Dziasko, MA; (2016) Localisation of corneal epithelial progenitors and characterization of cell-cell interactions in the human limbal stem cell niche. Doctoral thesis , UCL (University College London).

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Abstract

The cornea, the transparent tissue located at the front of the eye, is a highly specialized tissue that transmits and refracts light onto the retina. Maintenance of the corneal epithelium relies on a population of limbal epithelial stem cells (LESCs) that maintain transparency of the ocular surface that is essential for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. After observing a high population of basal epithelial cells expressing stem cell markers within the previously identified limbal crypts (LC), the first aim of this study was to demonstrate by in vitro clonal analysis that these structures provide a niche for the resident LESCs. High-resolution transmission electron microscopy has been further used to image the basal epithelial layer at the limbus. Cells with morphology consistent with stem cells were present within the basal layer of the limbal crypts but not within the basal layer of non-crypt limbal biopsies. Moreover, LESCs appeared proximal to limbal stromal cell extensions that suggested a possible route for direct cell-to-cell interaction. These observations were further confirmed by serial block-face scanning electron microscopy that revealed, for the first time, direct epithelial-stromal interactions in the LESC niche whereas limbal melanocytes maintained the LESC apically. In order to assess the role of limbal melanocytes (hLM) as niche cells for the maintenance of LESC, a novel co-culture system was developed in which hLM were used as a feeder layer for the expansion of limbal epithelial cells in vitro. Interestingly, hLM had the ability to support the clonal growth of LECs that maintained stem cell-like characteristics in 2D and 3D tissue equivalents. Taken together, these observations suggest an important role for melanocytes as niche cells in the native human limbal crypts.

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