Enhanced generation of human induced pluripotent stem cell (hiPSC)-derived corneal epithelium using extracellular matrix molecules

Wang, Yixin 2025. Enhanced generation of human induced pluripotent stem cell (hiPSC)-derived corneal epithelium using extracellular matrix molecules. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The eye is a very complicated organ with highly specialized constituent tissues derived from different primordial cell lineages. For example, when we consider the cornea, which is the front clear window of the eye, it is known that the corneal epithelium is descended from surface ectoderm, while corneal keratocytes and the endothelium derive from neural crest cells. Current research on human induced pluripotent stem cells (hiPSCs) is highly influential in the biosciences field, as well as in the translational eye research field. However, there are sizable gaps in our knowledge regarding the technologies of differentiating hiPSCs into different organoid tissues of the eye, which warrants further examination. This thesis considers the possible influence of proteoglycans (PGs)-a type of extracellular matrix (ECM) molecule typically consisting of a protein core with many covalently attached glycosaminoglycan (GAG) chains (largely consisting of sugars or polysaccharides) - on the pattern of hiPSC growth in culture into eye-like cells. GAG chains have specific sulphation motifs and variable distributional patterns along the polysaccharides within the chains. The sulphation motifs play an important role in the development and maintenance of various stem cell niches in a range of tissues. Certain GAG chains like keratan sulphate (KS), have been shown to have specific sulphate motifs or patterns on polysaccharides within the chains. This thesis describes a series of experiments which studied the possible influence of KS GAGs, along with GAGs of the chondroitin sulphate/dermatan sulphate (CS/DS) variety, on the growth of hiPSCs into what my collaborators in Osaka University, Japan, discovered as SEAMs (Self-formed, Ectodermal, Autologous, Multi-Zones) that are 2D assemblies of cells with four concentric zones, which each resembles a specific eye-type cell. It is known that exogenous factors, including those of the ECM (laminin, in particular) can direct the fate of hiPSCs and the type of SEAM formed. The hypothesis tested here is that GAGs used as substrates for hiPSC growth into SEAMs (with and without laminin present) might impact the development. The GAGs used to grow SEAMs were partially and fully purified ones that I extracted from porcine corneas, along with GAGs provided by experts in the National Glycoengineering Research Centre, China. First, procedures were fairly extensively explored to optimise the protocol of GAG extraction and purification, after which SEAM growth on the GAG as substrates was assessed via morphological inspection, immunofluorescence microscopy using monoclonal antibodies to variously sulphated GAGs and putative stem cell markers and RT-PCR. As an additional analysis (presented as an appendix), results were assessed through some single-cell RNA sequencing data on SEAM growth provided by my colleague, Dr Laura Howard, and parallels were found. This novel approach has identified two candidate stem cell-associated sulphation motifs, 5D4 and 1B4, which could potentially facilitate the expansion of iPSC-derived corneal epithelial tissue following extraction and purification. Specific details are presented with clear indication that GAGs do influence the growth of hiPSCs into SEAMs, with further work required to understand the mechanism of action and GAG/laminin combinations best suited for various types of SEAM formation.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Optometry and Vision Sciences
Subjects:	R Medicine > RE Ophthalmology
Date of First Compliant Deposit:	16 June 2025
Last Modified:	26 Jun 2025 08:45
URI:	https://orca.cardiff.ac.uk/id/eprint/179005

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