Search

Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5- cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues.

Corneal stromal stem cells (CSSCs) are of particular interest in regenerative ophthalmology, offering a new therapeutic target for corneal injuries and diseases. This review provides a comprehensive examination of CSSCs, exploring their anatomy, functions, and role in maintaining corneal integrity. Molecular markers, wound healing mechanisms, and potential therapeutic applications are discussed. Global corneal blindness, especially in more resource-limited regions, underscores the need for innovative solutions. Challenges posed by corneal defects, emphasizing the urgent need for advanced therapeutic interventions, are discussed. The review places a spotlight on exosome therapy as a potential therapy. CSSC-derived exosomes exhibit significant potential for modulating inflammation, promoting tissue repair, and addressing corneal transparency. Additionally, the rejuvenation potential of CSSCs through epigenetic reprogramming adds to the evolving regenerative landscape. The imperative for clinical trials and human studies to seamlessly integrate these strategies into practice is emphasized. This points towards a future where CSSC-based therapies, particularly leveraging exosomes, play a central role in diversifying ophthalmic regenerative medicine.

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34 , ABCG2 , PAX6, NANOG, REX1, SOX2 , and THY1 , supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: AH declares a relationship with NuVision Biotherapies Ltd, Nottingham that includes employment and equity or stocks. All other authors declare no competing interests.

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

The cornea is frequently exposed to ultraviolet (UV) radiation and absorbs a portion of this radiation. UVB in particular is absorbed by the cornea and will principally damage the topmost layer of the cornea, the epithelium. Epidemiological research shows that the UV damage of DNA is a contributing factor to corneal diseases such as pterygium. There are two main DNA photolesions of UV: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs). Both involve the abnormal linking of adjacent pyrimide bases. In particular, CPD lesions, which account for the vast majority of UV-induced lesions, are inefficiently repaired by nucleotide excision repair (NER) and are thus mutagenic and linked to cancer development in humans. Here, we apply two exogenous enzymes: CPD photolyase (CPDPL) and T4 endonuclease V (T4N5). The efficacy of these enzymes was assayed by the proteomic and immunofluorescence measurements of UVB-induced CPDs before and after treatment. The results showed that CPDs can be rapidly repaired by T4N5 in cell cultures. The usage of CPDPL and T4N5 in ex vivo eyes revealed that CPD lesions persist in the corneal limbus. The proteomic analysis of the T4N5-treated cells shows increases in the components of the angiogenic and inflammatory systems. We conclude that T4N5 and CPDPL show great promise in the treatment of CPD lesions, but the complete clearance of CPDs from the limbus remains a challenge.

Ultraviolet (UV) irradiation induces DNA lesions in all directly exposed tissues. In the human body, two tissues are chronically exposed to UV: the skin and the cornea. The most frequent UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) that can lead to apoptosis or induce tumorigenesis. Lacking the protective pigmentation of the skin, the transparent cornea is particularly dependent on nucleotide excision repair (NER) to remove UV-induced DNA lesions. The DNA damage response also triggers intracellular autophagy mechanisms to remove damaged material in the cornea; these mechanisms are poorly understood despite their noted involvement in UV-related diseases. Therapeutic solutions involving xenogenic DNA-repair enzymes such as T4 endonuclease V or photolyases exist and are widely distributed for dermatological use. The corneal field lacks a similar set of tools to address DNA-lesions in photovulnerable patients, such as those with genetic disorders or recently transplanted tissue.

To discuss and evaluate new technologies for a better diagnosis of corneal diseases and limbal stem cell deficiency, the outcomes of a consensus process within the European Vision Institute (and of a workshop at the University of Cologne) are outlined. Various technologies are presented and analyzed for their potential clinical use also in defining new end points in clinical trials. The disease areas which are discussed comprise dry eye and ocular surface inflammation, imaging, and corneal neovascularization and corneal grafting/stem cell and cell transplantation. The unmet needs in the abovementioned disease areas are discussed, and realistically achievable new technologies for better diagnosis and use in clinical trials are outlined. To sum up, it can be said that there are several new technologies that can improve current diagnostics in the field of ophthalmology in the near future and will have impact on clinical trial end point design., (© 2020 S. Karger AG, Basel.)

