Suzanne Bertera

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Recovery of the Endogenous β Cell Function in the NOD Model of Autoimmune Diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of β cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft‐bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous β cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity‐free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

Delivery of a Cyclic Adenosine 3′,5′-Monophosphate Response Element-Binding Protein (CREB) Mutant to Seminiferous Tubules Results in Impaired Spermatogenesis1

FSH binding to Sertoli cells is required for optimal production of sperm in mammals. The cAMP response element-binding protein (CREB) is a major mediator of FSH-induced changes in gene expression. To determine whether CREB is required for spermatogenesis, an adenovirus encoding a phosphorylation-defective CREB mutant (AdCREBm1) was used to inhibit CREB activity in Sertoli cells. Addition of AdCREBm1 to primary rat Sertoli cell cultures completely abolished induction of the CREB-regulated c-fos gene. Injection of an adenovirus encoding ss-galactosidase into the rat testis seminiferous tubules in vivo demonstrated that predominately Sertoli cells were infected by adenovirus. AdCREBm1-directed expression of CREBm1 in seminiferous tubules did not affect Sertoli cell viability, but resulted in the apoptosis of meiotic spermatocyte germ cells within 4 days of adenovirus injection into seminiferous tubules. Disrupted spermatogenesis, defined by at least a 75% reduction of round spermatids, was observed in 42 +/- 5.8% of seminiferous tubules 14 days after AdCREBm1 infection, whereas using this criteria, testes injected with a control adenovirus did not display disrupted spermatogenesis. These data demonstrate that AdCREBm1 causes apoptosis and elimination of germ cells and suggest that CREB is required to produce a Sertoli cell-derived factor that is critical for germ cell survival.FSH binding to Sertoli cells is required for optimal production of sperm in mammals. The cAMP response element-binding protein (CREB) is a major mediator of FSH-induced changes in gene expression. To determine whether CREB is required for spermatogenesis, an adenovirus encoding a phosphorylation-defective CREB mutant (AdCREBm1) was used to inhibit CREB activity in Sertoli cells. Addition of AdCREBm1 to primary rat Sertoli cell cultures completely abolished induction of the CREB-regulated c-fos gene. Injection of an adenovirus encoding ss-galactosidase into the rat testis seminiferous tubules in vivo demonstrated that predominately Sertoli cells were infected by adenovirus. AdCREBm1-directed expression of CREBm1 in seminiferous tubules did not affect Sertoli cell viability, but resulted in the apoptosis of meiotic spermatocyte germ cells within 4 days of adenovirus injection into seminiferous tubules. Disrupted spermatogenesis, defined by at least a 75% reduction of round spermatids, was observed in 42 +/- 5.8% of seminiferous tubules 14 days after AdCREBm1 infection, whereas using this criteria, testes injected with a control adenovirus did not display disrupted spermatogenesis. These data demonstrate that AdCREBm1 causes apoptosis and elimination of germ cells and suggest that CREB is required to produce a Sertoli cell-derived factor that is critical for germ cell survival.

Suppression of Autoimmune Diabetes by Soluble Galectin-1

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the β-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic β-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of β-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4+ T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause β-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed β-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or β-cell toxicity in NOD mice, the use of gal-1 to control β-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.

PIG-TO-MONKEY ISLET XENOTRANSPLANTATION USING MULTI-TRANSGENIC PIGS

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3‐galactosyltransferase gene‐knockout background with ubiquitous expression of human CD46, with islet beta cell‐specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4‐lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin‐diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti‐CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi‐transgenic islet grafts did not demonstrate consistency in regard to long‐term success, with only two of five demonstrating function beyond 5 months.

Dendritic cells expressing transgenic galectin-1 delay onset of autoimmune diabetes in mice.

Type 1 diabetes (T1D) is a disease caused by the destruction of the β cells of the pancreas by activated T cells. Dendritic cells (DC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1−/− lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-γ-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-γ of T cells in response to β cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete β cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated.

Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies.

Abstract: Introduction:  Pig islets have been proposed as an alternative to human islets for clinical use, but their use is limited by rejection. The availability of genetically modified pigs devoid of α1,3‐galactosyltransferase might provide islets more suitable for xenotransplantation. To limit the costs involved in the logistics and health care of pigs for clinical xenotransplantation, we have studied whether younger, rather than older, pigs that are typically preferred can be used as islet donors.

Harmful Delayed Effects of Exogenous Isolation Enzymes on Isolated Human Islets: Relevance to Clinical Transplantation

The isolation process exposes human pancreatic islets to exogenous isolation enzymes. Exposure to these enzymes, as a result of intraductal injection in the pancreas or simple contact of islets with enzyme components, causes internalization into the islet cells of enzymes and their by‐products. Human islets exposed to Liberase‐HI exhibit a decreased insulin secretory ability that correlates with the time of exposure. This phenomenon is paralleled by increased expression of adhesion molecules (CD106 and CD62p) and activation of apoptotic pathways (Bax and Bcl‐2) in islet cells. Increased functional impairment is also observed after islet transplantation in diabetic immunodeficient mice. Experimental exposure of islet grafts to exogenous isolation enzymes causes intense inflammation (CD11b positive cells) at the transplant site and it was associated with sickness behavior and eventually death of mouse recipients. The extent of these adverse effects likely deceives the standard qualitative protocols currently in use to assess islet quality in vitro. Reducing the secondary effects of exogenous isolation enzymes on isolated human islets may be crucial to enhance the quality of islets as tissue grafts.

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. RESEARCH DESIGN AND METHODS The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. RESULTS Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. CONCLUSIONS Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.

Angiopoietin-1 Production in Islets Improves Islet Engraftment and Protects Islets From Cytokine-Induced Apoptosis

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a small fraction of implanted islets become engrafted. The underlying mechanisms remain elusive. To probe the mechanism of islet revascularization, we determined the effect of angiopoietin-1 (Ang-1), a proangiogenic and antiapoptotic factor, on the survival, function, and revascularization of transplanted islets using a syngeneic model. Islets were transduced with adenoviruses expressing Ang-1 or control LacZ, followed by transplantation under the renal capsule. Diabetic mice receiving a marginal mass of 150 islets pretransduced with Ang-1 vector exhibited near normoglycemia posttransplantation. In contrast, diabetic mice receiving an equivalent islet mass pretransduced with control vector remained hyperglycemic. At 30 days posttransplantation, mice were killed and islet grafts retrieved for immunohistochemistry. Islet grafts with elevated Ang-1 production retained significantly increased microvascular density, improved glucose profiles, and increased glucose-stimulated insulin release. Cultured islets expressing Ang-1 displayed improved viability and enhanced glucose-stimulated insulin secretion in the presence of cytokines. In contrast, control islets exhibited increased apoptosis and diminished glucose-stimulated insulin release in response to cytokine treatment. These results indicate that Ang-1 confers a cytoprotective effect on islets, enhancing islet engraftment and preserving functional islet mass in transplants.