Dora M. Berman

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.

Glutamate is a positive autocrine signal for glucagon release.

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.

Long‐Term Survival of Nonhuman Primate Islets Implanted in an Omental Pouch on a Biodegradable Scaffold

The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and five allogeneic diabetic monkey transplants under the cover of steroid‐free immune suppression (SFIS) were undertaken. Fasting blood glucose (FBG) and C‐peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/mL following transplant, a 66–92% posttransplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well‐granulated and well‐vascularized, insulin‐positive islets, surrounded by T‐cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n = 20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

Background. Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. Methods. Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 μg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10–25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. Results. Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. Conclusions. These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.

CD40–CD40 Ligand Interaction Activates Proinflammatory Pathways in Pancreatic Islets

Pancreatic islet transplantation is becoming an alternative to insulin therapy in patients suffering from brittle type 1 diabetes. A major obstacle to the procedure is the early graft loss caused by nonspecific inflammation at the site of implantation. We recently discovered that CD40, a member of tumor necrosis factor (TNF) receptor family, is expressed in pancreatic β-cells. CD40 expression in nonhematopoietic cells is generally associated with inflammation. Therefore, we investigated the potential proinflammatory role of CD40 in human and nonhuman primate islets. Islet β-cells responded to CD40L interaction by secreting interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein (MIP)-1β, the latter a chemokine first reported to be produced by islets. Induction of IL-8 and MIP-1β was confirmed at the transcriptional level by quantitative RT-PCR. MIP-1β expression in β-cells was verified by double-immunofluorescence staining. CD40-CD40L interaction activates extracellular signal–regulated kinase 1/2 and nuclear factor-κB pathways in insulinoma NIT-1 cells, and inhibitors of either pathway suppress cytokine/chemokine production in islets. Moreover, ligation of CD40 receptor upregulates intercellular adhesion molecule-1, associated with inflammation, at both transcriptional and translational levels. Our results in vitro indicate that the CD40 receptor expressed by β-cells could be activated in vivo, inducing proinflammatory responses contributing to early islet graft loss after transplantation.

Mesenchymal stem cell labeling and in vitro MR characterization at 1.5 T of new SPIO contrast agent: Molday ION Rhodamine-B™

In vivo detection of transplanted stem cells is requisite for improving stem cell-based treatments by developing a thorough understanding of their therapeutic mechanisms. MRI tracking of magnetically labeled cells is non-invasive and is suitable for longitudinal studies. Molday ION Rhodamine-B™ (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non-phagocytic cells. This investigation characterizes mesenchymal stem cell (MSC) labeling and MR imaging properties of this new SPIO agent. Effects of MIRB on MSC viability and differentiation as well as cellular loading properties were assessed for MSC labeled with MIRB at concentrations from 5 to 100u2009µg Fe/ml. Labeled MSC were evaluated, in vitro, on a clinical 1.5 T MRI. Optimal scanning sequences and imaging parameters were determined based on contrast-to-noise ratio and contrast modulation. Relaxation rates (1/T(2)*) for gradient-echo sequences were approximated and an idealized limit of detection was established. MIRB labeling did not affect MSC viability or the ability to differentiate into either bone or fat. Labeling efficiency was found to be approximately 95% for labeling concentrations at or above 20u2009µg Fe/ml. Average MIRB per MSC ranged from 0.7 pg Fe for labeling MIRB concentration of 5u2009µg Fe/ml and asymptotically approached a value of 20-25 pg Fe/MSC as labeling concentration increased to 100 µg Fe/ml. MRI analysis of MIRB MSC revealed long echo time, gradient echo sequences to provide the most sensitivity. Limit of detection for gradient echo sequences was determined to be less than 1000 MSC, with approximately 15 pg Fe/MSC (labeled at 20 µg Fe/ml). These investigations have laid the groundwork and established feasibility for the use of this contrast agent for in vivo MRI detection of MSC. Properties evaluated in this study will be used as a reference for tracking labeled MSC for in vivo studies.

Macroporous three-dimensional PDMS scaffolds for extrahepatic islet transplantation.