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.

In the past decade, novel lamellar keratoplasty techniques such as Deep Anterior Lamellar Keratoplasty (DALK) for anterior keratoplasty and Descemet stripping automated endothelial keratoplasty (DSAEK)/Descemet membrane endothelial keratoplasty (DMEK) for posterior keratoplasty have been developed. DALK eliminates the possibility of endothelial allograft rejection, which is the main reason for graft failure after penetrating keratoplasty (PK). Compared to PK, the risk of endothelial graft rejection is significantly reduced after DSAEK/DMEK. Thus, with modern lamellar techniques, the clinical problem of endothelial graft rejection seems to be nearly solved in the low-risk situation. However, even with lamellar grafts there are epithelial, subepithelial and stromal immune reactions in DALK and endothelial immune reactions in DSAEK/DMEK, and not all keratoplasties can be performed in a lamellar fashion. Therefore, endothelial graft rejection in PK is still highly relevant, especially in the "high-risk" setting, where the cornea's (lymph)angiogenic and immune privilege is lost due to severe inflammation and pathological neovascularization. For these eyes, currently available treatment options are still unsatisfactory. In this review, we will describe currently used keratoplasty techniques, namely PK, DALK, DSAEK, and DMEK. We will summarize their indications, provide surgical descriptions, and comment on their complications and outcomes. Furthermore, we will give an overview on corneal transplant immunology. A specific focus will be placed on endothelial graft rejection and we will report on its incidence, clinical presentation, and current/future treatment and prevention options. Finally, we will speculate how the field of keratoplasty and prevention of corneal allograft rejection will develop in the future., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Cyclosporine A (Cs) has been used as effective topical therapy for inflammatory dry eye disease since more than a decade. However, due to its lipophilic character, Cs is formulated as emulsions or oily solutions for topical application. This experimental study aimed to test if the use of semifluorinated alkanes (SFAs) as a preservative-free, well-tolerated non-stinging or burning vehicle maintains or even improves the benefits of Cs in the topical therapy of dry-eye disease., Methods: Desiccating stress was applied to C57BL/6 mice for 14 consecutive days to induce experimental dry-eye. Cs dissolved in SFA (perfluorobutylpentane = F4H5with 0.5% Ethanol), F4H5 with 0.5% ethanol only, 0.05% Cs (Restasis®), and dexamethasone (Monodex®) were applied three times daily beginning either at day 4 or day 11 of desiccating stress for up to 3 weeks after end of dry-eye induction., Results: In comparison to other groups, Cs/F4H5 demonstrated high efficacy and earlier reduction of corneal staining. In this study, Cs/F4H5 had the ability to maintain conjunctival goblet cell density once applied on day 4. Flow cytometry analysis from cervical lymphnodes demonstrated a significantly lower CD4+ and CD8+ T-cells in the Cs/F4H5 group following 3 weeks of therapy than at baseline, but no difference in regulatory T cells from regional lymphnodes were seen., Conclusions: Overall, compared to a commercially available Cs formulation (Restasis®) and dexamethasone, Cs/F4H5 was shown to be equally effective but with a significantly faster therapeutic response in reducing signs of dry-eye disease in an experimental mouse model.

