Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their ß-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of ß-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

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 ß1) 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.

The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure.

The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman's membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.

Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.

The chemokine receptor CCR7 is essential for migration of mature dendritic cells (DCs) to the regional lymph nodes, and it has been shown that blocking of CCR7 improves graft survival after high-risk corneal transplantation in vascularized recipient corneas. However, it is so far unknown whether blocking of CCR7 reduces migration of DCs from the avascular cornea to the draining lymph nodes and whether this leads to improved graft survival also in the low-risk setting of corneal transplantation, which accounts for the majority of perforating transplantations performed. Therefore, in this study, pellets containing Freund's adjuvant and bovine serum albumin (BSA) conjugated to Alexa488 fluorescent dye were implanted into the corneal stroma of BALB/c mice to analyze antigen uptake by corneal DCs and their migration to the regional lymph nodes. After pellet implantation, mice were either treated by local administration of a CCR7 blocking fusion protein that consisted of CCL19 fused to the Fc part of human IgG1 or a control-IgG. In vivo fluorescence microscopy showed uptake of Alexa488-conjugated BSA by corneal DCs within 8 h. Furthermore, analysis of single cell suspensions of draining lymph nodes prepared after 48 h revealed that 2.1 ± 0.3% of CD11c(+) cells were also Alexa488(+). Importantly, DC migration was significantly reduced after topical administration of CCL19-IgG (1.2 ± 0.2%; p < 0.05). To test the effect of CCR7 blockade on graft rejection after allogeneic low-risk keratoplasty, corneal transplantations were performed using C57BL/6-mice as donors and BALB/c-mice as recipients. Treatment mice received two intraperitoneal loading doses of CCL19-IgG prior to transplantation, followed by local treatment with CCL19-IgG containing eye drops for the first two weeks after transplantation. Control mice received same amounts of control-IgG. Kaplan-Meier survival analysis showed that in the CCL19-IgG treated group, 76% of the grafts survived through the end of the 8 week observation period, whereas 38% of the grafts survived in the control group (p < 0.05). Taken together, our study shows that blockade of CCR7 reduces the migration of mature corneal DCs to the draining lymph nodes and leads to improved graft survival in low-risk corneal transplantation.

The impact of age on the physical and cellular properties of the human limbal stem cell niche.

The limbal niche in the corneoscleral junction of the eye, habitat of the limbal epithelial stem cells (LESC), facilitates corneal epithelial regeneration by providing physical support and chemical signalling. Anatomical structures within the limbus, namely, limbal epithelial crypts and focal stromal projections, are believed to function as a putative niche for LESCs. In this study, the impact of age on the topography of this niche was investigated. Also, the relationship between niche topography and limbal epithelial cell phenotype was assessed. Ex vivo imaging of the limbus in cadaveric tissue of donors aged from infancy to 90 years was carried out using electron and confocal microscopy. The data suggested that the area occupied by the crypts was sharply reduced after the age of 60 years. The niche microstructures also became smoother with donor age. The phenotypic assessment of cultured limbal epithelial cells harvested from donors of different ages showed that the levels of putative stem cell markers as well as telomerase activity and telomere length remained unchanged, regardless of niche topography. However, the colony forming efficiency of the cultures was significantly reduced with age (p < 0.05). This is the first comprehensive study of the effect of age on the structural and phenotypic characteristics of the human limbal niche. The results have a significant biological value as they suggest a correlation of limbal architecture with decline of re-epithelialisation rate in older patients. Overall, the data also suggest that LESCs harvested from younger donors may be more suitable for cultured LESC therapy production.

In sickness and in health: Corneal epithelial stem cell biology, pathology and therapy.

Our window to the world is provided by the cornea on the front surface of the eye. The integrity and functionality of the outermost corneal epithelium is essential for vision. A population of limbal epithelial stem cells (LESCs) are responsible for maintaining the epithelium throughout life by providing a constant supply of daughter cells that replenish those constantly lost from the ocular surface during normal wear and tear and following injury. LESC deficiency leads to corneal opacification, inflammation, vascularization and discomfort (Daniels et al., 2001, 2007). Cultured LESC delivery is one of several examples of successful adult stem cell therapy in patients. The clinical precedence for use of stem cell therapy and the accessibility of the transparent stem cell niche make the cornea a unique model for the study of adult stem cells in physiological conditions as well as in disease.

Conjunctival epithelial cells maintain stem cell properties after long-term culture and cryopreservation.

AIM: Transplantation of tissue-engineered conjunctival epithelial cell sheets has proven to be a promising technique for conjunctival reconstruction. The ability to cryopreserve conjunctival epithelial cells and maintain their stem cell population would improve their availability for clinical use. The aim of this study was to evaluate whether cryopreservation and long-term in vitro culture has an effect on the proliferative capacity and the progenitor-like cell characteristics of conjunctival epithelial cells. METHOD: Human conjunctival cells from bulbar biopsies were isolated and expanded on a growth arrested 3T3 feeder layer. The cells were evaluated for cytokeratin (CK4/CK19) expression by immunostaining. An aliquot with half of the cells from the initial culture was frozen in liquid nitrogen and stored for 14 days and, in addition, donor cells were cryopreserved for more than 6 months (202.7 +/- 13.0 days). Both cryopreserved and noncryopreserved cells were serially cultivated over four passages. For each passage the colony-forming efficiency and the cell population doubling rates were evaluated, and expression of putative progenitor cell markers, p63alpha and ABCG2, was assessed by immunostaining and reverse transcription PCR. RESULTS: Both noncryopreserved and cryopreserved cells demonstrated a high colony-forming capacity that decreased with passage. Cells from both groups underwent approximately 20 cell population doublings before senescence. Immunoreactivity to p63alpha and ABCG2 was found in both groups until passage 4 and their presence was also confirmed by reverse transcription PCR. No difference in cell viability, colony-forming efficiency and immunoreactivity to p63alpha and ABCG2 was observed between cells cryopreserved for 14 days, and more than 6 months (202.7 +/- 13.0 days). CONCLUSION: Conjunctival epithelial cells with progenitor cell-like characteristics can be efficiently cryopreserved and can subsequently maintain their function in vitro over several culture passages. The option to cryopreserve conjunctival cells prior to in vitro expansion would be an advantage when cells have to be cultivated for clinical transplantation.

Cytocompatibility and hemocompatibility of a novel chitosan-alginate gel system.

Two chitosan-alginate gel systems in the form of membranes were produced and evaluated. The first membrane was produced by a novel gel system formed after blending N-(methylsulfonic acid) chitosan with ammonium alginate (CAG1) and the second was an N-(methylsulfonic acid) chitosan-sodium alginate blend cross-linked with glutaraldehyde and calcium chloride (CAG2). The cytocompatibility and hemocompatibility of the gels were examined by assessing the cell viability of 3T3 Swiss mouse fibroblasts, whole blood hemolysis, and platelet activation. Cell viability was not significantly different by exposure to these gels compared to the controls. Both gel types had minimal effect on hemolysis of whole heparinized rabbit blood after 1-h exposure. Further platelet activation by the surfaces was also minimal. These results indicate that these novel gels merit further investigation for blood contact applications.