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.

Development of a conjunctival tissue substitute on the basis of plastic compressed collagen.

Ocular surface disorders, such as pterygium, cicatricial pemphigoid and external disruptions, can cause severe inflammation, scarring, fornix shortening as well as ankyloblepharon. Current treatments do not resolve these conditions sufficiently. The aim of this study was to evaluate clinical applicability and suitability of plastic compressed collagen to serve as a substrate for the expansion of human conjunctival epithelial cells in order to develop an epithelialized conjunctival substitute for fornix reconstruction. Human conjunctival epithelial cells were expanded on plastic compressed collagen gels. Epithelial cell characteristics were evaluated by haematoxylin and eosin staining, electron microscopy and cytokeratin expression. The expression of putative epithelial progenitor cell markers p63α, ABCG2 and CK15 was assessed by immunostaining. The proliferative capacity and clonal growth of the cells was evaluated before (P0) and after expansion (P1) on the plastic compressed collagen gels by colony forming efficiency assay. The potential clinical applicability of this gel substitutes was evaluated by assessment of their biomechanical properties as well as their surgical handling. Human conjunctival epithelial cells cultured on plastic and plastic compressed collagen gels formed a confluent cell layer and expressed CK19. The cells showed expression of the putative epithelial progenitor cell markers p63α, ABCG2 and CK15 and sustained colony forming ability. The compressed collagen gels showed a high ultimate tensile strength and elasticity and the surgical handling of gels was comparable to amniotic membrane. An epithelialized conjunctival tissue construct on the basis of compressed collagen might therefore be a promising alternative bioartificial tissue substitute for conjunctival reconstruction. Copyright © 2015 John Wiley & Sons, Ltd.

Current prospects for adult stem cell-based therapies in ocular repair and regeneration.

Recent advances in stem cell biology have led to the exploration of stem cell-based therapies to treat a wide range of human diseases. In the ophthalmic field, much hope has been placed on the potential use of these cells to restore sight, particularly in those conditions in which other established treatments have failed and in which visual function has been irreversibly damaged by disease or injury. At present, there are many limitations for the immediate use of embryonic stem cells to treat ocular disease, and as more evidence emerges that adult stem cells are present in the adult human eye, it is clear that these cells may have advantages to develop into feasible therapeutic treatments without the problems associated with embryonic research and immune rejection. Here we discuss the current prospects for the application of various adult ocular stem cells to human therapies for restoration of vision.

Temporal stimulation of corneal fibroblast wound healing activity by differentiating epithelium in vitro.

PURPOSE: To determine whether differentiating corneal epithelium can temporally stimulate fibroblast activity. METHODS: Corneal epithelial cells were cultured to confluence and then stimulated to mature into multilayered epithelia with addition of serum-containing medium. Differentiation was assessed morphologically and immunocytochemically using a monoclonal antibody to cytokeratin 3. At intervals after onset of differentiation serum-free conditioned medium was collected up to 28 days. Preliminary experiments deduced the optimum concentration of conditioned medium to be used for assessing fibroblast activity. Conditioned medium (25% vol/vol) was added to donor-matched corneal fibroblasts in migration chambers, WST-1 reagent proliferation assays, and fibroblast-populated collagen gel contraction assays. Platelet-derived growth factor (PGDF)-AB and interleukin (IL)-1beta in conditioned media were quantified by enzyme-linked immunosorbent assay (ELISA). Fibroblast migration and collagen contraction assays were performed with concentrations of PDGF-AB. RESULTS: Conditioned medium collected from differentiating epithelium stimulated fibroblast migration and collagen gel contraction, with activity peaks occurring with medium collected on day 14 (P < 0.05). No significant difference was detected between fibroblast growth rates in each of the conditioned media. Levels of PDGF-AB increased during epithelial culture up to 22 days (up to approximately 360 pg/ml) with a subsequent decrease by 28 days. IL-1beta inversely correlated with fibroblast activity induced by conditioned medium, with a trough in concentration (2 pg/ml) occurring at 14 days. Both fibroblast migration and collagen contraction were stimulated in a dose-dependent manner by PDGF-AB. CONCLUSIONS: Corneal epithelium is capable of temporally stimulating fibroblast wound-healing characteristics during its differentiation. One of the growth factors potentially involved in this epithelial-stromal interaction is PDGF. This work demonstrated that developing epithelium (possibly similar to repairing epithelium in vivo) regulated fibroblast behavior and may indicate a mechanism of fibroblast recruitment to a wound after epithelial closure.

Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells.

PURPOSE: Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. METHODS: Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase-based assay. RESULTS: The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were-except for natural saliva-less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. CONCLUSIONS: This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes.

Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior.

PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production. METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment. The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels. The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment. RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF. Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls. Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment. Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period. The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period. The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period. CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate. These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.

Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.

Understanding and controlling the scarring response: the contribution of histology and microscopy.

In response to injury, the body usually initiates a full and swift wound healing response resulting in reconstructed, repaired tissue. In certain instances, due to a variety of factors, this may not happen, an example being chronic granulating venous leg ulcers. At the other extreme, the wound may heal excessively, producing disabling hypertrophic scarring such as can occur following large, deep burn injuries. Our group is interested in the surgical treatment of the eye disease glaucoma. As will be explained, the successful surgical treatment of this disease depends on a reduced scarring response at the end of wound healing. The purpose of this article is to give an overview of our microscopic and histological experimental work which has furthered our understanding of tissue repair, particularly the scarring response and its potential modification for successful glaucoma surgery.

MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule.

PURPOSE: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. METHODS: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. RESULTS: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p<0.05), and a reduction in capsule contraction was observed (p<0.05). CONCLUSIONS: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.

T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts.

AIMS: To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon's capsule fibroblasts. METHODS: IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. RESULTS: T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). CONCLUSION: T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically.

Autologous serum eyedrops for dry eyes and epithelial defects: clinical and in vitro toxicity studies.

BACKGROUND/AIMS: Autologous serum drops have been reported to be beneficial in keratoconjunctivitis sicca (KCS) and persistent epithelial defects (PED). A clinical pilot study was carried out to examine these potential uses and in vitro toxicity testing on corneal epithelial cell cultures was performed to compare the effect of serum drops with unpreserved hypromellose (hydroxypropylmethylcellulose 0.3%). METHODS: Patients with KCS and PED, unresponsive to conventional treatment were recruited. Patients were examined before treatment, at 1 and 2 weeks after initiation, and then 2 weekly until treatment ceased. Symptoms were assessed at each visit. Clinical examination included Schirmer's test without anaesthesia, rose bengal staining, and fluorescein staining. Epithelial defects were measured with the slit beam. In the laboratory, cultured human corneal epithelial cells were exposed to serum drops and hypromellose, and their viability evaluated with fluorescent viability staining (Calcein AM ethidium homodimer) and an ATP assay. RESULTS: Autologous serum was used in 15 eyes of 13 patients with PED and 11 eyes of nine patients with KCS. In two patients serum drops were started after penetrating keratoplasty (PK). The PKs were performed for perforations secondary to PEDs. Of the 15 eyes with PED, nine healed at a mean of 29 days and six failed. The mean duration of PED before the use of serum drops was 48.2 days. Of the 11 eyes with KCS, six had improved subjective scores and fluorescein scores, and five had improved rose bengal scores after the use of serum drops. For the two patients who used serum eyedrops post-PK, there was a stable and intact epithelium at 1 week. Cessation of serum drops during the postoperative period led to deterioration in the subjective and objective scores in both patients. One developed a PED that responded to reinstitution of serum drops. The morphology and ATP levels of cultured epithelial cells exposed to serum were better maintained than those exposed to hypromellose. CONCLUSION: Autologous serum drops are useful for PED and KCS. This effect may be related to a number of active factors in serum including growth factors, fibronectin, vitamin A, and anti-proteases. In vitro toxicity testing demonstrated that serum drops have reduced toxicity compared with unpreserved hypromellose. Currently regulatory restrictions in the UK have prevented the establishment of a prospective randomised controlled trial examining the efficacy of autologous serum drops for the management of this group of ocular surface disorders.

A physiologist's view of running economy.

The relationship between VO2 and velocity of running (running economy) has been rather casually dealt with until very recently, and there still remains considerable disagreement as to the importance of this variable. Various factors which have been shown, or appear, to affect running economy include environment (temperature, altitude, running surface), fatigue, age, weight, state of fitness, and inherent differences. That differences between individuals and within individuals can and do exist seems clear; the questions which need to be addressed in future research are: (1) What type of training is most effective in bringing about changes in running economy? and (2) How much change in economy can be expected with optimum training? Furthermore, it is suggested that running economy be investigated as an entity, so that changes that may take place with time or training can be more accurately related to their cause.

