Strategies to Augment Survival and Engraftment in Islet Transplantation

Type 1 diabetes mellitus (T1DM) is characterized by the autoimmune destruction of insulin-producing beta (β)-cells within pancreatic islets of Langerhans. For many patients, frequent blood glucose monitoring and insulin administration are primary therapies aimed to curb hyperglycemia, though long-term complications may still occur. β-cell replacement therapy through islet transplantation has become an accepted treatment modality for select patients suffering from “brittle” T1DM. The establishment of the ‘Edmonton Protocol’ in 2000 was instrumental in renewing global interest in utilizing islet transplantation as an effective therapy for such patients. Over the course of the last two decades, considerable refinements in islet isolation and culture techniques, as well as the incorporation of anti-inflammatory and novel immunosuppressive therapies have improved long-term islet transplantation outcomes. Despite such refinements, obstacles associated with islet transplantation still exist, as single-donor insulin independence remains elusive, warranting further investigation. This thesis presents results from multiple studies aimed to augment in vitro islet survival to potentiate subsequent engraftment. I hypothesize that the administration of therapeutic agents during pancreas procurement, islet culture and/or in the acute post-transplant period can improve in vitro and in vivo islet function. The thesis is laid out in a paper-based format, based on manuscripts published or under review. Sufficient background is provided to gain an understanding of islet transplantation, beginning with a historical perspective, as well as current limitations and therapeutic interventions employed pre-clinically and clinically to circumvent such obstacles. The reader is also provided with details of various regulated cell death pathways, those long-existing, as well as newly defined pathways yet to be fully elucidated in islet transplantation. We present our research to enhance islet viability and potency in vitro through the administration of a novel manganese superoxide dismutase (SOD)-mimetic, the metalloporphyrin BMX-001, during organ procurement and 24-hour culture, as well as evaluate whether preserved potency enhances engraftment outcomes in a syngeneic, marginal mass model. We observed the ability of this SOD-mimetic to indeed augment in vitro islet function and viability when administering a physiologically relevant dose of 34μM BMX-001. BMX-001-treated islets also exhibited improved engraftment outcomes relative to non-treated control islet recipients. As a means to expand the availability of donor pancreases, we further developed a murine donation after circulatory death (DCD) model, and further assessed the utility of BMX-001. The findings from this model were striking, notably 15 minutes of warm ischemia (WI) significantly impaired islet isolation yields in mice. The administration of BMX-001 during pancreas procurement did not significantly increase islet yield or viability relative to control DCD islets. BMX-001-cultured DCD islets demonstrated a significant decrease in extra cellular ROS, suggesting some cytoprotection. DCD murine islets, regardless of BMX-001 administration were able to engraft at a similar rate to islets isolated from murine pancreata that did not experience WI. Shifting focus to cell death in islet transplantation, the novel pan-caspase inhibitor, F573 was investigated. Murine and human islets cultured for 2 or 24 hours, respectively with or without F573 exhibited a marked reduction in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 activation in vitro. Human islet engraftment was significantly enhanced in F573-treated recipients relative to non-treated controls. Moreover, F573 was able to augment engraftment in a significant proportion compared with non-treated control recipients in the modified subcutaneous, device-less site, further supporting the potential of this site as an alternative to intra-portal islet infusion. To expand on the current knowledge of regulated cell death mechanisms that may contribute to islet dysfunction and compromise subsequent engraftment, ferroptosis, a non-apoptotic cell death modality was investigated in human islets. Utilizing ferroptosis inducing agents, erastin and RSL3, islet viability and function was compromised in vitro. These effects were abrogated in the presence of the ferroptosis-specific inhibitor, ferrostatin-1, thus confirming that this cell death modality can contribute to islet demise. Further work evaluating alternative regulated cell death mechanisms in islet isolation and transplantation may lead to improved adjuvant therapies to deter islet dysfunction and subsequently improve long-term engraftment outcomes. Taken together, the results of this body of work reveals that preserving pre-transplant islet potency through the utility of novel agents administered during procurement or in culture can augment islet engraftment outcomes. Such agents hold promise as prospective adjuvant therapies to promote single-donor islet engraftment outcomes.

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