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Title: An in vitro investigation of human ocular surface epithelial stem cell homeostasis
Author: Ramos, T.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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The anterior surface of the eye is composed of the clear cornea centrally and the conjunctival and white sclera peripherally. The clarity of the cornea is of crucial importance for the transmission and focusing of light to the retina for visual perception. The limbus harbours the stem cells for corneal regeneration, the so-called limbal stem cells, and acts as a physical barrier preventing the conjunctiva and its blood vessels from encroaching onto the corneal surface. Upon injury or other inherited causes, the ocular surface homeostasis is impaired resulting in a migration of conjunctival epithelium onto the corneal surface, in a process called conjunctivalisation. Upon conjunctivalisation, which results in loss of corneal clarity and visual impairment, the conjunctival migrating cells can undergo a process of transdifferentiation towards cornea epithelial-like cells. This process of transdifferentiation seems to be incomplete, with the differentiated cells showing differences in glycogen metabolism, keratin profiling and tensile strength when compared to the native corneal epithelial cells. Limbal epithelial transplantation forms the mainstay of treatment in severe cases of such deficiency. However, two main hurdles stand for this treatment, including the requirement of large amounts of healthy tissue and the need of immunosuppression therapies in the case of allografts. Therefore, the emergence of cell-based therapies that potentially promote the complete transdifferentiation of conjunctival cells into corneal epithelial cells would overcome these two hurdles. This study aims to understand ocular surface and epithelial cell differentiation in response to the composition and morphogenic properties of the extracellular matrix (ECM). Cell cultures were treated with strong alkali solutions, by a process called "de-roofing", resulting in systems devoid of any cell and cell debris with only the respective ECM proteins attached to the cultureware. For the first-time, cells were driven to differentiate towards the lineage of the ECM depositing-cell. This process of differentiation required an intermediate step of cell dedifferentiation as suggested by the increased expression of early progenitor epithelial cell markers ?Np63 and ABCB5. The ECM composition and protein deposition profiles were extensively characterized to narrow the possible cues involved in the process of cell differentiation. Laminin-511 coated surface was shown to be a potential candidate to promote epithelial cell differentiation, with cell response shown to be concentration and time-dependent. Furthermore, the phosphorylation or cleavage levels of 18 different proteins, mainly involved in cell growth and differentiation, were assessed to understand the mechanisms behind this process. Additionally, and for the first-time, microvesicles were extracted from epithelial cells derived from human ocular epithelial cells. These microvesicles showed cargo containing several mRNA and miRNA molecules. Furthermore, the microvesicles were used to drive cell differentiation towards the lineage of the exosome-originating cells. Lastly, by replicating factors within the conjunctival and corneal epithelium environment, human embryonic stem cells were partially differentiated into conjunctival and corneal epithelial-like cells, respectively.
Supervisor: Ahmad, Sajjad ; Stewart, Rosalind ; Kaye, Stephen ; Hamill, Kevin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral