Limbal Epithelial Cells Research Articles

A stochastic model of corneal epithelium maintenance and recovery following perturbation.

Various biological studies suggest that the corneal epithelium is maintained by active stem cells located in the limbus, the so-called limbal epithelial stem cell hypothesis. While numerous mathematical models have been developed to describe corneal epithelium wound healing, only a few have explored the process of corneal epithelium homeostasis. In this paper we present a purposefully simple stochastic mathematical model based on a chemical master equation approach, with the aim of clarifying the main factors involved in the maintenance process. Model analysis provides a set of constraints on the numbers of stem cells, division rates, and the number of division cycles required to maintain a healthy corneal epithelium. In addition, our stochastic analysis reveals noise reduction as the epithelium approaches its homeostatic state, indicating robustness to noise. Finally, recovery is analysed in the context of perturbation scenarios.

Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2′-deoxyuridine (IdU), 5-chloro-2′-deoxyuridine (CldU) and 5-ethynyl-2′-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2–3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ± 10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24–48 h period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Effect of Cross-Linking Density on the Structures and Properties of Carbodiimide-Treated Gelatin Matrices as Limbal Stem Cell Niches.

Given that human amniotic membrane is a valuable biological material not readily available for corneal epithelial tissue engineering, gelatin is considered as a potential alternative to construct a cellular microenvironment. This study investigates, for the first time, the influence of cross-linking density of carbodiimide-treated gelatin matrices on the structures and properties of artificial limbal stem cell niches. Our results showed that an increase in the carbodiimide concentration from 1.5 to 15 mM leads to an upward trend in the structural and suture strength of biopolymers. Furthermore, increasing number of cross-linking bridges capable of linking protein molecules together may reduce their crystallinity. For the samples treated with 50 mM of cross-linker (i.e., the presence of excess N-substituted carbodiimide), abundant N-acylurea was detected, which was detrimental to the in vitro and in vivo ocular biocompatibility of gelatin matrices. Surface roughness and stiffness of biopolymer substrates were found to be positively correlated with carbodiimide-induced cross-link formation. Significant increases of integrin β1 expression, metabolic activity, and ABCG2 expression were noted as the cross-linker concentration increased, suggesting that the bulk crystalline structure and surface roughness/stiffness of niche attributed to the number of cross-linking bridges may have profound effects on a variety of limbal epithelial cell behaviors, including adhesion, proliferation, and stemness maintenance. In summary, taking the advantages of carbodiimide cross-linking-mediated development of gelatin matrices, new niches with tunable cross-linking densities can provide a significant boost to maintain the limbal stem cells during ex vivo expansion.

Exosomes from normal and diabetic human corneolimbal keratocytes differentially regulate migration, proliferation and marker expression of limbal epithelial cells

Limbal epithelial stem cells (LESC) maintenance requires communication between stem cells and neighboring stromal keratocytes. Extracellular vesicles (EVs) are important for intercellular communication in various stem cell niches. We explored the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos), an EV sub-population, in limbal epithelial cells (LEC) in normal and diabetic limbal niche and determined differences in Exo cargos from normal and diabetic LSC. Wound healing and proliferation rates in primary normal LEC were significantly enhanced upon treatment by normal Exos (N-Exos), but not by diabetic Exos (DM-Exos). Western analysis showed increased Akt phosphorylation in wounded LECs and organ-cultured corneas treated with N-Exos, compared to untreated wounded cells and DM-Exos treated fellow corneas, respectively. N-Exos treated organ-cultured corneas showed upregulation of putative LESC markers, keratin 15 (K15) and Frizzled-7, compared to the DM-Exos treated fellow corneas. By next generation sequencing, we identified differentially expressed small RNAs including microRNAs in DM-Exos vs. N-Exos. Overall, N-Exos have greater effect on LEC proliferation and wound healing than DM-Exos, likely by activating Akt signaling. The small RNA differences in Exos from diabetic vs. normal LSC could contribute to the disease state. Our study suggests that exosomes may serve as novel therapeutic tools for diabetic cornea.

Expression of retinoic acid signaling components ADH7 and ALDH1A1 is reduced in aniridia limbal epithelial cells and a siRNA primary cell based aniridia model

