Beta-cell therapy for daibetes :development of quality assured transport of human islets for transplantation at remote centres; investigation of augmentation of human islets ex vivo; and exploration of the potential for expansion and redifferentiation in vitro

PhD ThesisBeta cell replacement is the only therapy currently able to restore insulin independence in C-peptide negative Type 1 diabetes. This can be achieved through either vascularised whole pancreas transplantation or isolated islet transplantation. Whole pancreas transplantation can deliver long-term normalisation of blood glucose but nevertheless is associated with significant peri-operative morbidity and mortality. Islet transplantation is a much less invasive procedure which can provide complete resolution of disabling hypoglycaemia with potential insulin independence. However, at the outset of this research, this procedure was restricted to UK centres with islet isolation facilities. Two main hurdles prevent wide application of this treatment modality. First, a limit to the number of isolation facilities that can be established. These are sophisticated and very expensive laboratories and require extensive staff training and commitment. Second, there is significantly limited supply of suitable donor pancreases meeting less than 1% of the potential clinical need. The goal of this PhD project was to address, from the UK perspective, the limitations in transplantation sites and scarcity of suitable islets for clinical transplantation maintaining a clinically relevant focus through the study of primary human islets. The overall aims were: 1. To demonstrate safe and effective islet transport despite relatively long journey time in the UK without need for complete revalidation / repreparation for transplant at satellite site. 2. To evaluate potential for enhancing mass and function in intact human islets including the potential of in vitro incubation with pregnancy-related hormones 3. To evaluate potential for in vitro expansion of human pancreatic cells with determination of whether a functional beta-cell phenotype can be maintained over passage and whether putative pancreatic stem cells can be identified in culture. 4. To determine potential for redifferentiation of expanded human pancreatic cells towards a functional beta-cell phenotype by in vitro pseudo-islet formation. III A safe, practical and clinically viable islet transportation system was successfully established to suit the UK setting. This system enabled efficient utilisation of central islet isolation facilities for production of clinical grade high quality islets for transportation to several islet transplantation centres. Cooling a relatively small volume of high density islets in transport bags enabled transported islets to be transplanted at the satellite centres without any further manipulation and repreparation. This work underpinned government funding of a national islet transplantation program and led to the first successful UK islet transplantation of transported islets. Validated standard operating procedures (SOPs) created in this project were adopted nationally by all islet receiving centres. Augmentation of islet mass and function in intact primary human islets was attempted following treatment with pregnancy related hormones and a panel of other growth factors. In contrast to previously published reports on rodent islets or adherent human islets, these studies confirmed that growth factor treatment can only maintain as opposed to increasing islet mass or function in intact human islets. Nevertheless, prolactin induced a significant increase in insulin expression and potentiated insulin secretion in response to elevated glucose concentrations. Proliferative cultures from pancreatic islets were established and characterised achieving high proliferative capacity with potential to produce transplantable mass sufficient for several recipients from a single donor. Despite maintenance of differentiated phenotypic markers in early passages there was an accelerated loss of β-cell function. Nevertheless expression of pluripotency-associated markers was identified for first time suggesting stem cells residing in adult pancreas. Potential for enhanced β-cell function in islet proliferative culture with minimal manipulation was demonstrated through pseudo-islet formation. This was associated with down-regulation of pluripotency-associated markers and enhanced β-cell function in vitro IV and limited, but confirmed activity, in vivo. However, currently this remains limited and insufficient for clinical impact. In conclusion, validation of an effective and safe islet transport system was achieved underpinning unique National Health Service funding of a UK national clinical programme of islet transplantation and enabling the first successful UK transplantation of transported islets. Definitive studies to demonstrate functional enhancement in human islets in response to pregnancy related hormones and/or other factors would require toxic pre-conditioning. Significant islet mass expansion was attained in vitro with potential functional enhancement with pseudo-islet formation. However, further differentiation studies and validation of a practical large scale culture system remain an important requirement for any potential future clinical application

Validation of Islet Transport From a Geographically Distant Isolation Center Enabling Equitable Access and National Health Service Funding of a Clinical Islet Transplant Program for England

Islet transplantation has become established as a successful treatment for type 1 diabetes complicated by recurrent severe hypoglycemia. In the UK access has been limited to a few centrally located units. Our goal was to validate a quality-assured system for safe/effective transport of human islets in the UK and to successfully undertake the first transplants with transported islets. Pancreases were retrieved from deceased donors in the north of England and transported to King’s College London using two-layer method (TLM) or University of Wisconsin solution alone. Islets were isolated and transported back to Newcastle in standard blood transfusion or gas-permeable bags with detailed evaluation pre- and posttransport. In the preclinical phase, islets were isolated from 10 pancreases with mean yield of 258,000 islet equivalents. No significant differences were seen between TLM and University of Wisconsin solution organ preservation. A significant loss of integrity was demonstrated in islets shipped in gas-permeable bags, whereas sterility, number, purity, and viability were maintained in blood transfusion bags. Maintenance of secretory granules and glucose-stimulated insulin secretion was confirmed following transport. A Standard Operating Procedure enabling final pretransplant quality control from a simple side-arm sample was validated. Moreover, levels of insulin and cytokines in transport medium were low, enabling transplant without centrifugation/resuspension at the recipient site. Six clinical transplants of transported islets were undertaken in five recipients with 100% primary graft function and resolution of severe hypoglycemia. Safe and clinically effective islet transport has been established facilitating sustainable NHS funding of a clinical islet transplant program for the UK