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Gene expression profile of cells in successive stages of corneal stem cell lineage

Ballis, Andreas. 2006. Gene expression profile of cells in successive stages of corneal stem cell lineage. PhD Thesis, Cardiff University.

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Abstract

In this study the complete transcriptome of groups of cells at specific successive stages of the complete corneal stem cell hierarchy was revealed. The cornea presents a linear differential distribution of each type of cell of this hierarchy, with stem cells, transient amplifying cells and mature cells residing predominantly in the basal limbal, peripheral and central corneal epithelium respectively. In order to realise the complete set of genes that are up or down regulated at each stage of the corneal stem cell lineage, a Laser Micro-dissection and pressure Catapulting method was optimised, that allows for isolation of the desired type of cell from the specific areas they predominate, in a manner that would not challenge the integrity of their mRNA, as determined by 3'-5' relative ratios, estimated by semi-quantitative RT-PCR. To analyse the relative abundance of every gene in each of the cell types that were isolated, a linear amplification of mRNA method had to be optimised, as determined by comparing the relative abundances of specific endogenous and exogenous gene transcripts before and after the amplification reaction, using high density oligonucleotide arrays. In order to amplify the mRNA in such a manner and to such a degree that it could be analysed by high density oligonucleotide arrays an in-vitro transcription based amplification method was employed and optimised. The method entailed the generation of double stranded cDNA reverse transcripts carrying the T7 RNA polymerase promoter and subsequent in-vitro transcription that yielded large amounts of linearly amplified mRNA (aRNA) The midfield of data that was produced was analysed by appropriate mathematical methods such as Robust Multiarray Analysis and in order to obtain a set of genes that are up or down regulated specifically in each cell type. Principal Component analysis confirmed the validity of the hypothesis that the variance in gene expression arose from the fact that different types of cells were analysed The results were validated by semi-quantitative RT-PCR analysis, which confirmed the sensitivity of the arrays. Additionally several protein targets that were indicated by the array analysis were studied by immunohistochemical methods. The putative differential mechanisms regulating corneal epithelial stem and well as transient amplifying cell fate and corneal homeostasis are discussed. The results of this study are likely to augment the efforts of understanding corneal epithelial stem cells and possibly other adult stem cells and thereby assist in future research and therapeutic interventions involving stem cells.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Optometry and Vision Sciences
Subjects: Q Science > Q Science (General)
ISBN: 9781303205095
Date of First Compliant Deposit: 30 March 2016
Last Modified: 31 Jan 2020 07:42
URI: http://orca.cf.ac.uk/id/eprint/56073

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