Objective
Artificial insulin-secreting cells can be engrafted at different sites into the body, and it is still unknown which of these provides the best outcomes for patients with type 1 diabetes. Whilst engraftment of cells under the skin is usually chosen, the liver is also a popular choice. In either case, once in place, it becomes very difficult to know what has happened to the implanted material: did the cells fail to receive an adequate blood supply? Were they attacked by the immune system? Did they simply lose the ability to sense glucose and secrete insulin (“dedifferentiate”)? Usually, these questions can usually only be answered by surgically removing the graft and performing “histological” measurements (i.e. studies of the tissues under the microscope) to explore what has happened, thus ending the experiment, and losing the opportunity of knowing what would have happened had the graft remained in place. In other words, it is impossible to follow the loss of islet cells - or their activity - over time.
Our groups have, therefore, sought to find ways in which we can non-invasively image the engrafted material using existing technologies such as magnetic resonance imaging (MRI) or positron emission tomography (PET), and have developed approaches for this whose applicability to measure endogenous beta cell mass has been demonstrated in rodents. Leveraging this experience, in the present application, alongside substantial infrastructure funding in Montreal from the Canadian government for a new suite of imaging devices, we hope to better understand how (i.e. by what mechanisms), and when, failure occurs. We will explore this process, using the same non-invasive approaches, after engrafting in three different sites: under the skin, in the liver, and in the anterior chamber of the eye - a site where imaging is straightforward using a relatively simple light microscope. The latter strategy will allow us to corroborate our non-invasive strategies using simple visual inspection of the islets through the front of the eye.
Background Rationale
People affected by type 1 diabetes usually need to inject themselves up to six times a day with insulin to keep their blood sugar (glucose) levels stable. This provides a considerable challenge to the patient and is by no means a perfect solution since it carries a risk of causing blood sugar levels to fall too far (“hypoglycemia”). Consequently, for most patients, the normalization of blood glucose levels - which are needed to eliminate the risks of “hypoglycemia” - remains and can lead to complications such as blindness or kidney failure. New strategies are therefore needed to ensure that insulin release is more tightly controlled and that glucose remains in the narrow range which pertains to people without diabetes.
In order for the engraftment of stem cell-derived islets to be successful, multiple challenges exist. Firstly, for the cells to survive they must resist rapid destruction by the immune system: treatment with immunosuppressive drugs is usually used. Secondly, the cells must be adequately vascularised to receive nutrients and oxygen and release insulin accordingly. Finally, they must remain properly differentiated and responsive to glucose.
Our research aims to provide an innovative new method by which we can explore each of these processes, and how they occur at different transplant sites.
Description of Project
Insulin secretion becomes defective in people affected by type 1 diabetes since pancreatic beta cells are destroyed, or become inactive. A conceptually simple idea to restore the normal secretion of insulin, and hence blood glucose control, would be to transplant replacement beta cells, which then take over the role of the destroyed cells. However, this approach is fraught with complications associated with the rejection of the transplanted cells and the use of immunosuppressants, which can lead to further complications. Whilst engraftment of human islets was hailed in the early 2000s as a game-changing approach, the shortage of material for transplantation has severely limited the deployment of this technology. However, recently, it has become possible to generate, from largely undifferentiated cells called embryonic stem cells (ES cells) a variety of cell types in the test tube – including “beta-like” cells. Transplanted at a suitable location that permits adequate survival and the creation of a new blood supply, these will release insulin “on demand” and at almost exactly the right levels for the needs of the body. Unfortunately, grafts often fail either due to cell death, the fact that they are still attacked by the immune system, or that they do not receive an adequate blood supply – or a combination of all three. The fact that we do not understand what happens to the grafts is in part because it is impossible to “see” (that is, to visualize non-invasively) what is happening to the graft over time, in the same subject.
At present, two sites are usually used for the transplantation of stem-cell-derived islets into people with type 1 diabetes: under the skin or into the liver. At present, there are no "side by side" studies that have compared these sites either in people or in animal models and it is unclear which is likely to be the more successful. This is important since the production of the artificial cells "in the test tube" is still time-consuming and very costly. Here, we will use an imaging approach to compare these two sites, and a third - transplantation into the eye, where the islets can readily be seen with a light microscope - to further validate our findings.
Anticipated Outcome
The chief outcomes of our research will be (a) to provide a set of tools, including chemical "probes" (small, harmless molecules that are injected into a subject and which will "light up" the insulin-secreting cells in such a way that suitable instruments can "see them" through the skin in the same way that an X-ray machine can "see" a broken bone or a suitable a (CT) scanner can visualize the beating heart of fetus inside the mother's womb, and (b) a carefully controlled assessment of which of the two principal sites of engraftment currently used in the clinic is likely to provide greatest therapeutic benefit for patients. Since the generation of stem cell-derived islets is still costly, technically challenging, and time-consuming, this knowledge should further enhance the possibility of using these "artificial" cells in the clinic since it is expected to identify the site of engraftment that requires the fewest islets for effective treatment.
We will use our existing tools, and develop a suite of new and innovative approaches that combine synthetic chemistry with cell engineering, "genome editing", directed stem cell differentiation, transplantation, and in vivo imaging. To achieve these ambitious goals, we have assembled an international group of experts and highly qualified post-doctoral fellows with the relevant skills to carry out the proposed studies in London, U.K. and at the newly established center for in vivo imaging in Montreal, Canada (“Image T2D”). Our first aim is to determine which of the two existing synthetic probes that we have developed is the likelier to provide a robust readout of beta cell mass in the living animal. This work will involve stem-cell-derived islets deleted for the targeted receptors. Our second aim is to use these probes to monitor non-invasively the survival of human stem cell-derived beta cells after engraftment at different sites of suitable (non-immunocompetent) mice: under the skin, into the liver and in the anterior chamber of the eye. Finally, we wish to test whether we can extend the “generalisability” of our approach so that we can monitor two processes at the same time using distinct probes: firstly, beta cell number and the expression of markers of immune attack (in immune-competent animals). The latter approach should ultimately provide a means of understanding when, and to what extent, a blood supply to the graft develops – or fails.
Relevance to T1D
People affected by type 1 diabetes usually need to inject themselves up to six times a day with insulin in order to keep their blood sugar (glucose) levels stable. This provides a considerable challenge to the patient and is by no means a perfect solution since it carries a risk of causing blood sugar levels to fall too far (“hypoglycemia”). Consequently, for most patients, the normalization of blood glucose levels - which is needed to eliminate the risks of “hypoglycemia” - remains, and can lead to complications such as blindness or kidney failure. New strategies are therefore needed to ensure that insulin release is more tightly controlled and that glucose remains in the narrow range that pertains in people without diabetes.
For the engraftment of stem cell-derived islets to be successful, multiple challenges exist. Firstly, for the cells to survive they must resist rapid destruction by the immune system: treatment with immunosuppressive drugs is usually used. Secondly, the cells must be adequately vascularised to receive nutrients and oxygen and release insulin accordingly. Finally, they must remain properly differentiated and responsive to glucose.
Our research aims to provide an innovative new method by which we can explore each of these processes, and how they occur at different transplant sites.
Ultimately, anyone with type 1 diabetes may benefit from the more effective use of stem cell-derived beta cell engraftment. However, the continued requirement for immunosuppression in humans, when engrafting physically unprotected (i.e. “naked”) cells means that the approach is less useful in children and younger patients for whom the risks of infection may outweigh the benefits. Those most likely to benefit - as for alternative treatments including the use of closed-loop systems or human islet transplantation - are those whose disease is most poorly controlled (“brittle diabetes”) with the greatest risks of hypoglycemia.