Olle Korsgren’s projects – Transplantation of isolated islets to cure patients with the most severe TID

In collaboration with Gunnar Tufveson, Dept. of Surgical Sciences; Lars Johansson, Dept. of Radiology, Oncology and Radiation Sciences, and Peetra Magnusson, IGP.

Significant progress in the field of beta cell replacement therapy can only be made based on an increased understanding of the processes governing islet survival and engraftment. Two main routes are proposed to fill this gap in knowledge; 1) experimental studies in relevant animal models, and 2) establishment of new techniques to allow detailed characterization of the processes regulating islet survival and engraftment in humans.

Applying an efficient step-wise screening program, via a series of in vitro models, through small animal models and pre-clinical large animal models, for the identification of potential drugs suitable to promote islet survival and engraftment, we have developed several substances into novel drugs at present evaluated in clinical trials or awaiting final regulatory approval. Using the same step-wise screening program we have developed and validated powerful quantitative and qualitative diagnostic tools (PET-CT and MRI scans).

Hence, we feel confident that the established step-wise screening program constitute an indispensable tool for promoting development in clinical islet transplantation. The large size of the Nordic Network for Clinical Islet Transplantation (NNCIT) is a prerequisite for conducting randomized clinical trials to evaluate any novel approach in clinical islet transplantation.

Developing an intramuscular site for islet transplantation

Transplantation of pancreatic islets intraportally into the liver has become a treatment for selected patients with TID. One-year insulin-independence rate is comparable to that for whole pancreas transplantation. However, in contrast to whole-organ transplantation there seems to be a progressive decline in function, and few patients remain insulin-independent at 5 years post-transplantation. Since the histocompatibility barrier, the underlying autoimmune disease and the immunosuppressive agents used are the same for whole-pancreas transplants, it is most likely that issues related to the isolation of islets and/or adaptation of the transplanted islets to their new microenvironment play an important role in this context.

Islets within a pancreas transplant have an intact endogenous vascular system. In contrast, transplanted islets become disconnected from their vascular supply when isolated. During culture and immediately after transplantation they critically depend on diffusion of oxygen and nutrients from the surrounding environment. Massive islet cell death occurs during the immediate post-transplantation period due to inflammatory and hypoxic events. Subsequent, revascularization of transplanted islets is a slow process occurring over a period of several weeks.

Within NNCIT particular interest has been focused on development of a novel endocrine organ at the intramuscular site, cf. the feasibility of this site for autotransplantation of parathyroid glands. Also, in experimental studies the density of blood vessels and blood perfusion in islets transplanted to the musculature is remarkably high compared to islets at other implantation sites.

Encouraged by these results a clinical study was initiated in 2008 in Finland and Sweden comparing the efficacy of the intramuscular site to the liver with or without the support of autologous MSC (“Open Multi-Center Center Randomized Study to Compare Safety and Efficacy of Islet Transplantation Using the Intraportal or Intramuscular Site in Simultaneous Islet and Kidney Transplantation”; PI Kaija Salmela Helsingfors Universitet, Finland; www.clinicaltrials.gov). In clinical trials we have shown that autotransplanted islets into the musculature can function with high and stable c-peptide production for many years. The musculature has the inherent advantage of a resting high oxygen tension of ~30 mmHg, possibly limiting beta cell death due to hypoxia during the engraftment period.

Determination of islet survival, vascular leakage and revascularization after clinical intramuscular transplantation using the high resolution MR technique

We have over the last years developed MRI-based structural and functional methods aiming at longitudinal monitoring of islets transplanted intramuscularly. The developed MRI-technique yields a very high in-plane resolution of 200 μm in vivo allowing high-resolution imaging of the small volume of islets transplanted and spread over several strings into the arm. The technique can also be used to monitor both inflammation and the effects of anti-rejection treatment. Fractional plasma volume, which corresponds to capillary density of the scanned area, was investigated through kinetic modeling.

