a future of glucoseresponsive secretion: bionics versus nature

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1 science at the cutting edge a future of glucoseresponsive insulin secretion: bionics versus nature Pratik Choudhary, John Pickup, Peter Jones, Stephanie A Amiel people with diabetes constantly walk a tight rope between strict glucose control to prevent complications and hypoglycaemia. Too often, slight miscalculations result in hypoglycaemia, which can be at best embarrassing and irritating, and may cause injury or even death. new drugs for diabetes and new ways of measuring blood glucose bring hope of easing the burdens of diabetes but the dream for many (and for the researchers trying to help them) is a treatment that restores glucose-responsive insulin secretion so that normal blood glucose concentrations can be achieved with no risk of hypoglycaemia and perhaps even with no need to think about diabetes all the time. There are two obvious routes towards this holy grail: one, for the pure scientist, is the development of the perfect insulin delivery device, driven by the perfect biochemical glucose sensor the artificial pancreas or bionic solution ; the other accepts that nature got it right the first time and that all we need to do is restore the beta-cells in the islet structures that diabetes destroyed. a group of authors from the UK take a look at the new technologies in diabetes that are bringing us, step by step, closer to one or both of these goals. 22 DiabetesVoice June 2011 Volume 56 Special Issue 1

2 science at the cutting edge Towards the bionic solution From urine tests, to finger prick blood samples and now to subcutaneously implanted online glucose sensing, our ability to monitor diabetes control has increased enormously. Sensors implanted into the subcutaneous tissue can record either for later downloading (in the manner of 24-hour ECG monitoring) or for display in near real-time. As work progresses towards linking the new glucose monitors to new insulin delivery devices, what has science in store for us? The altered properties of nanoscale structures are being used to develop glucose-responsive insulin delivery. Nanotechnology and nanomedicine are set to make increasing contributions in diabetes care in the coming years, and this will be particularly true of regulated insulin delivery. Nanos is the Greek for dwarf. Nanotechnology involves making or measuring things on a very small scale, usually nm (a nanometre is 1x10-9 metres) larger than an atom but smaller than a cell. At this small scale, nanoscale structures and devices often have fundamentally altered properties, including metabolite sensing, controlled porosity, biocompatibility and the ability to target tissues and molecules in the body. Some of these properties are being used to develop glucose-responsive insulin delivery. Despite the progress alluded to above, we still lack a completely reliable and accurate glucose sensor. Alternative technologies to the electrochemical sensors used in current systems are urgently needed. One nano approach that is showing promise is based on fluorescence. Several groups around the world, including in the laboratory at Guy s Hospital, London (UK), are researching a glucose-binding protein from bacteria, which changes shape on linking to glucose and, when tagged with a fluorescent probe, can be used to monitor glucose concentrations. Protein engineering is used to make the molecule operate in the range of glucose levels found in people with diabetes. Detecting nanosecond changes in the fluorescence lifetime (the time taken for the fluorescence to decay after excitation) upon glucose binding, rather than depending on measuring the intensity of fluorescence, has the advantages of stability and lack of interference. Prototypes of the first product, a fibre optic-based sensor implanted in the subcutaneous tissue, have already been tested. researchers are thinking of ways to close-couple glucose sensing and insulin delivery in what might be called an artificial nanopancreas. Could the glucose-binding protein sensor become part of a non-invasive glucose monitor? Researchers at Guy s have encapsulated the protein in nano-thickness polymer films, making micro-sensors that potentially could be implanted or impregnated in the skin as a kind of smart tattoo with a portable meter held over the glucose tattoo to excite the fluorescence and detect light changes as the tissue glucose changes. Many problems need to be solved before such a system can be commercialized and enter clinical practice but the possibilities are exciting. The distant nano future Coupling detection to treatment in one molecule or nanoscale device is called theranostics, and researchers are already thinking of ways to close-couple glucose sensing and insulin delivery in June 2011 Volume 56 Special Issue 1 DiabetesVoice 23

