Effect of low dose honey on the apoptosis and inflammation gene expression in corneal limbal stem cells and keratocytes and its efficacy as an ophthalmic formulation in the treatment of dry eye: in-vitro and clinical study.

Background: The use of honey as an eye treatment encounters challenges due to its high osmolarity, low pH, and difficulties in sterilization. This study addresses these issues by employing a low concentration of honey, focusing on both in-vitro experiments and clinical trials for treating dry eye disease in corneal cells. Methods: In the in-vitro experiment, we investigated the impact of a 1% honey-supplemented medium (HSM) on limbal stem cells (LSCs) and keratocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and real-time polymerase chain reaction (PCR) for BCL-2, BAX, and IL-1ß gene expression. Simultaneously, in the clinical trial, 80 participants were divided into two groups, receiving either a 1% w/v honey ophthalmic formulation or a placebo for 3 months. Study outcomes included subjective improvement in dry eye symptoms, tear break-up time (TBUT), and Schirmer's test results. Results: MTT results indicated that 1% HSM did not compromise the survival of corneal cells and significantly reduced the expression of the IL-1ß gene. Additionally, participants in the honey group demonstrated a higher rate of improvement in dry eye symptoms and a significant enhancement in TBUT values at the three-month follow-up. However, there was no significant difference between the study groups in terms of Schirmer's test values. No adverse events were observed or reported. Conclusion: In conclusion, 1% honey exhibits anti-inflammatory and anti-infective properties, proving effective in ameliorating dry eye symptoms and enhancing tear film stability in patients with dry eye disease.Clinical Trial Registration: https://irct.behdasht.gov.ir/trial/63800.

Mesenchymal stem cells derived-exosomes enhanced amniotic membrane extract promotes corneal keratocyte proliferation.

Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties. Although WJ-MSC-Exos and AME have been used separately for wound healing research, their combined synergistic effects have not been studied extensively. In this study, we evaluated the effects of both AME and WJ-MSC-Exos, individually and together, on the proliferation of corneal keratocytes as well as their ability to promote in vitro cell migration, wound healing, and their impact on cellular morphology. Our findings indicated that the presence of both exosomes (3 × 105 Exo/mL) and AME (50 µg/mL) synergistically enhance the proliferation of corneal keratocytes. Combined use of these solutions (3 × 105 Exo/mL + 50 µg/mL) increased cell proliferation compared to only 50 µg/mL AME treatment on day 3 (**** p < 0.0001). This mixture treatment (3 × 105 Exo/mL + 50 µg/mL) increased wound closure rate compared to isolated WJ-MSC-Exo treatment (3 × 105 Exo/mL) (*p < 0.05). Overall, corneal keratocytes treated with AME and WJ-MSC-Exo (3 × 105 Exo/mL + 50 µg/mL) mixture resulted in enhanced proliferation and wound healing tendency. Utilization of combined use of AME and WJ-MSC-Exo can pave the way for a promising foundation for corneal repair research.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Mini review: human clinical studies of stem cell therapy in keratoconus.

Treatment of keratoconus is one of the most interesting research fields for researchers in the world. Regenerative medicine based on human stem cells in the treatment of keratoconus has recently received attention. Despite extensive laboratory and animal studies in regenerative medicine of cornea, there are limited clinical studies in keratoconus. These studies showed promising results of stem cell therapy. In initial studies, the transplantation of these cells into stroma was associated with increased vision and improved corneal parameters without side effects. In this article, we tried to review different aspects of keratoconus stem cell therapy, including cell extraction and culture, surgical procedure, effectiveness and safety of this method in human clinical studies.

Studying the Proliferative Activity of Human Corneal Stromal Cell Populations.

The proliferative activity of populations of stromal cells (fibroblasts) obtained from human corneal lenticles under conditions of their differentiation into keratocytes was studied. It was shown that during differentiation, the number of dividing fibroblasts and the frequency of divisions, and motor activity of these cells (speed of movement along the cell trajectory and the length of the trajectory) sharply decreased. These findings indicate a decrease in the proliferative activity of fibroblasts under conditions of their differentiation and transformation into keratocytes. A period of 17 days is sufficient for differentiation of corneal fibroblasts into keratocytes.

Animal Models for the Study of Keratoconus.

Keratoconus (KC) is characterized by localized, central thinning and cone-like protrusion of the cornea. Its precise etiology remains undetermined, although both genetic and environmental factors are known to contribute to disease susceptibility. Due to KC's complex nature, there is currently no ideal animal model to represent both the corneal phenotype and underlying pathophysiology. Attempts to establish a KC model have involved mice, rats, and rabbits, with some additional novel animals suggested. Genetic animal models have only been attempted in mice. Similarly, spontaneously occurring animal models for KC have only been discovered in mice. Models generated using chemical or environmental treatments have been attempted in mice, rats, and rabbits. Among several methods used to induce KC in animals, ultraviolet radiation exposure and treatment with collagenase are some of the most prevalent. There is a clear need for an experimental model animal to elucidate the underlying mechanisms behind the development and progression of keratoconus. An appropriate animal model could also aid in the development of treatments to slow or arrest the disorder.