Purpose: We analyzed the effects of short-term ultraviolet A (UVA) irradiation on the putative limbal stem cell phenotype, limbal fibroblasts, corneal inflammation, and corneal (lymph)angiogenic privilege., Methods: Primary human limbal epithelial cells and fibroblasts were irradiated with 5.2 J/cm2 of UVA. The limbal epithelial cell phenotype was assessed using P63a, cytokeratin 15, integrin b1 (marking stem and transient amplifying cells), and cytokeratin 3 (a differentiation marker) as well as by a colony-forming efficiency (CFE) assay. An epithelial-fibroblast coculture model was used to compare the ability of irradiated and nonirradiated fibroblasts to support the putative limbal stem cell phenotype. The effects of the conditioned media of irradiated and nonirradiated cells on proliferation and tube formation of human lymphatic and blood endothelial cells also were tested. The levels of factors related to angiogenesis and inflammation were assessed in a protein array and using ELISA., Results: Ultraviolet A induced phenotypical changes of limbal epithelial cells, as their CFE and putative stem cell/transient amplifying marker expression decreased. Limbal epithelial cells cocultured with UVA-irradiated limbal fibroblasts also exhibited differentiation and CFE decrease. Conditioned media from irradiated limbal epithelial cells and fibroblasts inhibited lymphatic endothelial cell proliferation and tube network complexity. Levels of monocyte chemoattractant protein 1 (MCP1) were reduced following UVA irradiation of both cell populations, while levels of IFN-γ increased in irradiated limbal epithelial cells., Conclusions: These data imply a key role of cellular components of the limbal niche following short-term UVA irradiation. Overall, UVA irradiation leads to dysfunction of these cells and a anti(lymph)angiogenic and anti-inflammatory micromilieu.

Ultraviolet light B (UVB)-irradiation is linked to various ocular pathologies such as limbal stem cell defects in pterygium. Despite the large circumstantial evidence linking UVB irradiation and limbal epithelial stem cell damage, the precise molecular responses of limbal stem cells to UVB irradiation are unclear. Here the effect of UVB irradiation on the putative stem cell phenotype, limbal niche cells and the subsequent effects on corneal (lymph)angiogenic privilege were investigated. Primary human limbal epithelial stem cells and fibroblasts were irradiated with 0.02 J/cm(2) of UVB, a low dose corresponding to 3 min of solar irradiation. UVB irradiation caused significant reduction of limbal epithelial and limbal fibroblast proliferation for 24 h, but apoptosis of limbal epithelial stem cells only. Moreover, UVB induced stem-like character loss of limbal epithelial cells, as their colony forming efficiency and putative stem cell marker expression significantly decreased. Interestingly, limbal epithelial cells co-cultured with UVB-irradiated limbal fibroblasts also exhibited loss of stem cell character and decrease of colony forming efficiency. Conditioned media from limbal epithelial cells inhibited lymphatic endothelial cell proliferation and tube network complexity; however this effect diminished following UVB irradiation. In contrast, pro-inflammatory and macrophage-recruiting cytokines such as TNFα, IFNγ and MCP1 were significantly upregulated following cell irradiation of limbal fibroblasts. These data demonstrate the key role of the limbal stem cell niche in response to UVB and subsequent (lymph)angiogenic and inflammatory events. These data suggest that the known pro(lymph)angiogenic effect of UVB irradiation in pterygium is not linked to a direct up-regulation of pro-angiogenic cytokines, but rather to indirect macrophage-recruiting cytokines being upregulated after UVB irradiation., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Malignant melanomas of the ocular surface (conjunctival melanoma [CM]) and within the eye (uveal melanoma [UM]) show different types of metastatic behavior. While CM has a propensity to spread first to regional lymph nodes, UM metastasizes almost exclusively via the hematogenic route to the liver. We investigated whether these different metastatic patterns might be attributable to differential hem- and lymphangiogenic characteristics of CM and UM cells., Methods: Human CM (CM2005.1, CRMM1, CRMM2) and UM (Mel270, Mel290, OM431) cell lines were analyzed for VEGF-A, -C, and -D expression by RT-PCR and ELISA. The influence of CM- or UM-conditioned medium on blood (BEC) and lymphatic (LEC) endothelial cell proliferation and migration was measured using 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) and scratch assays, respectively., Results: Vascular endothelial growth factor-A, -C and -D mRNA, and VEGF-A and -D protein were expressed by all CM and UM cell lines, while VEGF-C protein was only expressed by UM cell lines. The CM- and UM-conditioned medium did neither differentially affect BEC (P = 0.86) and LEC (P = 0.90) proliferation, nor BEC (P = 0.56) and LEC (P = 0.90) migration., Conclusions: Conjunctival melanoma cell lines did not show a higher prolymphangiogenic potential, and UM cell lines did not show a higher prohemangiogenic potential. Accordingly, other mechanisms within the tumor microenvironment might account for the diverging metastatic patterns of conjunctival versus uveal melanomas.

Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer.

Most men over 50 experience some lower urinary tract symptoms of nocturia, poor stream, urgency and frequency for urination, due to hyperplastic enlargement of the prostate (benign prostate hyperplasia, BPH). BPH is thought to be a disease with multiple aetiologies including hormone signalling, disruption of proliferation and apoptosis dynamics and chronic inflammation with changes in the morphology and phenotype of the prostate stroma. It has been proposed, recently, that stromal stem cells in prostate may be caused by the development of BPH. This review focuses on this putative role of stromal stem or stem-like cells in the development of BPH and assesses the potential of targeting the stem cells for the treatment of BPH.

Aims: To investigate the optimum conditions for the differentiation of a mouse embryonic stem cell line towards corneal epithelial cell fate., Materials & Methods: The effect of conditioned media from both metabolically active (to produce lineage A) and growth-arrested limbal fibroblasts (lineage G) were compared with basal media (lineage N) in terms of morphology and marker expression, assessed by immunocytochemistry and reverse transcription PCR. Cultures were transplanted into a porcine ex vivo model to investigate their ability for wound healing and cornea repair., Results: Lineage N exhibited cobblestone morphology and expressed CK12 and p63α, while OCT4 and SSEA1 were downregulated. Post-transplantation, these cells were able to multilayer and heal after wounding while maintaining marker expression., Conclusion: Lineages with corneal epithelial-like characteristics, which are derived from embryonic stem cells, have potential for use in the study of corneal wound healing and therapy.

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ∼1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.

The corneal epithelium is maintained by the limbal epithelial stem cells (LESCs). In this study, an in vitro model is proposed for the investigation of cell-cell interactions involving LESC maintenance. Imaging of the limbal niche demonstrated close spatial arrangement between basal limbal epithelial cells within putative LESC niche structures and fibroblasts in the stroma. Interactions of the human limbal epithelial (HLE) cells and mitotically active human limbal fibroblasts (HLF) were studied for the first time in a serum-free in vitro model that simulated aspects of the limbal niche microenvironment. HLE cocultured in a ratio 3:1 with HLF exhibited enhanced expression of the putative stem cell markers ABCG2 and p63α and holoclones were preserved as shown by colony-forming efficiency assays, clonal analysis, and colony characterisation. Interleukin 6 (IL6) was found to be up-regulated in the 3.1SF system when compared to the HLE culture with growth-arrested fibroblasts and serum (gold standard system, GS). IL6 caused a time-dependent phosphorylation of STAT3 in HLE cells. STAT3 and IL6 inhibition in 3.1SF cultures significantly reduced HLE colony-forming efficiency, suggesting a previously undetected STAT3-mediated involvement of IL6 in the maintenance of HLE cells in a progenitor-like state., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aim: To evaluate a serum-free system where mitotically active subconjunctival fibroblasts were co-cultured with conjunctival epithelial cells to mimic a niche environment for conjunctival progenitor cells., Methods: Human conjunctival epithelial cells were expanded in vitro and evaluated for their colony-forming efficiency and clonal ability. The cells were then transferred to a serum-free co-culture system and cultured in the presence of mitotically active subconjunctival fibroblasts (human conjunctival epithelial cells and human bulbar subconjunctival fibroblasts [HCEC-HCF]). Cells were evaluated by Ki67 staining, total colony-forming efficiency and the number of colonies with a surface area of more than 10 mm(2). The expression of putative progenitor cell markers p63α, ABCG2 and CK15, and the presence of MUC5AC- and periodic acid-Schiff-positive cells was compared with standard culture conditions (HCEC-3T3)., Results: Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions demonstrated strong immunoreactivity to p63α and ABCG2. Co-localization of CK15 and p63α revealed a subpopulation of CK15-positive cells under HCEC-3T3 conditions compared with only a few CK15-positive cells found under HCEC-HCF conditions. MUC5AC- and periodic acid-Schiff-positive cells were much more common under HCEC-3T3 conditions than under HCEC-HCF conditions. These results were confirmed by reverse transcription-PCR. Cells in HCEC-HCF conditions demonstrated a significantly higher total colony-forming efficiency and a significantly higher percentage of colonies with holoclone-like morphology., Conclusions: The simulation of a niche environment in vitro by co-culturing mitotically active subconjunctival fibroblasts with conjunctival epithelial cells supports the maintenance of conjunctival cells with progenitor cell characteristics and therefore might be a useful tool to expand conjunctival epithelial progenitor cells in vitro for clinical use.