3 science at the cutting edge what might be called an artificial nanopancreas. This might comprise a glucosesensing molecule like glucose-binding protein embedded on the surface of an artificial cell that contains microvesicles of insulin and with a molecular link from the sensing to insulin delivery. Towards replacing the lost islets Since the publication in 2000 of a report from Edmonton, Canada, on seven people with type 1 diabetes who achieved insulin independence after receiving fresh human islets that had been isolated and donated by multi-organ donors using a novel digestion technique and steroid-free immunosuppression, more than 400 people have received an islet transplant. So where does islet cell transplantation stand today? Obtaining islets from a donor pancreas is a long and painstaking procedure. The organ undergoes enzymatic and then mechanical digestion before islets are separated from exocrine tissue. This process can take 12 to 15 hours. Islets are then cultured for up to 24 hours and assessed for purity and viability before they are transplanted introduced into the liver trans-portally under local anaesthetic. Prior to transplantation, recipients are given an induction treatment to reduce T-cell reactivity and increase immune tolerance to these foreign cells. Immune suppression is then maintained with a combination of drugs such as tacrolimus, sirolimus and mycophenolate mofetil. These are intended to prevent the rejection of the transplant and the recurrence of type 1 diabetes, and need to be continued throughout the recipient s life. Indeed, most of the risk from islet transplantation comes from the long-term risk of immune suppression. Challenges for the future In its present form, islet transplantation is not efficient enough for all. The International Collaborative Islet Transplant Registry reports that one year after transplant over 85% of all recipients show detectable graft function (which is associated with almost complete protection from severe hypoglycaemia), and 70% after four years. Nevertheless, only 70% of all recipients achieved insulin independence at some stage, with 55% still insulin independent after two years. Providing sufficient donor organs will become a major issue as the procedures and the immunosuppression improve. A major obstacle lies in preventing the gradual loss of islet function. This is probably due to a combination of factors: the loss of islets on first infusion; gradual islet death; the recurrence of auto-immunity; the gradual process of organ rejection. Providing sufficient donor organs to supply people with type 1 diabetes is another challenge. This will become a major issue as the procedures and the immunosuppression improve. Novel alternatives are being investigated, ranging from converting exocrine cells into beta cells to obtaining islets from animals. Stem cells are a possible source of new islets. Stem cells are defined by their by their capacity for self-renewal (proliferation) and their ability to differentiate into a number of specialized cell types (pluripotency), properties which make them excellent candidates from which to generate the large numbers of functional beta cells that are required for transplantation. There are three main sources: 24 DiabetesVoice June 2011 Volume 56 Special Issue 1

4 science at the cutting edge tissue stem cells found in foetal and adult tissues, where they repair and renew the host tissue embryonic stem cells generated from a blastocyst, which can form all differentiated cell types in the developing embryo induced pluripotent stem cells similar to embryonic stem cells but generated from adult cells via the forced expression of pluripotency genes. Tissue stem cells Stem cells isolated from many tissues may have the capacity to differentiate into insulin-expressing cells. However, experimental studies using tissue stem cells have so far failed to translate into reliable protocols for generating large numbers of functional beta cells in vitro. Pancreatic stem cells with the potential to generate beta cells have been identified in the exocrine pancreas the pancreatic ducts within endocrine islets. Isolating the stem cells from a human pancreas, expanding them ex vivo and differentiating them into functional beta cells is an attractive therapeutic option but it remains to be demonstrated whether this is technically feasible. Bone marrow stem cells are alternative candidates and are already used therapeutically. They offer the potential for autograft transplantation without immune rejection. Several studies have reported that bone marrow stem cells can be driven towards an insulin-expressing phenotype. Others, however, suggest that the therapeutic benefits of bone marrow stem cells are achieved primarily by their enhancing the regeneration and survival of endogenous beta cells rather than generating new ones. Liver cells can be induced by the forced overexpression of pancreatic genes to adopt some functional aspects of beta cells. However, it remains to be seen whether these experimental observations will translate to human tissues with sufficient efficiency to generate enough material for therapeutic purposes. Stem cells from a range of tissues including the central nervous system, intestinal epithelium, dermis, spleen, salivary gland and blood monocytes have also been reported to differentiate into insulin-expressing cells. But there is little convincing evidence that these cells are capable of the ex vivo expansion required to generate significant amounts of tissue for effective transplantation therapy. June 2011 Volume 56 Special Issue 1 DiabetesVoice 25

