Islet Culture Research Articles

Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling.

Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two ex vivo models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation.

Quantitative Proteomics Evaluation of Human Multipotent Stromal Cell for β-Cell Regeneration

Human multipotent stromal cells (hMSC) are one of the most versatile cell types used in regenerative medicine, due to their ability to respond to injury, combat infection and disease. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSC lines is donor-specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSC are lengthy, costly and higher throughput assays are needed. Therefore, a high-throughput quantitative proteomics assay was developed to screen β-cell regenerative potency of donor-derived hMSC lines. Using quantitative proteomics, coupled with machine learning, we identified 16 proteins contained within hMSC conditioned media that effectively identify β-cell regenerative hMSC. This protein signature was validated using in vitro human islet culture assay with multiparametric flow cytometry, ELISA and the potency was confirmed by in vivo recovery of hyperglycemia in STZ-treated mice. For the first time, we demonstrated that quantitative proteomics can determine sample-specific protein signatures that can be used to classify previously uncharacterized donor-derived hMSC lines for β-cell regenerative clinical applications.

Examining the potential for porcine-derived islet cells to harbour viral pathogens.

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal-derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture.

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

Aims/hypothesisGenetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available.MethodsWe used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells.ResultsDRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5–7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet−1 h−1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 μm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 μm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 μl min−1 [g pancreas]−1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats.Conclusions/interpretationThe present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Hypoxia-Induced Damage in Human Islets Is Reduced With the Use of Mesenchymal Stem Cell–Preconditioned Medium

BackgroundMesenchymal stem cells (MSCs) are protective for islets when cotransplanted in a hypoxic environment. However, the risk of neoplasia is increased when MSCs are transplanted into immunosuppressed patients. This initial study aimed to investigate whether the production of protective factors from MSC can be stimulated by different culture conditions to benefit human islets cultured in hypoxia. MethodsMSC were isolated from human adipose tissue and cultured for 2 days in supplemented Minimum Essential Media α (MEMα) and 21% (21%-MEMα) or 1% oxygen (1%-MEMα). Native MEMα served as control. After MSC harvesting, cell-depleted media were frozen at −20°C until use for human islet culture in 2% oxygen for 72–96 hours before islet characterization. Data were normalized to control islets cultured in native MEMα and 2% oxygen (mean ± SEM). ResultsAfter culture in 21%- or 1%-MEMα, islet recovery increased to 117 ± 12% (NS) and 138 ± 12% (P < .05), respectively. Viability did not change after culture in native MEMα (59 ± 2%), 21%-MEMα (59 ± 3%), or 1%-MEMα (61 ± 3%). Compared with control samples, the glucose stimulation index was increased after culture in 21%-MEMα (P < .05) or 1%-MEMα (P < .05). Overall survival was higher in 1%-MEMα (143 ± 14%) than in 21%-MEMα (119 ± 14%; NS) or native MEMα (P < .05). ConclusionsThis study demonstrates that MSC-preconditioned MEMα increases survival and in vitro function of hypoxic human islets. These findings indicate that hypoxic MSCs seem to produce factors that improve survival of islets suffering from hypoxia.