Unveiling Cellular Diversity in the Buffalo Corneal Stroma: Insights into Telocytes and Keratocytes Using Light Microscope, Transmission Electron Microscope, and Immunofluorescence Analysis.

Telocytes and keratocytes are important cells that maintain the structure and function of the cornea. The buffalo cornea, known for its resilience in harsh conditions, has not been extensively studied regarding the presence and role of telocytes and keratocytes. We used light microscopy, transmission electron microscopy (TEM), and immunofluorescence assays with platelet-derived growth factor receptor alpha (PDGFRα), CD34, and Vimentin markers to investigate their expression and localization in the cornea. TEM analysis confirmed the presence of spindle-shaped keratocytes with intercellular connections, while telocytes exhibited small spindle-shaped bodies with long, thin branches connecting to corneal keratocytes. Immunofluorescence findings showed that CD34 was more abundant near the endothelium, Vimentin was prominently expressed near the epithelium, and PDGFRα was uniformly distributed throughout the corneal stroma. Co-expression of CD34 and Vimentin, PDGFRα and Vimentin, as well as CD34 and PDGFRα, was observed in keratocytes and telocytes within the stroma, indicating the potential presence of mesenchymal cells. These results suggest the involvement of telocytes and keratocytes in corneal wound healing, transparency maintenance, and homeostasis. The co-expression of these markers highlights the critical role of telocytes and keratocytes in regulating corneal physiological functions, further enhancing our understanding of corneal biology in the buffalo model.

Calreticulin accelerates corneal wound closure and mitigates fibrosis: Potential therapeutic applications.

The processes involved in regeneration of cutaneous compared to corneal tissues involve different intrinsic mechanisms. Importantly, cutaneous wounds involve healing by angiogenesis but vascularization of the cornea obscures vision. Previous studies showed that topically applied calreticulin (CALR) healed full-thickness excisional animal wounds by a tissue regenerative process markedly enhancing repair without evoking angiogenesis. In the current study, the application of CALR in a rabbit corneal injury model: (1) accelerated full wound closure by 3 days (2) accelerated delayed healing caused by corticosteroids, routinely used to prevent post-injury inflammation, by 6 days and (3) healed wounds without vascularization or fibrosis/hazing. In vitro, CALR stimulated proliferation of human corneal epithelial cells (CE) and corneal stromal cells (keratocytes) by 1.5-fold and 1.4-fold, respectively and induced migration of CE cells and keratocytes, by 72% and 85% compared to controls of 44% and 59%, respectively. As a marker of decreased fibrosis, CALR treated corneal wounds showed decreased immunostaining for α-smooth muscle actin (α-SMA) by keratocytes and following CALR treatment in vitro, decreased the levels of TGF-ß2 in human CE cells and α-SMA in keratocytes. CALR has the potential to be a novel therapeutic both, to accelerate corneal healing from various injuries and in conjunction with corticosteroids.

Mouse Corneal Epithelial and Stromal Cell Isolation and Culture.

Corneal epithelium and stroma are the major cellular structures for ocular protection and vision accuracy; they play important roles in corneal wound healing and inflammation under pathological conditions. Unlike human, murine corneal and stromal fibroblast cells are difficult to isolate for cell culture. In our laboratory, we successfully used an ex vivo culture procedure and an enzymatic procedure to isolate, purify, and culture mouse corneal epithelial and stromal fibroblast cells. Key features • Primary cell culture models of a disease are critical for cellular and molecular mechanism studies. • Corneal tissues with the limbus contain stem cells to generate both epithelial and stromal cells. • An ex vivo corneal culture provides a constant generation of primary corneal cells for multiple passages. • The isolated cells are validated by the corneal epithelial cell markers Krt12 and Cdh1 and the stromal fibroblast marker Vim.

A review of the epithelial and stromal effects of corneal collagen crosslinking.

Induced corneal collagen crosslinking and mechanical stiffening via ultraviolet-A photoactivation of riboflavin (UVA CXL) is now a common treatment for corneal ectasia and Keratoconus. Some effects of the procedure such as induced mechanical stiffening, corneal flattening, and cellular toxicity are well-known, but others remain more controversial. Authors report a variety of contradictory effects, and provide evidence based on individual results and observations. A full understanding of the effects of and mechanisms behind this procedure are essential to predicting its outcome. A growing interest in modifications to the standard UVA CXL protocol, such as transepithelial or accelerated UVA CXL, makes analyzing the literature as a whole more urgent. This review presents an analysis of both the agreed-upon and contradictory results reported and the various methods used to obtain them.