In this study a spontaneously formed corneal epithelial cell line, namely HCE-S, was established and characterised. The cell line was karyotyped and corneal epithelial maker expression of the cell line was assessed by immunostaining and semi-quantitative RT-PCR. The morphological characteristics were investigated using SEM and TEM analyses. The functional response to EGF in terms of cell proliferation, wound healing and cell migration was tested using Alamar Blue, scratch wound and colony dispersion assays, respectively. The cells were maintained in culture for more than 100 divisions and 35 passages suggesting that an immortalised cell line had been established. HCE-S, has maintained an epithelial morphology and has not phenotypicaly changed through passages. SEM and TEM microscopy showed morphological similarities to primary corneal epithelial cells. HCE-S expressed a battery of characteristic markers of primary corneal epithelial cells including cytokeratin 3 and PAX 6 as well as the basal cell integrins beta1 and alpha9 and the putative corneal stem cell marker ABCG2. HCE-S cells were responsive to exogenous EGF as shown by proliferation, migration and scratch wound assays. HCE-S can be cultured in a simple DMEM and only serum-based media which gives them an advantage against available corneal epithelial cell lines. This fact, along with the often limited availability and variability of primary corneal epithelial cells and the similarities of the cell line with primary cell characteristics suggest that HCE-S could be a useful tool for the study of corneal epithelial cell biology, ocular surface toxicity studies and pharmacological testing., (Copyright 2009 Elsevier Inc. All rights reserved.)

Reconstruction of the conjunctiva is an essential part of ocular surface regeneration, especially if an extensive area or the whole ocular surface is affected, such as in patients with ocular cicatricial pemphigoid, Stevens-Johnson syndrome, toxic epidermal necrolysis, or chemical/thermal burns. In these situations, corneal reconstruction almost inevitably fails unless the conjunctival surface is first repaired and a deep fornix is restored. The growing field of tissue engineering and advances in stem cell research offer promising new alternatives for these challenges. This article reviews the present approaches for reconstruction of the conjunctival surface, considering the established strategies and new potential methodologies.

Our aim was to develop an improved cell transfer system for delivering laboratory-cultured human limbal epithelial cells to the cornea, which would be low risk for the patient and convenient to use for the surgeon. We took a standard contact lens and developed a plasma polymer layer for coating this for attachment of cells to the lens and subsequent transfer of cells to the cornea. A range of plasma polymer surfaces were examined for initial cell attachment using three different combinations of human and rabbit epithelial and stromal cells, initially expanding cells both with and without bovine serum. The most promising surfaces, based on acrylic acid, were then coated onto contact lenses. Cell transfer from the lenses to the denuded surface of a 3D rabbit organ culture model was then used to make a second selection of substrates, which permitted reliable cell transfer. Primary rabbit and human corneal cells attached and proliferated well on acrylic acid-coated surfaces. Reliable transfer of primary epithelial cells from the coated contact lenses to a rabbit cornea was achieved by coating lenses with acrylic acid at 5 W/10 cm(3)/min and using cell densities of 1 x 10(5)/lens and above.

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., (Copyright 2008 Wiley Periodicals, Inc.)

