatment of limbal stem cell deficiency...

Clinical outcomes of modified simple limbal epithelial transplantation for limbal stem cell deficiency in Chinese population: a retrospective case series

ObjectiveTo report the clinical outcomes of a novel surgical technique, namely simple limbal epithelial transplantation (SLET), for the treatment of limbal stem cell deficiency (LSCD).MethodsThirteen patients (13 eyes) with LSCD who underwent autologous (10 eyes) or allogeneic (3 eyes) modified SLET between 2018 and 2021 were enrolled in this study. Grades of symblepharon, corneal conjunctivalization, vascularization, opacification, and visual acuity (VA) were evaluated preoperatively and postoperatively. In 2 cases, in vivo confocal microscopy (IVCM) and impression cytology (IC) were performed to assess the proliferation and degeneration of limbal tissue.ResultsAt a postoperative follow-up of 6.5±5.3 (range, 2–20) months, 10 (10/13, 76.92%) eyes maintained a successful outcome. The grades of symblepharon, corneal conjunctivalization, vascularization, and opacification were significantly improved after SLET (P<0.05). Two-line improvement in VA was found in 6 (6/10, 60%) eyes of the successful cases. Recurrence of LSCD occurred in 3 (3/13, 23.08%) eyes, and conjunctival cyst occurred in 1 patient. After SLET, the morphology and structure of corneal epithelial cells and epithelial transition around the limbal tissue fragments were detected by IVCM and IC.ConclusionsOur findings suggest that the SLET is a safe and effective technique for the treatment of LSCD. The corneal stroma and hAM can provide protection and nutrition for the limbal stem cells (LSCs) without negatively influencing the clinical outcomes. IVCM and IC after SLET can evaluate the effectiveness of surgery and the transition of LSCs and corneal epithelial cells.

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.

Subconjunctival Injection of Transdifferentiated Oral Mucosal Epithelial Cells for Limbal Stem Cell Deficiency in Rats.

Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium-derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD.

Recent Advances in Stem Cell Therapy for Limbal Stem Cell Deficiency: A Narrative Review.

Destruction of the limbus and depletion of limbal stem cells (LSCs), the adult progenitors of the corneal epithelium, leads to limbal stem cell deficiency (LSCD). LSCD is a rare, progressive ocular surface disorder which results in conjunctivalisation and neovascularisation of the corneal surface. Many strategies have been used in the treatment of LSCD, the common goal of which is to regenerate a self-renewing, transparent, and uniform epithelium on the corneal surface. The development of these techniques has frequently resulted from collaboration between stem cell translational scientists and ophthalmologists. Direct transplantation of autologous or allogeneic limbal tissue from a healthy donor eye is regarded by many as the technique of choice. Expansion of harvested LSCs in vitro allows smaller biopsies to be taken from the donor eye and is considered safer and more acceptable to patients. This technique may be utilised in unilateral cases (autologous) or bilateral cases (living related donor). Recently developed, simple limbal epithelial transplant (SLET) can be performed with equally small biopsies but does not require in vitro cell culture facilities. In the case of bilateral LSCD, where autologous limbal tissue is not available, autologous oral mucosa epithelium can be expanded in vitro and transplanted to the diseased eye. Data on long-term outcomes (over 5 years of follow-up) for many of these procedures is needed, and it remains unclear how they produce a self-renewing epithelium without recreating the vital stem cell niche. Bioengineering techniques offer the ability to re-create the physical characteristics of the stem cell niche, while induced pluripotent stem cells offer an unlimited supply of autologous LSCs. In vivo confocal microscopy and anterior segment OCT will complement impression cytology in the diagnosis, staging, and follow-up of LSCD. In this review we analyse recent advances in the pathology, diagnosis, and treatment of LSCD.

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.