Variation in the aerobic demand of running among trained and untrained subjects.

Variation in the aerobic demand (VO2) of submaximal running was quantified among trained and untrained subjects stratified by performance capability. Based on a retrospective analysis of seven published studies, maximal aerobic power (VO2max), and submaximal VO2 values were analyzed in three groups of trained distance runners (Category 1 (C1) (elite runners; N = 22), Category 2 (C2) (sub-elite runners; N = 41), and Category 3 (C3) (good runners; N = 16), and one group (N = 10) of untrained subjects (Category 4; C4). Results indicated that VO2max differed significantly (P < 0.05) across groups, such that C1 > C2 > C3 > C4. Analysis of submaximal VO2 data also revealed that C4 was more uneconomical than C1, C2, and C3 and that C2 and C3 were less economical than C1. Average within-group variability in submaximal VO2 was similar across categories and a marked overlap of minimum, mean and maximal economy values existed across categories. These data suggest that 1) trained subjects are more economical than untrained subjects, 2) elite runners display better economy compared to less-talented counterparts, and 3) economical and uneconomical runners can be found in all performance categories.

Human keratinocyte isolation and cell culture: a survey of current practices in the UK.

A survey was conducted to establish current techniques for isolation and culture of human keratinocytes. A questionnaire was sent to all units thought to be involved in keratinocyte culture, a total of 34 individuals; 62 per cent of those surveyed responded to the questionnaires. The proportion of individuals using high-calcium medium to culture keratinocytes was 53 per cent, while 47 per cent used low-calcium serum-free medium. The majority of replies followed trends dependent on the culture method employed. Details of anatomical donor skin site, keratinocyte isolation and culture were compared. In particular, the problems associated with the use of commercially prepared low-calcium, serum-free medium were reported. The basic principles of keratinocyte culture reported in the literature were seen amongst all the replies received. It is interesting to note the variations in methods adopted as techniques are passed on and continuously modified to suit the requirements of the individual worker. This survey also highlights the difficulties that can occur when using mass-produced complex media.

An investigation into the potential of extracellular matrix factors for attachment and proliferation of human keratinocytes on skin substitutes.

The purpose of this investigation was to assess the potential of commercially available extracellular matrix proteins, as enhancers of human keratinocyte attachment and proliferation, with a view to their incorporation into a skin equivalent. The following substrates were studied: type I and type IV collagen, fibronectin, gelatin and laminin. Human keratinocytes were cultured in low-calcium, serum-free medium. The number of cells attached to each substrate, observed under phase-contrast inverted microscopy in randomly selected fields of view, were counted 2 h afterseeding. Measurements of growth rate and colony-forming efficiency were made at 24-h intervals. None of the substrates tested were found to have an effect significant enough to warrant further investigation or inclusion into skin equivalent.

Evaluation of ewe and lamb immune response when ewes were supplemented with vitamin E.

Fifty-two Targhee twin-bearing ewes were used in a factorial arrangement of treatments to investigate the role of supplemental vitamin E (vit E); 0 (NE) vs 400 IU of vit E x ewe x (-1)d(-1) (E) and parainfluenza type 3 (PI3) vaccination; none (NP) vs PI3 vaccination (P) in immune function. Parainfluenza type 3 vaccination was used to evoke an immune response. Ewes receiving PI3 were vaccinated at 49 and 21 d before the expected lambing date. Ewes receiving vit E were orally dosed daily, 32 to 0 d before lambing. Blood was collected from ewes at the time of the initial PI3 vaccination and 4 h postpartum. Blood was collected from lambs (n = 104) at 3 d postpartum. Ewe and lamb sera were analyzed for anti-PI3 antibody titers, immunoglobulin G (IgG) titers, and vit E concentrations. Colostrum was collected 4 h postpartum and analyzed for IgG. The model for ewe and lamb analysis included the main effects of vit E and PI3, sex (lambs model only), and their interactions. No interactions were detected (P > 0.20) for any ewe or lamb variables. Serum anti-PI3 titers were greater (P < 0.01) in P ewes and their lambs than NP ewes and their lambs. Serum vit E concentrations were greater (P < 0.01) in E ewes and their lambs than NE ewes and their lambs. Colostral IgG titers and serum anti-PI3 titers did not differ (P > 0.20) between E and NE ewes. Serum IgG titers in E ewes and their lambs did not differ (P > 0.15) from IgG titers in NE ewes and their lambs. Lamb anti-PI3 titers did not differ (P = 0.76) between lambs reared by E and NE ewes. These results indicate that, although supplemental vit E to the ewe increased lamb serum vit E concentration, it had no effect on measures used in this study to assess humoral immunity in the ewe or passive immunity to the lamb.

