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Title: A study of the variability in the molecular structure of the human pancreas with age and its effect on islet isolation and transplantation outcomes
Author: Spiers, Rebecca
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Pancreatic islet transplantation is a minimally invasive treatment that has the potential to reverse Type 1 Diabetes Mellitus in selected patients. Islets of Langerhans are isolated (extracted) from the pancreatic exocrine tissue using a collagenase and supplementary protease enzyme blend, which digests the extracellular matrix (ECM) scaffold surrounding human islets. However, the inefficiency of current enzyme blends to digest the pancreatic ECM from younger donors (those aged ≤35 years) has led to the underutilisation of donor pancreases from this age group. The hypothesis is that suboptimal digestion in younger donors is a likely consequence of age-related differences in the pancreatic ECM. However, this has not been previously studied in detail at the molecular level. The studies described in this thesis therefore aimed to define how the composition of the native pancreatic ECM is influenced by age and how this in turn affects the enzyme-mediated digestion of this structure. Initially, the UK donor data set was analysed to determine the impact of different donor factors on islet isolation and transplantation outcomes. Whilst the success of islet isolation was clearly shown to increase with donor age, islets from younger donors demonstrated enhanced post-transplantation graft function. High-throughput proteomics was then used to identify quantitative differences in native pancreatic matrix protein expression between younger (≤35 years old) and older (≥45 years old) donors. These novel studies identified substantial age-related differences in the pancreatic matrisome and demonstrated that a number of key matrix proteins, including several collagen and laminin chains, were significantly more highly expressed in younger donors. Subsequently, the impact of donor age on pancreatic matrix digestion was studied through the development of a slide-based digestion assay. This involved the treatment of pancreatic tissue sections with collagenase and neutral protease, at clinically relevant concentrations. Following enzyme treatment, immunostaining was used to assess the digestion of specific matrix proteins. Once assay reproducibility was confirmed, this technique successfully demonstrated that age significantly influenced the digestion of several key matrix proteins. These results were confirmed by using Raman microspectroscopy (RMS) as a novel label-free technology to study ECM digestion. Furthermore, RMS indicated that age affected the action of collagenase to a greater extent than neutral protease. The studies detailed in this thesis provide novel insights into the impact of donor age on the composition and digestion of the pancreatic ECM. Continued detailed analysis will aid in the development of targeted donor-specific enzyme blends to dramatically improve the overall efficiency of human islet isolation and transplantation.
Supervisor: Johnson, Paul R. V. ; Cross, Sarah E. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Extracellular matrix ; Collagenase ; Human islet transplantation ; Collagen ; Human islet isolation