Characterisation of the porcine corneal epithelial stem/progenitor cell niche

It is widely accepted that the surface epithelium of the cornea is maintained by a population of stem cells that resides in the corneoscleral limbus. However, the extracellular matrix and surrounding cells that maintain this stem cell population and constitute the niche of these cells are not fully understood. The aim of this thesis was to further characterise the extracellular matrix, specifically the chondroitin sulphate/dermatan sulphate distribution, and cellular components of the limbal epithelial stem cell niche using a porcine model. This was done using immunohistochemistry, transmission electron microscopy, scanning electron microscopy and creating 3D tissue reconstructions via serial block-face scanning electron microscopy (SBFSEM) and X-ray micro-computed tomography (microCT). This thesis was the first to investigate using microCT to image the limbal/corneal epithelium and found the porcine cornea has one or more continuous limbal troughs that span across all limbal quadrants, as opposed to small, discrete limbal crypts as found in humans. These elongated projections of the limbal epithelium into the stroma contain basal epithelial cells positive for the putative limbal stem cell markers cytokeratin 19 and ABCB5. The stroma immediately subjacent to the limbal trough is rich in the chondroitin sulphate sulphation motifs recognised by antibodies 6C3 and 3B3(+), as well as hyaluronic acid. The distribution of sulphated proteoglycans varies between the stroma and epithelial basement membranes of the porcine central cornea versus the limbus. SBFSEM reconstructions demonstrate direct cell-cell contact between stromal niche cells and limbal basal epithelial cells in the porcine cornea, the third mammalian species to show this. This thesis has shown marked differences in the limbal stem cell niche of the porcine eye from the human, but also confirming some shared characteristics. The newly identified limbal trough forms part of this niche, playing host to putative stem cells, where forms of chondroitin sulphate are in close proximity to these cells and stromal-epithelial cell interaction exists. These factors may play a role in stem cell maintenance in vivo in the porcine cornea and also could have uses in the culturing of limbal stem cells and future niche reconstruction.