Limbal epithelial stem cells (LESCs) are a population of stem cells responsible for maintenance and repair of the corneal surface. Injury and disease can result in a deficiency of these stem cells, the vision affecting condition called limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy was first described in 1997, and LESCs cultured from either patients or donors have been used to successfully treat LSCD. In this review, some of the challenges and controversies associated with cultured LESC therapy will be discussed including alternative stem cell sources.

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.

Aim: To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. Methods: HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. Results: HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. Conclusion: HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

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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.

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilises amniotic membrane as a cell support and/ or murine 3T3s as feeder fibroblasts. The aim of this study was to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real Architecture for 3D Tissue (RAFT) was used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) were used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents were produced and characterised: firstly, for patients with bilateral LSCD, an oral mucosal tissue

Supplementary data Table 1. Stevens Johnson syndrome patients extra data. Donor HOMF isolated? Medicines 1 Yes Not on any systemic treatment at time of biopsy but had previously been on treatment with Azathioptine, systemic steroid (oral prednisolone) and oral Mycophenolate. 2 No No systemic medicines related to eye condition. Had had a bone marrow transplant and also had graft versus host disease. Previous oral surgery. 3 No No systemic medicines of note.