Clinical islet transplantation has demonstrated success in treating type 1 diabetes. A current limitation is the intrahepatic portal vein transplant site, which is prone to mechanical stress and inflammation. Transplantation of pancreatic islets into alternative sites is preferable, but challenging, as it may require a three-dimensional vehicle to confer mechanical protection and to confine islets to a well-defined, retrievable space where islet neovascularization can occur. We have fabricated biostable, macroporous scaffolds from poly(dimethylsiloxane) (PDMS) and investigated islet retention and distribution, metabolic function, and glucose-dependent insulin secretion within these scaffolds. Islets from multiple sources, including rodents, nonhuman primates, and humans, were tested in vitro. We observed high islet retention and distribution within PDMS scaffolds, with retention of small islets (<100 μm) improved through the postloading addition of fibrin gel. Islets loaded within PDMS scaffolds exhibited viability and function comparable to standard culture conditions when incubated under normal oxygen tensions, but displayed improved viability compared to standard two-dimensional culture controls under low oxygen tensions. In vivo efficacy of scaffolds to support islet grafts was evaluated after transplantation in the omental pouch of chemically induced diabetic syngeneic rats, which promptly achieved normoglycemia. Collectively, these results are promising in that they indicate the potential for transplanting islets into a clinically relevant, extrahepatic site that provides spatial distribution of islets as well as intradevice vascularization.

The anterior chamber of the eye as a clinical transplantation site for the treatment of diabetes: a study in a baboon model of diabetes

Aims/hypothesisThe aim of this study was to provide evidence that the anterior chamber of the eye serves as a novel clinical islet implantation site.MethodsIn a preclinical model, allogeneic pancreatic islets were transplanted into the anterior chamber of the eye of a baboon model for diabetes, and metabolic and ophthalmological outcomes were assessed.ResultsIslets readily engrafted on the iris and there was a decrease in exogenous insulin requirements due to insulin secretion from the intraocular grafts. No major adverse effects on eye structure and function could be observed during the transplantation period.Conclusions/interpretationOur study demonstrates the long-term survival and function of allogeneic islets after transplantation into the anterior chamber of the eye. The safety and simplicity of this procedure provides support for further studies aimed at translating this technology into the clinic.

Automated, High-Throughput Assays for Evaluation of Human Pancreatic Islet Function

An important challenge in pancreatic islet transplantation in association with type 1 diabetes is to define automatic high-throughput assays for evaluation of human islet function. The physiological techniques presently used are amenable to small-scale experimental samples and produce descriptive results. The postgenomic era provides an opportunity to analyze biological processes on a larger scale, but the transition to high-throughput technologies is still a challenge. As a first step to implement high-throughput assays for the study of human islet function, we have developed two methodologies: multiple automated perifusion to determine islet hormone secretion and high-throughput kinetic imaging to examine islet cellular responses. Both technologies use fully automated devices that allow performing simultaneous experiments on multiple islet preparations. Our results illustrate that these technologies can be applied to study the functional status and explore the pharmacological profiles of islet cells. These methodologies will enable functional characterization of human islet preparations before transplantation and thereby provide the basis for the establishment of predictive tests for β-cell potency.

Immune cell populations in nonhuman primate islets

Islet transplantation is a promising cellular therapy for the treatment of type 1 diabetes (T1D). The immunogenicity of isolated islets has been of interest to the transplant community for many years, as upon transplantation, islets are damaged or destroyed through specific and nonspecific inflammatory and immune events. Antigen presenting cells (APC) are crucial intermediates in the generation of both innate and specific immune responses, and it has long been understood that some APC are resident in islets in situ, as well as after isolation. Our aim was to identify and characterize intraislet resident populations of APC and other immune cells in islets from nonhuman primates (Macaca fascicularis) in situ (pancreas biopsies obtained prerecovery) and after isolation using immunohistochemistry, confocal microscopy, and flow cytometry. The numbers of cells obtained in situ are similar to those in islets postisolation. Each isolated islet equivalent contains an average of 21.8 immune cells, 14.7 (67%) of which are APC. Many of these APC are dentritic cells and, surprisingly, 50% are B lymphocytes. The number of islet-resident immune cells increases with islet size, with greater numbers in large versus small islets (p < 0.001). The APC were localized around the exterior or spread evenly throughout the islets, with no definitive orientation identified. This knowledge will be useful to develop tailored modulation strategies to decrease immunogenicity, enhance engraftment, and ultimately prevent islet rejection.