In this review, we describe a population of adult stem cells that are currently being successfully used in the clinic to treat blinding ocular surface disease, namely limbal epithelial stem cells (LESC). The function and characteristics of LESC and the challenges faced in making use of their therapeutic potential will be examined. The cornea on the front surface of the eye provides our window on the world. The consistency and functionality of the outer-most corneal epithelium is essential for vision. A population of LESC are responsible for replenishing the epithelium throughout life by providing a constant supply of daughter cells that replace those constantly removed from the ocular surface during normal wear and tear and following injury. LESC deficiency results in corneal inflammation, opacification, vascularisation and severe discomfort. The transplantation of cultured LESC is one of only a few examples of the successful use of adult stem cell therapy in patients. The clinical precedence for the use of stem cell therapy and the ready accessibility of a transparent stem cell niche make the cornea a unique model for the study of adult stem cells in health and disease.

Aims: Murine 3T3 feeder cells are commonly used for stem cell expansion. Although 'feeder-free' systems are being developed for a variety of stem cells including embryonic, the use of feeder cells currently remains optimal for the expansion of epithelial stem cells. In this study, MRC-5, a human embryonic fibroblast cell line, has been investigated for its potential use as a feeder layer in human limbal epithelial (HLE) cell expansion under serum-free conditions, with the aim of developing a xenobiotic-free culture system for therapeutic corneal regeneration applications., Materials and Methods: MRC-5 feeder cells were compared with J2 3T3 mouse fibroblasts, in both serum-supplemented and serum-free conditions, in terms of their relative ability to support HLE cell metabolic activity, expression of the putative stem cell markers ABCG2 and P63 alpha, cell differentiation using the cornea-specific cytokeratin 3 antibody and colony-forming efficiency., Results: The proportion of HLE stem cells maintained was determined by functional colony-forming efficiency assays. The metabolic activity results showed that HLE cells cultured on MRC-5 fibroblasts under serum-free conditions proliferated as well as cells cultured on J2 cells under serum-free conditions. Moreover, the HLE cultured on MRC-5 fibroblasts under serum-free conditions expressed high levels of putative stem cell markers ABCG2 and P63 alpha and low levels of the differentiation marker CK3, indicating that they retained poorly differentiated 'stem cell-like' characteristics under those culture conditions. Clonal analysis of HLE cells cultured on growth-arrested feeder layers of J2 and MRC-5 fibroblasts showed that cells expanded on MRC-5 and J2 fibroblasts in serum-free conditions had a colony-forming efficiency of approximately 1.5%, indicating the maintenance of stem cells., Conclusions: These results demonstrate feasibility of expanding HLE cells for clinical purposes by using a human fibroblast cell line as a feeder layer, avoiding the use of bovine serum, while preserving the proliferative potential and stem cell characteristics of HLE cells.

The potential use of plasma polymer coatings as substrates for serum-free expansion of limbal epithelial cells was investigated. Preliminary studies using a human corneal epithelial cell line showed that acrylic acid-coated surfaces performed better than allyl amine and allyl alcohol coated surfaces in terms of cell metabolic activity and confluence as assessed using the MTT assay. Subsequently, the proliferation and maturity of primary human limbal epithelial cells in co-culture with growth arrested 3T3 fibroblasts on a range of acrylic acid plasma coated surfaces, octadiene plasma coated surfaces and tissue culture plastic was investigated using MTT and cytokeratin 3 immunostaining. The cells performed better in the presence of serum on all surfaces. However, the acrylic acid coated surfaces successfully sustained a serum-free fibroblast/epithelial cell co-culture. The metabolic activity of the epithelial cells was superior on the acrylic acid coated surfaces than on tissue culture plastic in serum-free conditions and their levels of differentiation were not significantly higher than in the presence of serum. These results suggest that these surfaces can be used successfully for the serum-free expansion of human limbal epithelial cells.

Restoring vision in patients suffering from previously intractable blinding ocular surface disease has become possible with the advent of techniques for ex vivo expansion and transplantation of limbal epithelial stem cells onto the cornea. This approach represents one of the few adult stem cell therapies presently in the clinic. This article highlights several key research areas where progress will be made to specifically understand the biology and therapeutic potential of limbal epithelial stem cells, which may have an applicability to the understanding of other adult stem cell populations.