Creation of a Humanized Mouse Model to Study Islet Graft Rejection
Creators: Shayeganpour, Kasra
Publisher: name: University of Alberta Library
Year: 2022

<p>Background<br>
Intraportal islet transplantation is a suitable method to treat type 1 diabetes. However, this treatment option is limited to a small subpopulation of patients with diabetes. For islet transplantation to be a more sustainable treatment option, major obstacles must be overcome. An alternative supply of islets must replace the unsustainable human islet source and an alternative method must be developed reduce the usage of systemic immunosuppressive medications. Before alternative islet sources and novel immunosuppressive strategies can be used in clinical practice, it is important to conduct immunological studies using a variety of islet sources in a reliable model that realistically mimics the human immune system.<br>
To achieve this, an appropriate humanized mouse model must be developed, and quantitative methods must support the rejection of a variety of islet sources. In addition, it is important for qualitative observations to further reinforce the quantitative analytical observations. The main objectives of this thesis are twofold: 1) to use NSG-MHC I/II double knockout (NSG) mice to support the survival of human (HI), neonatal porcine (NPI), and stem cell-derived islets (SC-β), and 2) to display islet graft rejection in these mice via a reversal to the hyperglycemic state, or through functional rejection of stimulated insulin secretion, after reconstitution with human peripheral blood mononuclear cells (PBMCs) without graft-vs-host disease (GVHD).</p>

<p>Methods<br>
A brief investigation was conducted to assess the native immune system in mice using Immunofluorescence (IF). A random selection of HI transplanted mice was stained using anti-mouse CD4, CD8, and CD68 T-cells and human insulin. Metabolic follow-up in streptozotocin- induced diabetic mice transplanted with HI and SC-βs was determined by examining blood glucose values pre- and post-reconstitution, and a reversal of blood glucose to hyperglycemic levels indicated islet graft rejection. Metabolic follow-up in naïve, non-diabetic mice transplanted with NPIs was determined by comparing a 4-week post-transplantation intraperitoneal glucose tolerance test (IPGTT) and at 2-, 4-, 5-, and 6-weeks post-reconstitution IPGTT and stimulated serum porcine insulin was obtained and analyzed. Stimulated porcine insulin values at or near the lowest limit of detection on the enzyme-linked immunosorbent assay (ELISA) curve for both basal (time 0) and stimulated (time 60) levels were considered as complete porcine islet graft rejection.<br>
To determine the possibility of GVHD in these mice, the weight values of naïve, non- diabetic NPI transplanted mice were taken three times weekly and compared to baseline values. Loss of more than 20% of initial weight indicated the presence of GVHD.<br>
To demonstrate the ability of SC-β transplanted mice to secrete insulin, an IPGTT was conducted at 8- and 12-weeks post-transplantation prior to reconstitution, and stimulated insulin values were obtained. Further exploration of the cell composition of SC-βs and outcome of transplantation were explored in Appendix A.<br>
Immunohistochemistry (IHC) and IF imaging was used to determine the presence of human immune cell infiltration in the graft region as well as co-localization of human immune cells in relation to insulin or chromogranin A using mice in all three groups (HI, NPI, SC-β).</p>

<p>Results<br>
Investigation of the native immune system displays an absence of mouse CD4+ and CD8+ T-cells. CD68+ T-cells were identified, though are stated to be functionally defective in these mice. Prior to reconstitution, mice transplanted with HI and SC-β demonstrated graft functionality through normalized blood glucose values. Post-reconstitution, HI and SC-β transplanted mice displayed a reversal to hyperglycemia between 5 to 18 days and 21 to 23 days, respectively. IHC and IF imaging confirmed the infiltration and co-localization of human immune cells in relation to insulin positive cells in the kidney graft. 53.8% of NPI transplanted mice displayed complete islet graft rejection at 6-weeks post-reconstitution. Mice did not decrease their weights by more than 20% from baseline values indicating the absence of this GVHD symptom. Co-localization studies confirmed the infiltration and human immune cells in relation to chromogranin A positive cells.</p>

<p>Conclusions<br>
These results display the feasibility of transplanting a wide variety of islet sources in NGS mice. Results demonstrate that reconstitution of mice with peripheral blood mononuclear cells exhibits islet graft rejection and that this mouse model can be adapted for future strategies, including the use of hypoimmunogenic stem cells and encapsulation strategies.</p>