Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. ARTICLE HIGHLIGHTS: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Desmoglein-2 is important for islet function and ß-cell survival.

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic ß-cells. Although ß-cell targeted autoimmune processes and ß-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports ß-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing ß-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced ß-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine ß-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of ß-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.

Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs.

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes.

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping.

Local Sphingosine Kinase 1 Activity Improves Islet Transplantation.

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive ß-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

Antigen-encoding bone marrow terminates islet-directed memory CD8+ T-cell responses to alleviate islet transplant rejection.

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by 'conventional' therapies, memory T-cell mediated attack is a substantial challenge in islet transplantation and this will extend to application of personalized approaches using stem-cell derived replacement ß cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement ß cells. Here we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions inactivates established memory CD8+ T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8+ memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigen-specific memory T-cell responses and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and importantly, the clinical application of personalized ß-cell replacement therapies using patient-derived stem cells.

Endothelial progenitor cells enhance islet engraftment, influence ß-cell function, and modulate islet connexin 36 expression.

The success of pancreatic islet transplantation is limited by delayed engraftment and suboptimal function in the longer term. Endothelial progenitor cells (EPCs) represent a potential cellular therapy that may improve the engraftment of transplanted pancreatic islets. In addition, EPCs may directly affect the function of pancreatic ß-cells. The objective of this study was to examine the ability of EPCs to enhance pancreatic islet transplantation in a murine syngeneic marginal mass transplant model and to examine the mechanisms through which this occurs. We found that cotransplanted EPCs improved the cure rate and initial glycemic control of transplanted islets. Gene expression data indicate that EPCs, or their soluble products, modulate the expression of the ß-cell surface molecule connexin 36 and affect glucose-stimulated insulin release in vitro. In conclusion, EPCs are a promising candidate for improving outcomes in islet transplantation, and their mechanisms of action warrant further study.

Incorporation of endothelial progenitor cells into mosaic pseudoislets.

Pancreatic islet transplantation is limited by extensive apoptosis and suboptimal function of the implanted islets in the longer term. Endothelial progenitor cells (EPC) may be ideal for enhancing both the survival and function of transplanted islets. Here, we describe for the first time the in vitro formation of rat mosaic pseudoislets comprised of pancreatic ß-cells with interspersed vasculogenic EPC. Bone marrow-derived EPC displayed a similar phenotype to non-adherent EPC, recently described in the human and mouse. Mosaic pseudoislet formation was enhanced by the use of an embryoid body forming medium (BPEL) and a spin protocol. Mosaic pseudoislets maintained function in vitro and may represent an enhanced cell therapy delivery approach to enhance the survival and revascularisation of transplanted islets.