Development of a Fully Synthetic Corneal Stromal Construct via Supramolecular Hydrogel Engineering.

Recent advances in the field of ophthalmology show great potential in the design of bioengineered constructs to mimic the corneal stroma. Hydrogels based on synthetic supramolecular polymers, are attractive synthetic mimics of the natural highly hydrated corneal stroma. Here, a fully synthetic corneal stromal construct is developed via engineering of an injectable supramolecular hydrogel based on ureido-pyrimidinone (UPy) moieties. The hydrogel displays a dynamic and tunable behavior, which allows for control of biochemical and mechanical cues. Two hydrogels are developed, a fully synthetic hydrogel functionalized with a bioactive cyclic arginine-glycine-aspartate UPy (UPy-cRGD) additive, and a hybrid hydrogel based on UPy-moieties mixed with collagen type I fibers. Both hydrogels supported cell encapsulation and associated cellular deposition of extracellular matrix (ECM) proteins after 21 days. Excitingly, the hydrogels support the activation of isolated primary keratocytes into stromal fibroblasts as well as the differentiation toward more quiescent corneal stromal keratocytes, demonstrated by their characteristic long dendritic protrusions and a substantially diminished cytokine secretion. Furthermore, cells survive shear stresses during an injectability test. Together, these findings highlight the development of an injectable supramolecular hydrogel as a synthetic corneal stromal microenvironment able to host primary keratocytes.

Transforming growth factor beta-3 localization in the corneal response to epithelial-stromal injury and effects on corneal fibroblast transition to myofibroblasts.

The purpose of this study was to evaluate the localization of TGF beta-3 in situ in unwounded rabbit corneas and corneas that had epithelial-stromal injuries produced by photorefractive keratectomy (PRK) in rabbits and to evaluate the in vitro effects of TGF beta-3 compared to TGF beta-1 on alpha-smooth muscle actin (α-SMA) protein expression and myofibroblast development in corneal fibroblasts. Forty-eight New Zealand white rabbits underwent either -3 diopter (D) or -9D PRK and were studied from one to eight weeks (four corneas in each group at each time point) after surgery with immunohistochemistry for TGF beta-3, laminin alpha-5, and alpha-smooth muscle actin (α-SMA). Rabbit corneal fibroblasts were treated with activated TGF beta-1 and/or TGF beta-3 at different concentrations and duration of exposure and studied with immunocytochemistry for myofibroblast development and the expression of α-SMA using Jess automated Western blotting. TGF beta-3 was detected at high levels in the stroma of unwounded corneas and corneas at one to eight weeks after -3D or -9D PRK, as well as in the epithelium and epithelial basement membrane (EBM). No difference was noted between corneas that healed with and without myofibroblast-mediated fibrosis, although TGF beta-3 was commonly associated with myofibroblasts. TGF beta-3 effects on corneal fibroblasts in vitro were similar to TGF beta-1 in stimulating transition to α-SMA-positive myofibroblasts and promoting α-SMA protein expression. The corneal stromal localization pattern of TGF beta-3 protein in unwounded corneas and corneas after epithelial-stromal injury was found to be higher and different from TGF beta-1 and TGF beta-2 reported in previous studies. TGF beta-3 had similar effects to TGF beta-1 in driving myofibroblast development and α-SMA expression in corneal fibroblasts cultured in medium with 1% fetal bovine serum.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia.

PURPOSE: We hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). METHODS: Corneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. RESULTS: Slit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased ∼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. CONCLUSION: Tgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.

Computational approaches for evaluating morphological changes in the corneal stroma associated with decellularization.

Decellularized corneas offer a promising and sustainable source of replacement grafts, mimicking native tissue and reducing the risk of immune rejection post-transplantation. Despite great success in achieving acellular scaffolds, little consensus exists regarding the quality of the decellularized extracellular matrix. Metrics used to evaluate extracellular matrix performance are study-specific, subjective, and semi-quantitative. Thus, this work focused on developing a computational method to examine the effectiveness of corneal decellularization. We combined conventional semi-quantitative histological assessments and automated scaffold evaluations based on textual image analyses to assess decellularization efficiency. Our study highlights that it is possible to develop contemporary machine learning (ML) models based on random forests and support vector machine algorithms, which can identify regions of interest in acellularized corneal stromal tissue with relatively high accuracy. These results provide a platform for developing machine learning biosensing systems for evaluating subtle morphological changes in decellularized scaffolds, which are crucial for assessing their functionality.