PAX6-related Aniridia is a sight-threatening disease involving progression of secondary glaucoma and aniridia related keratopathy (ARK). Change or loss of limbal epithelial progenitors causes epithelial surface defects. We analyzed the effect of PAX6 on mRNA expression changes with a two-step approach, as follows. First, we sequenced mRNA from limbal epithelial cells isolated from controls and aniridia patients. Second, we confirmed the bioinformatics and literature-based result list for a small interfering RNA (siRNA)-based primary aniridia cell model (PAX6 knockdown). With this approach, we expected that the genes directly influenced by PAX6 would be distinguishable from those affected secondarily by the ARK disease state. Therefore, epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in keratinocyte serum-free medium. Normal control cells were obtained from the limbus region of corneal donors. For the siRNA-based aniridia cell model, cells were transfected with Lipofectamine and 5 nM siRNA against PAX6 or control treatment. All cells were lysed to yield DNA, RNA, and protein. Reduction of PAX6 protein was assessed by western blot. Aniridia and control Poly-A–enriched RNA libraries were subjected to next-generation sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparison to NCBI GEO datasets, annotated with DAVID, and manually annotated using a literature search. Based on the resulting filtered gene list, qPCR primers were purchased, and candidate genes (TP63, ABCG2, ADH7, ALDH1A1, PITX1, DKK1, DSG1, KRT12, KRT3, KRT13, SPINK6, SPINK7, CTSV, SERPINB1) were verified by qPCR on the siRNA-based aniridia cell model. We identified genes that might be regulated by PAX6 and showed that SPINK7 mRNA, which codes for a protease inhibitor, is downregulated in patients as well as in our primary aniridia cell model. ALDH1A1 and AHD7 mRNA levels were reduced in limbal epithelial cells of aniridia patients, and both transcripts were downregulated by PAX6 knockdown in our cell model. This siRNA-based aniridia cell model is a valuable tool for confirming identified PAX6-affected genes that might promote ARK pathogenesis. The model recapitulated expression changes for SPINK7, ADH7, and ALDH1A1 that were also observed in patient samples. These results provide evidence that PAX6 might drive corneal epithelial differentiation by direct or indirect control of retinoic acid signaling processes through ADH7 and ALDH1A1.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

Sustained and Widespread Gene Delivery to the Corneal Epithelium via In Situ Transduction of Limbal Epithelial Stem Cells, Using Lentiviral and Adeno-Associated Viral Vectors.

Corneal epithelial dystrophies are typically characterized by symptoms such as pain, light sensitivity, and corneal opacification leading to impaired vision. The development of gene therapy for such conditions has been hindered by an inability to achieve sustained and extensive gene transfer, as the epithelium is highly replicative and has evolved to exclude foreign material. We undertook a comprehensive study in mice aiming to overcome these impediments. Direct injection of lentiviral vector within the stem cell niche resulted in centripetal streaks of epithelial transgene expression sustained for >1 year, indicating limbal epithelial stem cell transduction in situ. The extent of transgene expression varied markedly but at maximum covered 26% of the corneal surface. After intrastromal injection, adeno-associated viral (AAV) vectors were found to penetrate Bowman's membrane and mediate widespread, but transient (12-16 days), epithelial transgene expression. This was sufficient, when applied within a Cre/lox system, to result in recombined epithelium covering up to approximately 80% of the corneal surface. Lastly, systemic delivery of AAV2/9 in neonatal mice resulted in extensive corneal transduction, despite the relative avascularity of the tissue. These findings provide the foundations of a gene therapy toolkit for the corneal epithelium, which might be applied to correction of inherited epithelial dystrophies.

UV light-blocking contact lenses protect against short-term UVB-induced limbal stem cell niche damage and inflammation

UVB irradiation has been linked to pathogenesis of pterygium, a conjunctival tumor growing onto transparent cornea, the windscreen of the eye. Due to corneal anatomy, ambient UVB irradiation is amplified at the stem cell-containing nasal limbus. The aim of this study was to analyse the effect of a UV-blocking contact lens (UVBCL, senofilcon A, Class 1 UV blocker) on limbal epithelial cells and fibroblasts under UVB irradiation compared to a non-UVB-blocking contact lens. UVBCL prevented UVB-induced DNA damage (as assessed by cyclobutane pyrimidine dimer immunostaining) as well as a decrease in proliferation and scratch wound closure rate of both limbal epithelial and fibroblast cells. Similarly, UVBCL protected limbal epithelial cells from UVB-induced loss of their phenotype in terms of colony forming efficiency and stem cell marker expression (ABCB5, P63α, integrin β1) compared to controls. Moreover, with UVBCL pro-inflammatory cytokines such as TNFα and MCP1 remained unchanged. These data demonstrate the significance of UV-protection in preserving the limbal niche in response to at least short-term UVB. Our data support the use of UVBCL in protecting limbal niche cells, especially after limbal stem cell transplantation and in patients after pterygium surgery, to help prevent recurrences.

Differential Distribution of Laminin N-Terminus α31 Across the Ocular Surface: Implications for Corneal Wound Repair.

PurposeLaminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing.MethodsHuman, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell–associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging.ResultsLaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities.ConclusionsThese data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

Long-acting liposomal corneal anesthetics

Eye drops producing long-acting ocular anesthesia would be desirable for corneal pain management. Here we present liposome-based formulations to achieve very long ocular anesthetic effect after a single eye drop instillation. The liposomes were functionalized with succinyl-Concanavalin A (sConA-Lip), which can bind corneal glycan moieties, to significantly prolong the dwell time of liposomes on the cornea. sConA-Lip were loaded with tetrodotoxin and dexmedetomidine (sConA-Lip/TD), and provided sustained release for both. A single topical instillation of sConA-Lip/TD on the cornea could achieve 105 min of complete analgesia and 608 min of partial analgesia, which was significantly longer than analgesia with proparacaine, tetrodotoxin/dexmedetomidine solution or unmodified liposomes containing tetrodotoxin and dexmedetomidine. sConA-Lip/TD were not cytotoxic in vitro to human corneal limbal epithelial cells or corneal keratocytes. Topical administration of sConA-Lip/TD provided prolonged corneal anesthesia without delaying corneal wound healing. Such a formulation may be useful for the management of acute surgical and nonsurgical corneal pain, or for treatment of other ocular surface diseases.

Decellularized corneal lenticule embedded compressed collagen: toward a suturable collagenous construct for limbal reconstruction

Recently, compressed collagen has attracted much attention as a potential alternative for a limbal epithelial stem cell (LESC) carrier to treat limbal stem cell deficiency (LSCD), in that it can provide mechanically improved collagen fibrillar structures compared to conventional collagen hydrogel. However, its clinical efficacy as an LESC carrier has not yet been studied through in vivo transplantation due to limited mechanical strength that cannot withstand a force induced by surgical suturing and low resistance to enzymatic degradation. This study firstly presents a suturable LESC carrier based on compressed collagen in the form of a biocomposite. The biocomposite was achieved by integrating a decellularized corneal lenticule, which is a decellularized stromal tissue obtained from corneal refractive surgery, inside a compressed collagen to form a sandwich structure. A suture retention test verified that the biocomposite has a much higher suture retention strength (0.56 ± 0.12 N) compared to the compressed collagen (0.02 ± 0.01 N). The biocomposite also exhibited more than 3 times higher resistance to enzymatic degradation, indicating long-term stability after transplantation. In vitro cell culture results revealed that the biocomposite effectively supported the expansion and stratification of the LESCs with expressions of putative stem cell and differentiated corneal epithelial cell markers. Finally, the biocomposite verified its clinical efficacy by stably delivering the LESCs onto an eye of a rabbit model of LSCD and effectively reconstructing the ocular surface.

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein.

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

Effect of Transportation on Cultured Limbal Epithelial Sheets for Worldwide Treatment of Limbal Stem Cell Deficiency

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

Emerging Therapeutic Strategies for Limbal Stem Cell Deficiency.

Identification and characterization of the limbal epithelial stem cells (LESCs) has proven to be a major accomplishment in anterior ocular surface biology. These cells have been shown to be a subpopulation of limbal epithelial basal cells, which serve as the progenitor population of the corneal epithelium. LESCs have been demonstrated to play an important role in maintaining corneal epithelium homeostasis. Many ocular surface diseases, including intrinsic (e.g., Sjogren's syndrome) or extrinsic (e.g., alkali or thermal burns) insults, which impair LESCs, can lead to limbal stem cell deficiency (LSCD). LSCD is characterized by an overgrowth of conjunctival-derived epithelial cells, corneal neovascularization, and chronic inflammation, eventually leading to blindness. Treatment of LSCD has been challenging, especially in bilateral total LSCD. Recently, advances in LESC research have led to novel therapeutic approaches for treating LSCD, such as transplantation of the cultured limbal epithelium. These novel therapeutic approaches have demonstrated efficacy for ocular surface reconstruction and restoration of vision in patients with LSCD. However, they all have their own limitations. Here, we describe the current status of LSCD treatment and discuss the advantages and disadvantages of the available therapeutic modalities.

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.

Current approaches to the problem of carrier selection for limbal stem cells cultivation in the treatment of limbal stem cell deficiency

Diseases and damages of the ocular surface are one of the common causes of decreased vision and blindness. Dysfunction or death of limbal epithelial stem cells (LESC) plays an important role in the development of pathological processes in these conditions, which leads to the development of the limbal stem cell deficiency (LSCD). Currently, one of the methods to treat LSCD is a transplantation of cultured ex vivo LESC. The most common carriers for the cultivation of LESC in the world is the amniotic membrane (AM). However, the presence of certain disadvantages in using AM for the cultivation of LESC compels to search new types of carriers made from biological or synthetic materials. In this review, we have analyzed various types of carriers: collagen, fibrin, chitosan with gelatin, silk fibroin, keratin, contact lenses, polylactide-co-glycolide, polycaprolactone, and the possibility of their application as carriers for the LESC cultivation followed by transplantation on the ocular surface is considered.

Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking.

Purpose To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. Methods Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. Results There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63+ limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. Conclusions Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.