Research Highlights

Defining Total-body AIDS Virus Burden With Implications for Curative Strategies Estes JD, Kityo C, Ssali F, et al. [published online October 2, 2017]. Nat Med. doi: 10.1038/nm.4411. Human immunodeficiency virus (HIV) reservoirs have an important impact on the potential cure for the disease and will continue to be important in HIV transplant practices, especially when using HIV-positive donors. Despite having undetectable viral loads in serum, patients carry a significant amount of virus in lymphoid tissue (LT) which could result in reactivation of latent viral pools, thus making it very difficult to eradicate the virus. Furthermore, anti-retroviral therapy (ART) penetration into LT is poor. Therefore, viral load may be undetectable in serum; however, a significant amount of virus may still exist in LT, solid organs, and the brain. Interestingly, the penetration for ART into the brain is also limited, and many patients who have undetectable levels of virus in the serum will have high viral loads in the brain. The authors examined HIV reservoirs in humans as well as in rhesus macaques and looked for the virus in LT, gut, brain, heart, kidney, liver, lung, and spleen. To diagnose reservoirs, they looked for DNA, RNA, as well as virus-producing cells. They illustrated that the primary reservoir site was LT, where 98% of the RNA cells resided. Another organ with a high frequency of RNA-positive cells was the lung. Interestingly, the levels of ART in LT were significantly less than that in peripheral blood mononuclear cells and well below optimal concentration for viral suppression. Furthermore, there was a very large residual reservoir of DNA cells in individuals on ART, even in the presence of undetectable plasma viral loads. In the gut, there was almost no decrease of the DNA cell reservoir subsequent to ART. The authors demonstrated that even during effective ART, virus-producing cells persisted in the tissue, despite plasma viremia being less than 50 copies/mL. Ongoing viral production or reactivation of latently infected cells could be potential sources of virus that may reignite infection after treatment interruption. Inhibition of Y1 Receptor Signaling Improves Islet Transplant Outcome Loh K, Shi YC, Walters S, et al. Nat Commun. 2017;8(1):490. Clinical outcomes of pancreatic islet cell transplantation remain unsatisfactory with low survival rate of donor islets and compromised efficiencies of these cells. The authors investigated the effect of Y1 receptor blockers on islets after transplantation. Moreover, they tested the functional capacity of native beta cells at a time when type 1 diabetes becomes apparent, although patients are not yet fully insulin dependent, a condition referred by many as the “honeymoon period” for type 1 diabetics. The addition of Y1 receptor antagonists not only extended the period of normoglycemia in autoimmune-prone diabetic mice in their “honeymoon period,” but also improved outcomes in mice who received an islet cell transplant. Mechanistically, Y1 receptor stimulation reduced the intracellular cyclic adenosine monophosphate level resulting in reduced insulin secretion of pancreatic islet cells. Therefore, Y1 receptor blockers have the potential to increase insulin production, not only in transplanted islet cells but also in native pancreatic beta cells. The authors also investigated whether Y1 deficient islet transplants had a better outcome than wild type islet transplants in diabetic mice by manipulating Y1 receptors in islet cells before transplantation. Mice were either transplanted with a sufficient or a suboptimal number of islets (300 vs 60, respectively). An additional group of mice were transplanted with a minimal number of islets (60) where the Y1 receptor had been deleted. In the last group, the outcome was just as good as in the group of mice who received an adequate number of islets. In the other group who received a deficient number of islets, the outcome was poor, and the mice never became normoglycemic. This finding was repeated by blocking the Y1 receptor with a pharmacological agent (BIB03304). Interestingly, a transient inhibition over 5 days resulted in glycemic control in mice with a minimal islet mass (60). In mice receiving a minimal islet transplant (60) with an absent initial glycemic control could not be obtained initially, normoglycemia had been restored with a delayed Y1 receptor blockade starting from day 9. Similarly, human islet cultures enhanced insulin secretion when BIBO3304 was added to the culture, significantly improving glucose control. Thus, adding Y1 antagonists to the clinical treatment of islet cell transplants may reduce the number of beta cells to achieve normoglycemia.

Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes

Long-term graft survival is an ongoing challenge in the field of islet transplantation. With the growing demand for transplantable organs, therapies to improve organ quality and reduce the incidence of graft dysfunction are of paramount importance. We evaluated the protective role of a recombinant DNA repair protein targeted to mitochondria (Exscien I-III), as a therapeutic agent using a rodent model of pancreatic islet transplantation. We first investigated the effect of therapy on isolated rat islets cultured with pro-inflammatory cytokines (interleukin-1 β, interferon γ, and tumor necrosis factor α) for 48 h and documented a significant reduction in apoptosis by flow cytometry, improved viability by immunofluorescence, and conserved functional potency in vitro and in vivo in Exscien I-III-treated islets. We then tested the effect of therapy in systemic inflammation using a rat model of donor brain death (BD) sustained for a 6-h period. Donor rats were allocated to 4 groups: (non-BD + vehicle, non-BD + Exscien I-III, BD + vehicle, and BD + Exscien I-III) and treated with Exscien I-III (4 mg/kg) or vehicle 30 min after BD induction. Sham (non-BD)-operated animals receiving either Exscien I-III or vehicle served as controls. Islets purified from BD + Exscien I-III-treated donors showed a significant increase in glucose-stimulated insulin release in vitro when compared to islets from vehicle-treated counterparts. In addition, donor treatment with Exscien I-III attenuated the effects of BD and significantly improved the functional potency of transplanted islets in vivo. Our data indicate that mitochondrially targeted antioxidant therapy is a novel strategy to protect pancreas and islet quality from the deleterious effects of cytokines in culture and during the inflammatory response associated with donation after BD. The potential for rapid translation into clinical practice makes Exscien I-III an attractive therapeutic option for the management of brain-dead donors or as an additive to islets in culture after isolation setting.

Generating Mature Islet Cells In Vitro Using Decellularized Murine Pancreas

Generating new islets from a patient's own induced pluripotent stem cells (iPSCs) could address the shortcomings of islet transplantations for the treatment of type 1 diabetes. The maturation process, however, can take months in vivo and does not occur efficiently in vitro indicating that factors are missing from traditional two-dimensional (2D) cultures. The interaction between cells and the extracellular matrix (ECM) is one such factor. Three-dimensional (3D) culturing using ECM can address this deficit. The removal of cells from an organ, through decellularization, can be performed to obtain the ECM. Our aim is to develop a 3D culture system using decellularized murine pancreas to better support the growth and differentiation of iPSC derived islets. Specifically, two decellularization protocols were tested to determine the optimal detergent for removal of all cellular material without loss of ECM structure and associated proteins. Two detergents were tested, Triton X-100 or 3-[(3-Cholamidopropyl) dimethylammonio]-1-Propanesulfonate (CHAPS)). Results showed that both protocols produced a visibly decellularized ECM. Immunohistochemistry (IHC) was performed for key ECM proteins as well as the nuclear stain 4,6-diamidino-2-phenylindole (DAPI). Results of the IHC for the Triton X-100 protocol showed retention of the ECM proteins without the presence of nuclear material. Results for CHAPS are currently being analyzed. Our preliminary results indicate that the generation of decellularized murine pancreas using Triton X-100 and CHAPS is a promising method for 3D islet culturing.

Tocotrienols Stimulate Insulin Secretion of Rat Pancreatic Isolated Islets in a Dynamic Culture.

Tocotrienols (T3) are the naturally occurring vitamin E derivatives that possess antioxidant properties and therapeutic potential in diabetic complications. The bioactivities of the derivatives are determined by the number and arrangement of methyl substitution on the structure. The objective of this study was to determine the effects of T3 derivatives, σ-T3, γ-T3 and α-T3 on insulin secretion of rat pancreatic islets in a dynamic culture. Pancreatic islets isolated from male Wistar rats were treated with T3 for 1 h at 37°C in a microfluidic system with continuous operation that provided a stable cell culture environment. Glucose (2.8 mM and 16.7 mM, as basal and stimulant, respectively) and potassium chloride (KCl) (30 mM) were added to the treatment in calcium free medium. The supernatant was collected for insulin measurements. Short-term exposure (1 h) of σ-T3 to β cells in the stimulant glucose condition significantly potentiated insulin secretion in a dose-dependent manner. γ-T3 and α-T3 also displayed dosedependent effect but were less effective in the activation of insulin secretion. Essentially, KCl, a pancreatic β cell membrane depolarizing agent, added into the treatment further enhanced the insulin secretion of σ-T3, γ-T3 and α-T3 with ED50 values of 504, 511 and 588 µM, respectively. The findings suggest the potential of σ-T3 in regulating glucose-stimulated insulin secretion (GSIS) in response to the intracellular calcium especially in the presence of KCl.

Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival.

BackgroundType 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency.MethodologyThe oxygen tension inside isolated human islets of different sizes was simulated under varying oxygen environments using a computational in silico model. In vitro human islet viability was also assessed after culturing in different oxygen conditions. Correlation between simulation data and experimentally measured islet viability was examined. Using these in vitro viability data of human islets, the effect of islet diameter and oxygen tension of the culture environment on islet viability was also analyzed using a logistic regression model.Principal findingsComputational simulation clearly revealed the oxygen gradient inside the islet structure. We found that oxygen tension in the islet core was greatly lower (hypoxic) than that on the islet surface due to the oxygen consumption by the cells. The hypoxic core was expanded in the larger islets or in lower oxygen cultures. These findings were consistent with results from in vitro islet viability assays that measured central necrosis in the islet core, indicating that hypoxia is one of the major causes of central necrosis. The logistic regression analysis revealed a negative effect of large islet and low oxygen culture on islet survival.Conclusions/SignificanceHypoxic core conditions, induced by the oxygen gradient inside islets, contribute to the development of central necrosis of human isolated islets. Supplying sufficient oxygen during culture could be an effective and reasonable method to maintain isolated islets viable.

Ex vivo Pretreatment of Islets with Mitomycin C

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

Induction of IAPP amyloid deposition and associated diabetic abnormalities by a prion-like mechanism.

Although a large proportion of patients with type 2 diabetes (T2D) accumulate misfolded aggregates composed of the islet amyloid polypeptide (IAPP), its role in the disease is unknown. Here, we show that pancreatic IAPP aggregates can promote the misfolding and aggregation of endogenous IAPP in islet cultures obtained from transgenic mouse or healthy human pancreas. Islet homogenates immunodepleted with anti-IAPP-specific antibodies were not able to induce IAPP aggregation. Importantly, intraperitoneal inoculation of pancreatic homogenates containing IAPP aggregates into transgenic mice expressing human IAPP dramatically accelerates IAPP amyloid deposition, which was accompanied by clinical abnormalities typical of T2D, including hyperglycemia, impaired glucose tolerance, and a substantial reduction on β cell number and mass. Finally, induction of IAPP deposition and diabetic abnormalities were also induced in vivo by administration of IAPP aggregates prepared in vitro using pure, synthetic IAPP. Our findings suggest that some of the pathologic and clinical alterations of T2D might be transmissible through a similar mechanism by which prions propagate in prion diseases.

Biomimetic Surfaces Supporting Dissociated Pancreatic Islet Cultures

This study describes a method to screen biomimetic surfaces based on intracellular insulin content of either fully or partly dissociated primary endocrine islet tissue. It is challenging to maintain endocrine pancreatic islets and more so, dissociated ones. Physiological activity of isolated islet cells in vitro declines due to loss of cell-to-cell and cell-to-extracellular matrix interactions. An in vitro model was developed to evaluate specific extracellular binding components potentially affecting islet biology, with the intention to identify in vivo-like peptides promoting survival and function. Synthetic peptides were bound to low-fouling carboxy-methyl-dextran surfaces, effectively presenting defined surfaces while minimizing non-specific interactions. These biomimetic surfaces were screened based on intracellular insulin content of applied mouse primary islet tissue by analysis with an anti-insulin cell-ELISA. Three active biomimetic surfaces were identified, two laminin- (IKLLI and PDSGR) and one cadherin (HAVDI)-derived, which supported adhesion and survival of insulin-containing cultures for 5days, respectively suggesting a benefit from both cell-extracellular matrix and cell–cell interactions. Cells from dissociated islets show progression over 10days on the HAVDI-biomimetic for the insulin immunoreactivity and cell density. The three surfaces did not act additively or synergistically. A favorable reaction to glucose-stimulated insulin secretion on the cadherin-biomimetic indicated the cultures were physiologically functional. This supportive role of biomimetic peptides represents initial progress in defining minimal extracellular binding requirements influencing islet cell physiology. This will influence further optimization of growth surfaces and promote the basic understanding of islet biology. Low-fouling biomimetics are predicted to be applicable to additional diverse culture systems.

Amyloid precursor protein in pancreatic islets.

