Research Highlights

Pancreatic islet isolation and transplantation following the Edmonton Protocol offer life-altering results for diabetes patients, but injury and stress to cells during the process are major obstacles for achieving effective post-transplant functional performance. One of the issues is oxidative stress due to low antioxidant capacity in islets, highly detrimental when cells are challenged with ischemia and reperfusion injury. In an effort to overcome this, Dr Gina Rayat and a collaborative team that included former Alberta Diabetes Institute Founding Scientific Director Dr Ray Rajotte and fruit scientists from Zhejiang University in China, explored the use of cyanidin-3-O-glucoside (C3G), an anthocyanin with powerful antioxidant properties derived from the Chinese bayberry that has been also used in other medical applications. The group had previously shown that isolated mouse islets cultured with C3G increased expression of various genes related to enhanced survival, including HO-1, ERK 1/2 and P13K/Akt, with knockout of these genes nullifying the advantage. In a more recent study they isolated islets from mouse pancreases and cultured them with and without C3G for 24 hours before transplanting the islets under the kidney capsule of recipient mice that had been rendered diabetic with streptozotocin. They found that mouse recipients of 400 or 200 C3G-treated islets achieved normoglycemia significantly faster than mouse recipients of untreated islets - 10 days vs. 13 days and 10 days vs. 18 days, respectively. Even more, animals that received just 100 C3G-treated islets achieved normoglycemia within 27 days, whereas mice transplanted with the same number of untreated islets failed to show lowering of blood glucose. When the investigators repeated the experiment using the portal vein as site for transplantation they found that islets were less efficient in attaining normoglycemia. For example, 40% and 20% of mice transplanted with 200 and 100 C3G-treated islets, respectively were able to achieve normoglycemia, when the transplant was into the portal vein of the liver. This compares with 100% success when similar numbers were transplanted under the kidney capsule. While the latter site may not be practical in human applications, it does demonstrate the importance of transplantation site for islet survival, even with antioxidant treatment. Regardless of site though, their results show that pre-treatment with C3G enhances both viability and function of islets after transplantation and encourages further research into the use of antioxidants (Transplantation, 99:508-514, 2015). Dr Rayat and her group are currently testing the effect of C3G on pig and human islets.

Autoimmunity results from abnormal recognition of self-antigens by both B- and T-cells, which can lead to autoantibody production and cell-mediated attack. The relative importance of the two acquired immune types and their interplay in the pathogenesis of T1D is an ongoing area of investigation, but understanding T-cell response is still considered central to managing the autoimmune disease itself as well as achieving allotransplant tolerance. An area of focus of several Alberta Diabetes Institute scientists is on regulatory T-cells (Tregs), the cell type involved in moderating T-cell mediated immune reactions along with helper T cells (Th cells) - essentially the cells that put the brakes on a cell-mediated immune reaction. Immunologist Dr Colin Anderson has made inroads into understanding how Tregs are generated, with earlier work suggesting the co-inhibitory receptor PD-1 played a role in controlling the proliferation of peripheral Tregs from newly formed/immature T cells known as 'recent thymic emigrants'. This modulation is critical for keeping balance and promoting self-tolerance during transient states, but whether or not PD-1 was acting directly was not clear: conclusions were derived indirectly from looking at the ligand PDL1, not PD-1 itself. Since PD-L1 is known to interact with receptors other than PD-1, Anderson wanted to perform a more direct in vivo experiment to determine if PD-L1 modulates pTreg cell conversion by something other than PD-1. They compared the response of wild-type and PD-1 knockout T cell populations, but did so in the same animals - a strategy which avoided a number of confounding experimental issues that have led to conflicting results from other scientists. What they showed was that conversion of RTEs to pTregs was not impeded by a lack of PD-1; in fact, conversion to pTregs was actually higher for PD-1 KO cells populations. In a mouse model of lymphopenia-driven autoimmunity, PD-1 deficiency did not lead to Th17 cell mediated autoimmunity. Taken together, the data suggests that PD-1 does not intrinsically control the conversion of RTEs to pTreg cells, but rather that the primary role of PD-1 is to restrain the expansion of T cell activation and proliferation to self-antigens (European Journal of Immunology, doi:10.1002/eji.201444688, 2014). Meanwhile pediatric cardiologist/immunologist Dr Lori West is looking at the feasibility of harvesting Tregs from thymuses discarded during infant cardiac surgery as a means of immunosuppressive cell therapy during both allo- and xenotransplantation. Treg cells sequestered from peripheral blood and expanded in culture have yielded promising results in various clinical trials, but there are challenges with this approach. One issue is that peripheral Treg numbers are typically low, requiring massive ex vivo expansion. Another problem is unavoidable contamination with effector T cells. A further challenge is limited stability and loss of FOXP3+ expression following repeated ex vivo stimulation. West and post-doctoral fellow Dr Esme Dijke were able to collect and expand large numbers of Tregs from single thymuses that maintained stable function and phenotype including FOXP3+ expression, even under inflammatory conditions. They were able to use these to suppress proliferation and cytokine production of activated allogenic T cells in vitro. They were further able to demonstrate delayed development of xenogeneic graft versus host disease (GVHD) in an in vivo suppression assay with efficacy that was superior to that achieved using blood Tregs. Results of their research have set the stage for possible preclinical and clinical trials in the future using thymus-derived Tregs that overcome current therapeutic shortfalls (American Journal Transplantation, doi:10.1111/ajt.13456, 2015).

