Kokkinopoulos, loannis; (2008) Characterisation of neural progenitors from the adult retina and ciliary epithelium. Doctoral thesis , University of London.

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Abstract

The mammalian central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. However, retinal stem cells have been isolated from the ciliary epithelium of the mammalian retina. ChxW is a paired-like homeobox transcription factor gene expressed in the presumptive neural retina of the invaginating optic vesicle. The Chx10 gene is expressed in the proliferating retinal progenitor cell population throughout retinal development hence is one of the earliest characterised RPC-specific markers. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, it was showed that in the ocular retardation mouse model lacking Chx10 (Chx1(frJ/orJ), dividing cells persist in the adult CNR, suggesting the existence of a dormant stem/progenitor population. The neurosphere-forming assay is a tool which has allowed scientists to study the behaviour of neural stem/progenitor cells in vitro. Here, I show that cells deriving from the CNR of the adult microphthalmic retina are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Muller glial cell markers. My results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR possible deriving from the abnormal appearance of GFAPpos Muller glia in late embryonic stages of the Chx1(frJ/orJ retina. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors. One of the cardinal features of stem cells is their differentiation potential and multipotency. My experiments illustrate that Chx10 rJ/orJ CNR-derived neurospheres are able to differentiate in a similar fashion to wildtype CE-derived neurospheres. Furthermore, when neurospheres lacking Chx10 are placed in conditions that promote differentiation, they significantly up-regulate the expression of photoreceptor genes in comparison to wildtype. Hitherto, the developmental origin of CE-derived neurosphere-forming retinal stem cell is unclear. The ciliary body, where the CE is located in adult mammals, includes cells of mesodermal, neural crest and neural ectodermal origin. Here, data collected from lineage tracing analysis and in vivo Brdll-tagging experiments suggest that neurospheres are formed from BrdUpos cells observed in vivo, and that these cells originate from the embryonic anterior forebrain. The comparative analysis of the microphthalmic CNR retinal progenitors and CE-derived progenitors provides valuable information on cell properties relevant for potential cell-based replacement therapies, as well for retinal regeneration potential in mammals.

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