The amyloid precursor protein (APP) has been extensively investigated for its role in the production of amyloid beta (Aβ), a plaque-forming peptide in Alzheimer's disease (AD). Epidemiological evidence suggests type 2 diabetes is a risk factor for AD. The pancreas is an essential regulator of blood glucose levels through the secretion of the hormones insulin and glucagon. Pancreatic dysfunction is a well-characterized consequence of type 1 and type 2 diabetes. In this study, we have examined the expression and processing of pancreatic APP to test the hypothesis that APP may play a role in pancreatic function and the pathophysiology of diabetes. Our data demonstrate the presence of APP within the pancreas, including pancreatic islets in both mouse and human samples. Additionally, we report that the APP/PS1 mouse model of AD overexpresses APP within pancreatic islets, although this did not result in detectable levels of Aβ. We compared whole pancreas and islet culture lysates by Western blot from C57BL/6 (WT), APP-/- and APP/PS1 mice and observed APP-dependent differences in the total protein levels of GLUT4, IDE and BACE2. Immunohistochemistry for BACE2 detected high levels in pancreatic α cells. Additionally, both mouse and human islets processed APP to release sAPP into cell culture media. Moreover, sAPP stimulated insulin but not glucagon secretion from islet cultures. We conclude that APP and its metabolites are capable of influencing the basic physiology of the pancreas, possibly through the release of sAPP acting in an autocrine or paracrine manner.

Presence of diabetes autoantigens in extracellular vesicles derived from human islets

Beta-cell (β-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. We hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors. Here we report the caspase-independent production of EV by human islets in culture, and the characterization of DAA glutamic acid decarboxylase 65 (GAD65) and zinc transporter 8 (ZnT8), as well as the β-cell resident glucose transporter 2 (Glut2), present within the EV.

Use of additives, scaffolds and extracellular matrix components for improvement of human pancreatic islet outcomes in vitro: A systematic review

ABSTRACTPancreatic islet transplantation is an established treatment to restore insulin independence in type 1 diabetic patients. Its success rates have increased lately based on improvements in immunosuppressive therapies and on islet isolation and culture. It is known that the quality and quantity of viable transplanted islets are crucial for the achievement of insulin independence and some studies have shown that a significant number of islets are lost during culture time. Thus, in an effort to improve islet yield during culture period, researchers have tested a variety of additives in culture media as well as alternative culture devices, such as scaffolds. However, due to the use of different categories of additives or devices, it is difficult to draw a conclusion on the benefits of these strategies. Therefore, the aim of this systematic review was to summarize the results of studies that described the use of medium additives, scaffolds or extracellular matrix (ECM) components during human pancreatic islets culture. PubMed and Embase repositories were searched. Of 5083 articles retrieved, a total of 37 articles fulfilled the eligibility criteria and were included in the review. After data extraction, articles were grouped as follows: 1) “antiapoptotic/anti-inflammatory/antioxidant,” 2) “hormone,” 3) “sulphonylureas,” 4) “serum supplements,” and 5) “scaffolds or ECM components.” The effects of the reviewed additives, ECM or scaffolds on islet viability, apoptosis and function (glucose-stimulated insulin secretion - GSIS) were heterogeneous, making any major conclusion hard to sustain. Overall, some “antiapoptotic/anti-inflammatory/antioxidant” additives decreased apoptosis and improved GSIS. Moreover, islet culture with ECM components or scaffolds increased GSIS. More studies are needed to define the real impact of these strategies in improving islet transplantation outcomes.

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation.

BackgroundNo optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.MethodsHepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed.ResultsThe survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups.ConclusionsTD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

Amyloid formation disrupts the balance between interleukin-1β and interleukin-1 receptor antagonist in human islets