Collagen Hydrolysate Effects on Photodynamic Efficiency of Gallium (III) Phthalocyanine on Pigmented Melanoma Cells.

The conjugation of photosensitizer with collagen seems to be a very promising approach for innovative topical photodynamic therapy (PDT). The study aims to evaluate the effects of bovine collagen hydrolysate (Clg) on the properties of gallium (III) phthalocyanine (GaPc) on pigmented melanoma. The interaction of GaPc with Clg to form a conjugate (GaPc-Clg) showed a reduction of the intensive absorption Q-band (681 nm) with a blue shift of the maximum (678 nm) and a loss of shape of the UV-band (354 nm). The fluorescence of GaPc, with a strong emission peak at 694 nm was blue shifted due to the conjugation which lower intensity owing to reduce quantum yield (0.012 vs. 0.23, GaPc). The photo- and dark cytotoxicity of GaPc, Glg and GaPc-Clg on pigmented melanoma cells (SH-4) and two normal cell lines (BJ and HaCaT) showed a slight decrease of cytotoxicity for a conjugate, with low selectivity index (0.71 vs. 1.49 for GaPc). The present study suggests that the ability of collagen hydrolysate to form gels minimizes the high dark toxicity of GaPc. Collagen used for conjugation of a photosensitizer might be an essential step in advanced topical PDT.

Derivation of Human Corneal Keratocytes from ReLEx SMILE Lenticules for Cell Therapy and Tissue Engineering.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

The Cornea: No Difference in the Wound Healing Response to Injury Related to Whether, or Not, There's a Bowman's Layer.

Bowman's layer is an acellular layer in the anterior stroma found in the corneas of humans, most other primates, chickens, and some other species. Many other species, however, including the rabbit, dog, wolf, cat, tiger, and lion, do not have a Bowman's layer. Millions of humans who have had photorefractive keratectomy over the past thirty plus years have had Bowman's layer removed by excimer laser ablation over their central cornea without apparent sequelae. A prior study showed that Bowman's layer does not contribute significantly to mechanical stability within the cornea. Bowman's layer does not have a barrier function, as many cytokines and growth factors, as well as other molecules, such as EBM component perlecan, pass bidirectionally through Bowman's layer in normal corneal functions, and during the response to epithelial scrape injury. We hypothesized that Bowman's layer represents a visible indicator of ongoing cytokine and growth factor-mediated interactions that occur between corneal epithelial cells (and corneal endothelial cells) and stromal keratocytes that maintain the normal corneal tissue organization via negative chemotactic and apoptotic effects of modulators produced by the epithelium on stromal keratocytes. Interleukin-1 alpha, produced constitutively by corneal epithelial cells and endothelial cells, is thought to be one of these cytokines. Bowman's layer is destroyed in corneas with advanced Fuchs' dystrophy or pseudophakic bullous keratopathy when the epithelium becomes edematous and dysfunctional, and fibrovascular tissue commonly develops beneath and/or within the epithelium in these corneas. Bowman's-like layers have been noted to develop surrounding epithelial plugs within the stromal incisions years after radial keratotomy. Although there are species-related differences in corneal wound healing, and even between strains within a species, these differences are not related to the presence or absence of Bowman's layer.

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

In Vitro Expression Analysis of Cytokines and ROS-Related Genes in Human Corneal Fibroblasts and Keratocytes of Healthy and Keratoconus Corneas.

PURPOSE: To investigate expression of cytokines and ROS-related genes in stromal cells of healthy and keratoconus (KC) corneas. METHODS: Expression analysis was performed for cytokines including several interleukins (IL), Tumor necrosis factor-α (TNF-α), Transforming growth factor-ß1 (TGF-ß1), Interferon-γ (IFN-γ) and ROS-related genes such as Catalase, Glutathione peroxidase 1, NADPH oxidase 1, superoxide dismutase 1 in corneal fibroblasts (HCFs/KC-HCFs) or keratocytes (Keratocytes/KC-Keratocytes) by qPCR and ELISA. RESULTS: Gene and protein expression of most inflammatory markers was decreased in keratocytes compared to fibroblasts, whereas no differences were found between healthy and keratoconus cells for the majority of cytokines measured. TNF-α expression was increased at gene (KC keratocytes) and protein levels (supernatant of Keratocytes/KC-Keratocytes) compared to corneal fibroblasts. No differential expression of ROS-related genes was detected between healthy and diseased cells in both fibroblasts and keratocytes. CONCLUSION: Increased expression of several inflammatory markers described as altered in KC was not evident in KC cells in vitro.

Chondroitin Sulphate/Dermatan Sulphate Proteoglycans: Potential Regulators of Corneal Stem/Progenitor Cell Phenotype In Vitro.

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea's stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues.