Development of a topical therapeutic strategy using corneal derived mesenchymal stromal cells for the treatment of ocular surface disorders

Beeken, Lydia (2023) Development of a topical therapeutic strategy using corneal derived mesenchymal stromal cells for the treatment of ocular surface disorders. PhD thesis, University of Nottingham.

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Ocular surface inflammatory disorders can lead to serious complications, including vision loss. Current treatments, including eye drops and steroids, are not always effective and can have negative side effects. Mesenchymal stromal cells (MSCs) have shown potential as a regenerative medicine strategy due to their ability to suppress inflammation and promote wound healing. While bone marrow derived MSCs (BM-MSCs) are considered the "gold standard" for MSC therapies, they have limited availability and require invasive harvesting. Alternatively, MSCs from the corneal limbus (CMSCs) can be obtained from corneal transplant surgery waste tissue or eye banks. However, these cells lack necessary characterisation data for clinical translation. This research aimed to extensively characterise CMSCs and assess methods for topical administration of the cells for ocular surface disorders, through functionalisation of the contact lens material poly(HEMA-co-EGDMA).

Changes in CMSCs over passages were assessed to determine the effects of cell culture over time. Flow cytometry with the BD Human Cell Surface Lyoplate was used to assess donor-to-donor variation. Markers homogeneously expressed (low Median Fluorescent Intensity (MFI) variation and > 95% of CMSCs) included CD59, CD81, CD13, CD90, CD63, HLA-A,B,C, CD9, CD147, CD140b, CD47, CD73, CD105, CD49b, B2-MG, CD26, CD55, CD46, and CD49e which could be used to identify CMSCs. Markers heterogeneously expressed across donors may be linked to differences in cell safety and efficacy, and could be used for donor screening.

The CMSC phenotypic results were compared to BM-MSCs, identifying CD49b, CD49e, CD81, CD9, CD151, CD140b, CD99, CD47, CD147, CD63, CD95 and CD98 as homogeneously expressed (low MFI variation and > 85% expression in CMSCs and BM-MSCs). Significant differences in expression were observed for CD40, CD121a, CD108, CD49d, CD142 and HPC. Medium throughput genotypic profiling was performed using the Qiagen RT2 Profiler PCR Array, finding significantly higher gene expression in BM-MSCs for multiple markers including those associated with angiogenesis and coagulation and growth factors. Differentially expressed markers provide a reference point for future directions investigating safety and efficacy of CMSCs.

CMSCs were then exposed to inflammatory conditions to mimic the toxic environment of an inflamed ocular surface. Surviving CMSCs secreted an increase in multiple factors, including growth factors HGF, FGF2 and TGFß, which were not secreted by corneal epithelial cells, highlighting a higher specificity to CMSCs. Gene expression of extracellular matrix (ECM) structural constituents and cytoskeleton regulators were significantly reduced in cytokine exposed cells, potentially beneficial to avoid fibrosis. Phenotypic and genotypic profiles of CMSCs with a three-day recovery period following cytokine exposure provided an insight into the response of CMSCs to priming, with an increase in the expression of MIC A/B highlighting a risk of increased immunogenicity.

Finally, topical administration options were investigated, using techniques including rheology and equilibrium water content assessment. Successful cell attachment was observed through immunocytochemistry (ICC) and confocal imaging of CMSCs seeded on poly(HEMA-co-EGDMA) hydrogels functionalised for the first time with a resilin like polypeptide layer.

Overall, CMSC characterisation data identified a group of markers that can be used as a template for selecting directions for future functional investigations. This robust characterisation has advanced the biological understanding of CMSCs, in addition to being a substantial basis to aid successful translation of the cells to the clinic. Furthermore, this research demonstrated a novel method for topical administration of CMSCs to the ocular surface, through the development of a topical cell therapy for ocular surface disorders.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Sidney, Laura
Rose, Felicity
Alexander, Cameron
Keywords: Cornea, mesenchymal stromal cells, cell therapy, regenerative medicine
Subjects: W Medicine and related subjects (NLM Classification) > WW Ophthalmology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 73948
Depositing User: Beeken, Lydia
Date Deposited: 31 Jul 2023 04:41
Last Modified: 31 Jul 2023 04:41

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