The enhanced generation of corneal epithelium from iPSCs using chondroitin sulphate as a substrate

Ashworth, Sean 2022. The enhanced generation of corneal epithelium from iPSCs using chondroitin sulphate as a substrate. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Proteoglycans are a type of extracellular matrix molecule consisting of a protein core with several covalently attached glycosaminoglycan chains (negatively charged polysaccharides). Certain glycosaminoglycan chains, for example chondroitin sulphate (CS) and dermatan sulphate (DS), can be sulphated in specific patterns that confer unique properties. These sulphation patterns and their distributions play an important role in the development and maintenance of stem cell niches in a wide range of tissues, including the cornea. Many tissue engineering approaches use native extracellular matrix cues extracted from the resident tissue to enhance generation and maintenance of therapeutic cells. However, there are few studies detailing specific glycosaminoglycan species in this process. This is especially true in the study of the generation of corneal epithelial cells from induced human pluripotent stem cells (hiPSCs). The aim of this thesis was to examine the effect of native glycosaminoglycan sulphation patterns on the generation of corneal epithelial cells from hiPSCs, using a defined differentiation method. The objectives of this thesis were to, firstly, identify specific CS and DS sulphation patterns present in the stem cell niche of the cornea and those associated with the corneal epithelium using CS/DS antibodies and immunohistochemistry. Following this, identified corneal epithelium-associated sulphation patterns were extracted from different tissue sources and enriched for sulphation patterns of interest using density centrifugation and anion exchange chromatography. Finally, these substrates were applied to hiPSCs and subsequently differentiated toward corneal epithelial cells and the gene expression was examined using RT-qPCR, along with morphological and immunohistochemical evaluation. From this study, it appeared a mixed substrate consisting of highly sulphated keratan sulphate, and CS/DS enriched in sulphation patterns recognised by the antibodies 5D4 and 7D4, yielded best potential enhancement of corneal epithelial differentiation as identified by the methods described herein. Other native sulphation patterns also revealed significant effects on gene expression.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Optometry and Vision Sciences
Subjects:	R Medicine > RE Ophthalmology
Uncontrolled Keywords:	cornea, induced pluripotent stem cell, corneal epithelium, proteoglycans, glycosaminoglycans
Date of First Compliant Deposit:	23 January 2023
Last Modified:	20 Jan 2024 02:30
URI:	https://orca.cardiff.ac.uk/id/eprint/156121

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