Characterisation of limbal stem cells

Abstract

The cornea is the anterior most structure at the front of the eye and its most important function is to transmit light to the retina for a clear vision. The external surface of the cornea is composed of an epithelium which is continuous with the surrounding conjunctiva. The corneal limbus forms the narrow transition zone between the corneal and conjunctival epithelia and is believed to harbour the cornea stem cells. Limbal stem cell (LSC) deficiency (LSCD) is a painful and blinding condition of the eye. The recent technique of ex vivo expansion of healthy autologous limbal epithelia for transplantation is the mainstay of cellular therapy for this condition. Successful outcome of ex vivo expanded LSC transplantation is dependent on the quality of the transplanted tissues. The main aims of this thesis are; i) to characterise human LSC from both primary LEC (LEC) and from a human telomerase-immortalised corneal epithelial cell (HTCEC) line, ii) to enrich for LSC by utilising the side population (SP) discrimination assay. Further, adult stem cells are promising candidates for promoting donor-specific tolerance and adult stem cells have immunosuppressive mechanisms to protect them from immunological reactions which are damaging to the survival of the transplanted tissues. Therefore, another aim of the study was iii) to investigate the immunobiological aspects of LSC including HLA expression and cellular migration which can promote the success of cellular transplantation. LSC markers were characterised in both LEC and HTCEC. Using an optimised protocol, SP were identified in LEC and HTCEC. Isolated limbal mesenchymal stem cells (MSC) fulfil the minimum requirement of a human MSC. These limbal MSC (LMSC) exhibited plasticity, could maintain the expression of limbal markers and demonstrated viable growth on a biological substrate, qualities making them a suitable alternative to cultured limbal explants for clinical transplantation. SP cells in HTCEC and LMSC expressed known common limbal markers, stem cell antigens and the chemokine CXCR4. The presence of CXCR4 and CXCL12-mediated cellular migration were demonstrated in HTCEC. Further, HTCEC constitutively expressed HLA Class I antigens, while HLA Class II expressions were induced by Interferon-γ stimulation. In this study, HTCEC HLA-typing was presented and that HTCEC were in many ways comparable to LEC, therefore suitable as a LSC replacement or as a robust model for further understanding of LSC biology.