5 science at the cutting edge Embryonic stem cells The differentiation of mouse embryonic stem cells into insulin-expressing cells was first reported more than decade ago. Attention then was focused on how best to drive pluripotent, undifferentiated embryonic stem cells towards a functional beta-cell phenotype. Nowadays, we have detailed knowledge of the sequence of events and developmental cues in the formation of the endocrine pancreas, and this information is being applied to devise differentiation protocols based on the sequential exposure of embryonic stem cells to growth factors and mitogens. These protocols are designed to recapitulate in vitro the important in vivo signals that drive pluripotent cells first towards endoderm, then to an endocrine progenitor and finally to fully differentiated pancreatic endocrine cells. By measuring important staging markers (usually transcription factors) we can assess the effectiveness of the differentiation protocols. This type of research has produced encouraging results but cells generated by these in vitro protocols are in general functionally restricted; they produce either a weak glucose-induced insulin secretory response or none at all. Several studies have demonstrated improvements after transplantation into a person, which suggests that something important is lacking from current in vitro protocols. Identifying the factors involved in the development of the cells in the human body might inform the last stages of an entirely in vitro differentiation protocol for functioning beta cells. Inconsistencies have also been reported in the development in the body of embryonic stem cells. One likely cause is the disparities in the initial differentiation potential between various lines of embryonic stem cells. This highlights the importance of a systematic evaluation of available human embryonic stem cell lines in order to identify the most suitable starting material. Induced pluripotent stem cells Using induced pluripotent stem cells that are autologous (generated from a recipient s own body) has the obvious additional benefit that it avoids the use of cells derived from human blastocysts. Directed differentiation protocols have generated insulin-expressing cells from human fibroblast-derived induced pluripotent stem cells. However, these cells are subject to functional limitations similar to those described above for embryonic stem cells. Moreover, there remain other barriers to the clinical use of cells derived from induced pluripotent stem cells, including recent evidence of alterations in gene expression caused by epigenetic modifications of induced pluripotent stem cells heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence. Other obstacles include the accumulation of genetic coding mutations in induced pluripotent stem cells and the autoimmune rejection of transplanted cells generated from autologous induced pluripotent stem cells. It remains to be seen whether these obstacles can be overcome to allow induced pluripotent stem cells to achieve their therapeutic potential. The usefulness of substitute beta cells will depend in part on the development of fool-proof methods of ensuring their safety after transplantation. There are safety issues to consider. The pluripotency of stem cells and their capacity to grow and multiply raise the risk of uncontrolled cell proliferation and the formation of tumours. This is important when considering the transplantation of human islets through intra-portal administration into the liver (the most widely used method at the moment) because the transplanted material is rendered essentially irretrievable in the event of an adverse outcome a major safety issue. But we should be wary of overoptimism when interpreting human embryonic stem cell studies using directed differentiation protocols. In terms of differentiation, the effectiveness of similar in vitro protocols varies widely. 26 DiabetesVoice June 2011 Volume 56 Special Issue 1

6 science at the cutting edge on the left, a section of pancreas seen under the microscope shows a central clump of cells, with dense dark nuclei: the endocrine islet in a sea of dark staining exocrine pancreas cells. on the right, islets have been stained red with a dye that binds to insulin, showing intact islets being separated from the exocrine tissue of the pancreas, before infusion into a patient. The future clinical usefulness of substitute beta cells derived from stem cells will depend not only on their functional competence but also on the development of fool-proof methods of ensuring their safety after transplantation. Problems remain It has become clear that beta cells are gregarious they work best when collected together if not in formal islets at least in clumps of cells, so-called ` pseudo-islets'. When beta cells are isolated, they are not nearly as efficient in terms of secreting insulin in response to glucose as they are when clustered together. And the new beta-cell clusters or islets need to be protected from the recipient s immune system which will try to reject them as foreign and destroy them as beta cells as happened to cause the type 1 diabetes in the first place. Many strategies are being explored to overcome this rejection and nanotechnology might help. In the Guy s laboratory, nanofilms applied layer by layer using alternating layers of positively and negatively charged polymers are being used to encapsulate pancreatic islet cells to improve their chances of survival after transplantation. Using capsules to isolate the islets from immune cells and proteins, preventing rejection, while allowing glucose in and insulin out, has been the subject of research for many decades. Real hope for the future The conventional encapsulation technologies used in the past restricted oxygen and nutrients to the cells and provided incomplete immune protection, severely restricting the islets chances of survival. Now, biocompatible nanofilms applied very close to the islet cell surfaces are showing great promise with their adjustable permeability, fast response times and improved ability to admit nutrients. The researchers at Guy s have already seen significantly improved survival with such nano-encapsulated animal islets transplanted into animals with diabetes. The adoption of new technologies for diabetes will depend on them being demonstrably as safe and effective as the current therapy, administration of exogenous insulin, which has been used for almost a century. Working together, scientists from different backgrounds are helping to bring us ever closer to such goals. pratik Choudhary, John pickup, peter Jones, stephanie a amiel Pratik Choudhary is Senior Lecturer in diabetes at King's College Hospital, London, UK. John Pickup is Professor of Diabetes and Metabolism at King's College London, UK. Peter Jones is Professor of Endocrine Biology at King's College London, UK. Stephanie A Amiel is the RD Lawrence Professor of Diabetic Medicine at King's College London, UK. June 2011 Volume 56 Special Issue 1 DiabetesVoice 27