Objectivesβ-cell dysfunction and apoptosis associated with islet inflammation play a key role in the pathogenesis of type 2 diabetes (T2D). Growing evidence suggests that islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to islet inflammation and β-cell death in T2D. We recently showed the role of interleukin-1β (IL-1β)/Fas/caspase-8 apoptotic pathway in amyloid-induced β-cell death. In this study, we used human islets in culture as an ex vivo model of amyloid formation to: (1) investigate the effects of amyloid on islet levels of the natural IL-1 receptor antagonist (IL-1Ra); (2) examine if modulating the IL-1β/IL-1Ra balance can prevent amyloid-induced β-cell Fas upregulation and apoptosis. MethodsIsolated human islets (n = 10 donors) were cultured in elevated glucose (to form amyloid) with or without a neutralizing human IL-1β antibody for up to 7 days. Parallel studies were performed with human islets in which amyloid formation was prevented by adeno-siRNA-mediated suppression of hIAPP expression (as control). β-cell levels of IL-1Ra, Fas, apoptosis as well as islet function, insulin- and amyloid-positive areas, and IL-1Ra release were assessed. ResultsProgressive amyloid formation in human islets during culture was associated with alterations in IL-1Ra. Islet IL-1Ra levels were higher at early stages but were markedly reduced at later stages of amyloid formation. Furthermore, IL-1Ra release from human islets was reduced during 7-day culture in a time-dependent manner. These changes in IL-1Ra production and release from human islets during amyloid formation adversely correlated with islet IL-1β levels, β-cell Fas expression and apoptosis. Treatment with IL-1β neutralizing antibody markedly reduced amyloid-induced β-cell Fas expression and apoptosis, thereby improving islet β-cell survival and function during culture. ConclusionsThese data suggest that amyloid formation impairs the balance between IL-1β and IL-1Ra in islets by increasing IL-1β production and reducing IL-1Ra levels thereby promoting β-cell dysfunction and death. Restoring the IL-1β/IL-1Ra ratio may provide an effective strategy to protect islet β-cells from amyloid toxicity in T2D.

The effect of islet culture on the biphasic pattern of fuel-induced insulin secretion

The insulin secretion pattern by mouse islets is reported to differ from that of human and rat islets in that a strong first phase is followed by an elevated plateau instead of a slowly ascending second phase. This has led to the argument that mouse islets or beta cells may not be a good experimental model. Here, we have tested the role of islet culture on shaping the kinetics of insulin secretion.

Characterization of porcine endogenous retrovirus expression in neonatal and adult pig pancreatic islets.

Pig islets represent an alternative to the current modes of treatment for patients with diabetes. However, the concerns over pathogen transmission including that of PERV limit their immediate, widespread usage in humans. It has been previously demonstrated that PERV copy number and particularly expression levels can vary considerably between individuals and within different tissues of a single animal. In general, expression levels have been found to be particularly low in the pancreas compared to other porcine tissues suggesting a reduced risk associated with the use of this tissue. Data regarding this crucial aspect, however, remain limited and little is known about PERV status of islets themselves, which represent the final product to be transplanted. In addition, comparative analysis of the PERV status of neonatal piglets with adults is important as they are increasingly considered as potential islet donors for xenotransplantation. Tissue samples from 51 neonatal piglets (age between 14 and 21days) and 29 adult pigs were collected from Belgian landrace pigs used for pancreas procurement and islet isolation. Tissue biopsies were used to extract DNA for PERV copy number quantification by qPCR and RNA for PERV expression by qRT-PCR. As expected, PERV expression demonstrated great variation and was significantly lower in pancreas compared to other tissues. More importantly, PERV RNA expression was found to be specifically enriched in pancreatic islets reaching values similar to those found in other tissues such as liver and kidney. Interestingly, this expression was not coupled with the detection of reverse transcriptase in islet cultures or indeed detection of PERV virus. Lung, spleen, and lymph node consistently showed the highest levels of PERV expression. Comparison of PERV in neonatal and adult pigs showed that copy number did not vary significantly from birth to adulthood. PERV expression on the other hand was significantly lower in neonatal pig islets compared to adult islets and did not increase over the period of culture. Our study confirms the low level of PERV expression in whole pancreas in a large population of both neonatal and adult pigs (n=80). The level of PERV expression was however higher in the endocrine tissue than in the exocrine cells. There was no correlation between PERV status in donor PBMCs and islet cells, and no evidence of active replication in in vitro regardless of PERV expression in islet cells. Moreover, neonatal pig islets were found to have significantly lower PERV expression compared to adult islets. Neonatal islets have been suggested as the best choice for xenotransplantation in terms of economic and procurement considerations; the PERV status reported here would also potentially support their use.