Reprogramming of Redundant Human Exocrine Tissue to Endocrine Tissue for Transplantation in the Treatment of Type 1 Diabetes

Type 1 diabetes affects 0.4 to 0.5% of the population and it is one of the commonest long term conditions in children and adolescents. A 70% increase in prevalent cases of type 1 diabetes in those aged under 15 is predicted in Europe between 2005 and 2020. Despite advances in the care of people with type 1 diabetes, the condition continues to be associated with substantial mortality and morbidity with an estimated shortening of lifespan on average of 15 years. Rates of cardiovascular disease are increased 3.6 fold in men and 7.7 fold in women. The avoidance of complications, such as visual loss or end stage renal failure, would be extremely cost effective, but even more importantly would contribute greatly to quality of life. Efforts to reduce such morbidity and mortality associated with type 1 diabetes are hampered by the limitations of current treatments which cannot reflect physiological insulin secretion from intact beta cells.
Pancreatic islet cell transplantation offers hope for patients with Type 1 Diabetes by replacing the insulin producing tissue and therefore lowering blood sugar levels. The islet isolation laboratory in Edinburgh is a dedicated facility which is staffed 24/7 for human cadaveric islet isolation and has processed over 20 human pancreata in the last 6 months. The clinical programme and isolation facility is fully funded by the Scottish Government and has been set up to provide both a clinical service and to act as a catalyst for islet related stem cell research through links with Edinburgh University, Aberdeen University and the Scottish Centre for Regenerative Medicine. The first transplant was performed in February 2011 with four more in a short period of time. At present multiple donor pancreases are required to treat a single patient as the effectiveness of the initial transplant falls with time and further transplants are required. Only a small fraction of the pancreas has the ability to produce insulin with the remainder producing enzymes, substances which allow the gut to digest food. Using recent research we aim to reprogramme the enzyme producing pancreatic tissue so that instead it produces insulin. Potentially this will allow multiple transplants over a period of years from a single pancreas reducing the requirement for more than one donor.

Transplantation of isolated islets of Langerhans is viewed as the only effective treatment for type 1 diabetes that will normalise blood glucose levels without the attendant risk of hypoglycaemic. However, a major problem with current transplantation protocols is that up to three donor pancreases are required for each recipient, and in many cases the function of the graft deteriorates over time. During the isolation procedure, the bulk of the pancreatic material, i.e. the exocrine tissue is discarded. In our preliminary studies we have shown that the exocrine enriched tissue can be reprogrammed using 4 transcription factors (Pdx1, MafA, Ngn3 and Pax4) in combination with 3 growth factors (Betacellulin, Exendin-4 and nicotinamide) and that the efficiency (and direction) of reprogramming is enhanced by inducing the cultured exocrine cells to undergo a mesenchymal to epithelial transition (MET). Our preliminary studies provide proof of concept that we can efficiently reprogramme the exocrine enriched cells of the pancreas. We now propose to take the next step towards developing a novel cell therapy by characterising the cells in detail with a view to generating cells with properties as close as possible to that of an adult human islet cell.
The overall aim is to enhance the production of functional islet tissue. In aim 1 we will optimise the use of growth factors and small molecules. This will be done by ICC and ELISA using insulin and glucagon expression as an end-point. In aim 2 we will optimise further the transcription factor cocktail used in reprogramming. In aim 3 we will characterise in detail the fully reprogrammed cells, using standard in vitro insulin secretion protocols, and in aim 4 we will test the ability of the reprogrammed to reverse the symptoms of diabetes in an animal model.

The research proposed is likely to benefit people with type 1 diabetes. There are about 300,000 people with type 1 diabetes in the UK. The disease is treated with multiple daily injections of insulin combined with blood glucose monitoring and sensible dietary advice as per the DAFNE (dose adjustment for normal eating) course promoted by Diabetes UK. The major challenges in treating people with diabetes are preventing the long term complications of the disease and hypoglycaemia. A proportion of people with diabetes have huge problems controlling their disease particularly with respect to excessive daily excursions in blood glucose levels combined with an unawareness of the onset of hypoglycaemia. This is the group of patients that would benefit most from an islet transplant. However, at present the number of donors would provide tissue for <1% of these people. The problem is exacerbated by the requirement for up to three transplants per patient. A further problem is that the graft tends to fail over a period of time. Our project is aimed at addressing this problem of tissue availability and graft failure. Our preliminary data provide proof of principle that we can get regular access to the human material (8 pancreata between February and November 2011) and that we can efficiently reprogramme it into cells that express the major islet hormones, i.e. insulin, glucagon and somatostatin. With a view to taking this work towards the clinic we now need to characterise the cells in detail and improve the levels of insulin (and glucagon) that are expressed.
The project will also be of benefit to those companies involved in generating a replenishable supply of human Beta cells derived from ES/iPS cells. Our strategy towards identifying the concentration and temporal effects of transcription factors may help inform strategies targeting the differentiation of ES/iPS cells. The identification of growth factors and small molecules that act individually or in combination to reprogramme towards Beta cells would also be of great value to companies working in this area.