While continuing to direct the world's largest clinical islet transplant program based on the groundbreaking Edmonton Protocol, Dr James Shapiro is also principal investigator for research that may lead to the next generation of islet cell replacement therapy for T1D. The technology, developed by San Diego-based ViaCyte, features a combination of pancreatic endoderm stem cells (PEC-01) loaded in a device implanted subcutaneously called Encaptra®, with the combination product called VC-01™. Preclinical testing of this device has repeatedly demonstrated reversal of diabetes in over 1500 mice made diabetic with streptozotocin. In 2015 Edmonton became the second site for Phase 1/2 clinical trials called STEP ONE (Safety, Tolerability and Efficacy of VC-01™ Combination Product in T1D), where Shapiro and co-investigator Dr Peter Senior have led the transplantation of the devices in several patients. The VC-01™ offers a number of potential advantages over current islet transplantation protocols involving injection of islets into the liver. The device is easily retrievable with a minimally-invasive, outpatient procedure. The cell line produces other pancreatic regulatory hormones and co-factors, increasing the health benefits to the patient. The combination of stem cells with the encapsulation device boosts vascularization around the cells, improving oxygenation and the delivery of nutrients to the developing cells. And perhaps most significantly, the Encaptra® is designed to overcome immuno-rejection by the host by means of a semi-permeable membrane that allows the movement of glucose, proteins, oxygen and hormones but is impermeable to immune cells. Shapiro remarked in a recent press release that "this is a remarkable opportunity that could free T1D patients from severe complications and health issues such as hypoglycemia, eye, kidney and cardiovascular diseases, all without the requirement of powerful lifelong immunosuppression drugs." Once safety is demonstrated in early trials, additional transplants will be done with higher doses of stem cells that will produce therapeutic levels of insulin and other products while safety is further evaluated. STEP ONE is being supported by a $5-million Collaborative Research and Innovation Opportunities (CRIO) grant from the Alberta Government, and through a Juvenile Diabetes Research Foundation (JDRF) Clinical Trials Network grant.

Dr Greg Korbutt has pioneered research exploring the feasibility of using neonatal porcine islets (NPIs) as an alternative to the more limited supply of human islets for cell replacement therapy in T1D patients. Similar to the transplantation of human islets, there are drawbacks to transplanting NPIs into the portal vein of the liver based on the Edmonton Protocol (which Korbutt helped develop), including hepatic thrombosis and intraperitoneal bleeding. Like other scientists who are looking to improve the sustainability of human islet transplants, Korbutt is exploring the use of alternative transplant sites as well as utilizing supporting materials that promote the viability and longevity of transplanted NPIs. The goal is a supporting matrix that promotes vascularization of the graft for oxygen and nutrient delivery, while also providing protection against immune attack. Korbutt and PhD student Cara Ellis previously developed a collagen-based matrix containing a combination of chondroitin-6-sulfate, chitosan and laminin that proved to be efficacious for improving islet survival in preclinical transplant models. More recent research was aimed at 'tuning' this matrix with different cross-linkers to optimize functional characteristics like biodegradation rate, degree of vascularization and swelling ratio. The concentration of two linking compounds, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, were varied with the resulting matrix tested for functionality in both in vitro and in vivo models. In general, mechanical strength of the matrix was sufficient to support both transplantation and retrievability, important considerations for future clinical application. They were able to demonstrate that the matrix properties could be manipulated in a highly reproducible manner; for example, swelling ratio was significantly and negatively correlated with cross-linker concentration, important for controlling the rate of insulin diffusion and the rate of release of drugs that might be incorporated in the matrix. Biodegradation was tested using collagenase, with the rate determined to be significantly and inversely correlated with the concentration of cross-linking. The number and size of blood vessels (i.e., vascularization) was positively correlated with cross-link concentration; electron microscopy revealed that faster neovascularization was correlated with specific topographical features that are likely related to higher surface energy. Results of their research lend themselves to customizing matrices in the future for application in specific tissues (Bioresearch Open Access, 4:188-197, 2015). Korbutt's progress in this area has set the stage for his upcoming research focus: combining the collagen-based matrix with a novel poly-caprolactone scaffolding that will effectively incorporate growth factors and immunoprotective components. Korbutt is also director of Alberta Cell Therapy Manufacturing, Western Canada's only Good Manufacturing Practice (GMP) facility for cell-based therapeutic products for clinical application, including xenotransplantation.