Maria Notara

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.

IL6 and the human limbal stem cell niche: A mediator of epithelial–stromal interaction

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.

Tissue Engineering for Conjunctival Reconstruction: Established Methods and Future Outlooks

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.

Biological principals and clinical potentials of limbal epithelial stem cells

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.

Limbal epithelial stem cell therapy

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.

Characterisation and functional features of a spontaneously immortalised human corneal epithelial cell line with progenitor-like characteristics.

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.

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

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

Simulation of an in vitro Niche Environment That Preserves Conjunctival Progenitor Cells

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.

Characterization of the phenotype and functionality of corneal epithelial cells derived from mouse embryonic stem cells

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.