In subjects studied so far, the capillary density was assessed through dynamic contrast enhancement of the equivalent parameter, the plasma volume (Vp), and was found to be 2.5–3.4 times higher in the implanted islets when compared to the surrounding muscle tissue (fig. 1).

By introducing the imaging techniques described we believe that a unique opportunity to non-invasively monitor islet graft revascularization, survival and function after clinical intramuscular transplantation has been established. This opportunity will be applied in our ongoing and future clinical trials to assist in the establishment of the intramuscular site for clinical islet transplantation Similarly, the development of a novel endocrine organ using composite EC/MSC/islets and new biomaterials as described below will be facilitated and evaluated by applying the MR techniques described.

Optimization and standardization of large-scale production of composite cellular grafts

The overall aim of this project is to develop a “new endocrine organ” at the site of transplantation, applying modern tissue engineering technologies to improve islet engraftment and revascularization. Composite cellular grafts consisting of human islets, human endothelial cells (EC) and human mesenchymal stem cells (MSC) will be evaluated after transplantation in different resorbable scaffolds releasing defined growth factors at the sites of implantation.

When blood vessels are assembled, endothelial cells produce platelet-derived growth factor, which attracts supportive MSC to differentiate into pericytes. This process stabilizes the newly formed vessel, which than can exist for years. In addition, MSC secrete a large number of growth factors and contribute to migration of EC.

We have developed a method to coat human islets with both MSC and EC, fig 2. The addition of MSC to islets improves the binding of the EC and guide the EC to form vessel like structures (sprouts) penetrating into the islets as well as into the surrounding matrix or tissues, (fig 3). We hypothesize that composite grafts of islets, MSC and EC would provide the optimal setting for islet survival, engraftment and formation of new vessels in the graft. We aim to develop large-scale procedures to produce composite grafts consisting of islets/EC/MSC to be used in clinical islet transplantation.

Several microscope images showing sprouting from islets co-covered with mesenchymal stem cells and endothelial cells
Sprouting from islets co-covered with MSC and EC
Islet coated with MSC (green) and EC (red) in 3-D fibrin gel (figure indicates
enlarged pictures to the right; 1: MSC at the base of an endothelial sprout,
2: MSC and EC in close contact, 3: MSC and EC grown side by side,
4: EC at sprout tip, 5: MSC at sprout tip, 6: naked, unconnected EC sprout).

Structural support might improve engraftment

Engraftment and islet survival may be further facilitated with the use of various matrices. Biodegradable scaffolds composed of polyesters, collagen/gelatine or HyA/fibrin has the possibility to provide early structural support to implanted islets. The scaffolds can be decorated with growth factors, oxygen carriers such as fluorodecalins or polymerized hemoglobin, or drug carriers to optimize early functional support and improve engraftment and immunoprotection of the islets.

Special instruments have been developed to atraumatically implant the composite islet/EC/MSC grafts intramuscularly. The functional capacity of the graft will be evaluated by IVGTT after which the graft will be surgically removed after 2, 4 or 8 weeks. Evaluation of the cellular composition of the grafts will be performed using immunohistochemistry to detect human EC, MSC and cells positive for insulin, glucagon, somatostatin and PP. The Luciferase imaging system IVIS 100 (Xenogen Inc) and transduced EC, MSC and islets using different luciferases (firefly and renilla) and GFP, will be used to monitor the survival, location and interaction between the different celltypes. Islet graft revascularization will be examined using deeply penetrating in vivo multiphoton laser scanning confocal microscopy, as described above.

In the ongoing clinical study (see above) a small number of islets or islets/MSC will be implanted at a location separated from the main graft and marked by a suture to allow a biopsy to be taken without risking the main graft. The amount of surviving islets, remaining MSC in the graft, the formation of new stroma and revascularization will be determined using immunohistochemistry. The specific aim of this project is to understand the interaction between the islets/EC/MSC and the surrounding tissues at the site of implantation in order to optimize clinical islet transplantation at an alternative site and to validate the herein described experimental models.