Corneal degenerative diseases and opacity are leading causes of corneal impairment and blindness worldwide. Like many epithelial tissues, the constant renewal of transparent corneal epithelial cells is essential for a lifelong healthy cornea and optimal vision. The limbus (the boundary between the cornea and the conjunctiva) is believed to be the site that harbours adult stem cells responsible for corneal maintenance and repair after injury, referred to as limbal epithelial stem cells (LESCs). In the basal limbal epithelium, an active LESC subset divides to yield progenitor cells that migrate centripetally into the corneal epithelium for cell renewal. This asymmetric division however, is assumed to be regulated by a balance between cell renewal and loss of cells from the corneal surface. The search for specific LESC molecular markers has been difficult and to date there are few if any candidate markers that unambiguously identify LESCs but not their immediate progeny. Consequently, LESC clonality, activity and proliferative dynamics have remained poorly understood. In addition, the nature of the regulatory molecular pathways involved during LESC activity is still an open key question. In this research project, we identified stem cells on the ocular surface of the eye, assayed their activity and demonstrated quantitively for the first time how the cornea responds to damage. The retention of DNA labelling reagents in control and wounded corneas was combined with clonal analyses of cell division and migration using mice mosaic for reporter LacZ expression. Corneal transplant in LacZ reporter transgenic mice showed migration of LacZ-positive cells into the donor corneal button, with long-term disruption of patterns of migration. Corneal epithelial scrape wounds at the periphery also showed long– term disruption. Label retention suggested a small but statistically significant proliferation response of LESCs to injury, but this was attenuated or absent in aging mice and Pax6 mutants. The Hippo signalling pathway has been shown to have promising results in regulating stem cell activity and proliferation in many organs, however, its effect on LESC proliferation is unknown. Here, we investigated the regulatory role of the Hippo−YAP signalling pathway during cell proliferation, and determined whether the label retention assay in a uniform population of dividing cells is a real indicator of slow-cycling cells in vivo. Cell-cycling kinetics, Abstract v proliferation rate, and label retention were determined in a spontaneous human corneal epithelial (HCE-S) cell line, using double-labelling IdU and EdU thymidine analogues. During homeostasis, HCE-S cells underwent approximately one cell cycle per day, however, cells in which YAP-dependent signalling was activated by 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (Hsp90), showed slower proliferation rate and longer cell-cycle time. In vitro label-retention assay in confluent cultures estimated number of ~3-4 cell cycles needed to dilute out the label from slow-cycling cells in vivo. The data are suggestive that the Hippo pathway has a potential regulatory role in proliferative corneal epithelium, and that label-retention assay is a real indicator of slow-cycling cells. Furthermore, this research observed the proliferative dynamics of conjunctival stem cells. Clonal analysis of patterns of cell growth in the conjunctiva were analysed following tamoxifen-induction of LacZ transgene activity. The long−term presence of coherent patches of LacZ-positive cells suggested the presence of long-lived conjunctival stem cells but that the turnover time for the bulbar conjunctival epithelium may be more than 16 weeks. The key results of this research are that the limbus is the niche for stem cells, that there is a unidirectional migration of cells from the limbus to the corneal epithelium during homeostasis, but this is disrupted, perhaps permanently, by wounding. We find only a mild response of limbal epithelial stem cells to acute corneal injury, which is reduced to no response with age and is dependent on genetic background.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:731630 |
| Date | January 2017 |
| Creators | Sagga, Nada A. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=235418 |
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