Serum alpha-tocopherol and immune function in yearling ewes supplemented with zinc and vitamin E.

Yearling Targhee ewes (n = 24; not pregnant or lactating) were used in a 2 x 2 factorial arrangement of treatments to determine the effects of supplemental vitamin E (0 IU [0vitE] vs 330 IU vitamin E x ewe(-1) x d(-1) [+vitE]) and Zn (0 mg [0Zn] vs 140 mg Zn x ewe(-1) x d(-1) [+Zn]) on serum alpha-tocopherol concentrations, antibodies to parainfluenza type 3 (PI3), ewe BW, Zn liver concentrations, and serum alkaline phosphatase activity. Ewes were managed as one group, grazed native pasture, and had ad libitum access to white salt and water. Ewes that received supplemental vitamin E were orally dosed every other day with 660 IU of DL-alpha-tocopherol acetate in a gelatin capsule beginning on d 1 and continuing to d 63 of the study. Ewes that received Zn supplement were orally dosed every other day with 280 mg of Availa-Zn 100 (Zinpro Corp., Eden Prairie, MN, IFN 6-32-054) in gelatin capsules for 63 d. All ewes were vaccinated with killed PI3 on d 22 and 42. No interactions were detected (P > 0.35); however, serum alpha-tocopherol concentrations and PI3 antibody titer dilutions changed (P = 0.001) over the length of the study. Ewe BW change, serum alkaline phosphatase activity, and liver Zn concentrations did not differ (P > 0.22) between 0Zn and +Zn or 0vitE and +vitE ewes. Serum a-tocopherol tended to be higher (P = 0.08) in +vitE than 0vitE ewes and was numerically higher (P = 0.16) in +Zn than 0Zn ewes. Antibody titer dilutions were higher (P = 0.06) in 0Zn than +Zn ewes and did not differ (P = 0.83) between 0vitE and +vitE ewes. These results indicate that high levels of supplemental Zn may have a tendency to improve serum alpha-tocopherol concentrations but may have negative impacts on humoral immune function.

Preparing Alberto Salazar for the Heat of the 1984 Olympic Marathon.

In brief: Observations were made on US marathon record holder Alberto Salazar during a climatic chamber trial, heat acclimatization training, and the 1984 Olympic Marathon. The purpose was to evaluate his physiological responses to heat tolerance testing and to advise him regarding heat acclimatization, thermoregulation, and fluid discipline. Salazar was advised to avoid the showers along the race course during the marathon. Although fine-mist sprays, showers, and other skin-wetting techniques may make runners feel good, they have little effect on rectal temperature or sweat rate during a race. This applies to casual joggers as well as elite competitors.

Wnt signalling in an in vitro niche model for conjunctival progenitor cells.

Mimicking an environment in vitro that is more similar to the stem cell niche in vivo, by co-culture of mitotically active conjunctival fibroblasts (HCF) with human conjunctival epithelial cells (HCECs), improves the maintenance of epithelial cells with progenitor cell characteristics during in vitro expansion. However, little is known about the pathways controlling the fate of the epithelial progenitor cells during in vitro culture. In this study, differences in gene expression between this in vitro 'niche' model and standard culture conditions, in which growth-arrested 3 T3 feeder cells and fetal calf serum are used, were explored using a genome level microarray platform, quantitative (q)RT-PCR and western blot. The microarray analysis revealed significant alterations of biological processes involved in cell proliferation, differentiation and cell death. The analysis of stem cell-related pathways indicated changes in expression of genes involved in the Wnt signalling pathway, and further investigation by qPCR revealed significant downregulation of the Wnt ligands Wnt3, Wnt4, Wnt7B and Wnt10A, Wnt receptor proteins FZD1, LRP5, LRP6, ß-catenin and TCF7L1 and important Wnt target genes, such as CCND1, also confirmed by western blot and immunocytochemistry. The results indicate that epithelial cell expansion in the HCEC-HCF co-culture system is accompanied by significant changes in expression of genes involved in the Wnt signalling pathway. This altered pathway activation might be involved in the enhanced maintenance of epithelial progenitor cells in this in vitro 'niche' model.

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.