Neonatal Porcine Islet Xenotransplantation: Non-invasive in vivo imaging and basic islet biology
Creators: Purich, Kieran C.
Publisher: name: University of Alberta Library
Year: 2022

<p>Islet transplantation has been proven as a successful clinical therapy for diabetes; however, its widespread use is limited by multiple factors including a shortage of human donors, the need for long term immunosuppression and difficulty with graft monitoring post transplantation. Pig islet xenotransplantation is a promising solution to several problems faced by islet transplantation as it would directly address the limited number of islets available for transplantation and provide a tissue source deemed more appropriate for genetic experimentation. Significant research has been completed on genetic modification of pigs to optimize their tissues for transplantation; however, minimal previous work has been completed looking at the basic biology and development of pig islets, limiting the transition from animal models to the clinical realm.<br>
    In this thesis, we perform two series of experiments exploring some of  the aforementioned challenges. In Chapter 2 we study a novel synthetic polymer, polyvinylpyrrolidone (PVP), which is used in the imaging of islets following transplantation. Polyvinylpyrrolidone has previously proven useful in the coating of superparamagnetic iron oxide (SPIO) nanoparticles (deemed PVP-SPIO nanoparticles) which can then be utilized in the labelling of cells, including mouse islets. These labelled islets allow for greater resolution on MRI, allowing the ability to follow islet grafts post transplantation in animal models. We are the first to experiment with PVP-SPIO in pig islets, and we show that it can be used to label neonatal pig islets, improving their contrast on MRI imaging. We also demonstrate that PVP-SPIO can be used to improve contrast on MRI imaging when the islets are situated under the kidney capsule in a diabetic mouse model, while not impacting the islet’s ability to reverse diabetes. Furthermore, our data demonstrates an expansion of PVP-SPIO labelled islet grafts on MRI following graft rejection, suggesting PVP-SPIO nanoparticles may hold promise in the ability to non-invasively detect graft rejection.<br>
    In Chapter 3, the primary chapter of this thesis, we assess the gene and protein expression of neonatal pig islets in the early post-natal period by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western immunoassay. Primary pathways of interest span cell-cell adhesion and insulin secretion. Specific molecules of interest include: adhesion molecules E and VE-cadherin as well as glucose transporter GLUT-2, GTPase RAC1 and SNAP25 a protein involved in insulin exocytosis. We perform an exploratory analysis identifying trends seen in different ages of neonatal pigs across different days in culture.<br>
    Following gene and protein assessment, we assess islet function, demonstrating differences in the insulin secretory capacity of islets obtained from different ages of neonatal pigs. We examine islet function in vitro as well as in vivo using a diabetic mouse model. Specifically, we compare islets obtained from 1-day-old neonatal pigs to those obtained from 3 and 7-day-old neonatal pigs, identifying differences in function, highlighting the potential of dysregulated function in islets obtained from 1-day-old pigs, suggesting that they may not be the optimal age for neonatal pig islet recovery. Finally, in an attempt to connect cell-cell adhesion and the insulin secretion pathway, we demonstrate the loss of islet function at high glucose conditions when islets are treated with anti E-cadherin monoclonal antibody, suggesting its importance in stimulated insulin secretion. As far as we are aware, the importance of E-cadherin in neonatal pig islets has not been previously reported.<br>
    This study serves as a starting point, and further studies to investigate the physiologic pathways which direct early neonatal pig islet cell development are warranted.</p>