Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.     

Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic beta cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between beta cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of beta cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect beta cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect beta cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.     

Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.     

Erratum to: Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Aims/hypothesis  

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process.     

Morphology, cytochemical features, and membrane phenotype of HLA-DR+ interstitial cells in the human pancreas

The morphology, histological distribution, surface, and enzymatic phenotype of pancreatic HLA-DR+ cells were studied on seven human pancreata, removed from cadaver donors. Frozen and paraffin-embedded pancreas sections were stained with a battery of monoclonal antibodies by indirect immunofluorescence, immunoperoxidase, and immunophosphatase techniques. Two type of cells were found to express HLA-DR surface molecules: endothelial cells and nonfibroblastic non-B and non-T interstitial elements. The latter cells, which were localized both in the exocrine and endocrine portions of the organ, were distinguished in two main families (macrophagic and dendritic) according to their morphology, surface phenotype, and lysosomal enzymatic activities. The phenotype of cells belonging to macrophagic cell family was the following: Leu M1+, Leu M2+, Leu M3+, OKM1+, and OKT6-. In addition these cells were positive for the expression of lysosomal enzymes such as alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP). The "dendritic" cell family comprised, among others, cells that were characterized by the presence of numerous finger-like projections, the absence of Leu M1, Leu M2, Leu M3, OKM1, OKT6 surface antigens, and by the negativity for ANAE and AP. These "dendritic looking cells" (DLC) constituted the most represented DR+ cell type within pancreatic islets. The demonstration of dendritic cells within human islets may justify, in humans also, in vitro procedures of intra-islet dendritic cell removal prior to transplantation, in the attempt of islet rejection prevention.     

Beta‐cell replacement strategies for diabetes

Diabetes is characterized by elevated levels of blood glucose as a result of insufficient production of insulin from loss or dysfunction of pancreatic islet β‐cells. Here, we review several approaches to replacing β‐cells that were recently discussed at a symposium held in Kyoto, Japan. Transplant of donor human islets can effectively treat diabetes and eliminate the need for insulin injections, supporting research aimed at identifying abundant supplies of cells. Studies showing the feasibility of producing mouse islets in rats support the concept of generating pigs with human pancreas that can serve as donors of human islets, although scientific and ethical challenges remain. Alternatively, in vitro differentiation of both human embryonic stem cells and induced pluripotent stem cells is being actively pursued as an islet cell source, and embryonic stem cell‐derived pancreatic progenitor cells are now in clinical trials in North America in patients with diabetes. Macro‐encapsulation devices are being used to contain and protect the cells from immune attack, and alternate strategies of immune‐isolation are being pursued, such as islets contained within long microfibers. Recent advancements in genetic engineering tools offer exciting opportunities to broaden therapeutic strategies and to probe the genetic involvement in β‐cell failure that contributes to diabetes. Personalized medicine might eventually become a possibility with genetically edited patient‐induced pluripotent stem cells, and the development of simplified robust differentiation protocols that ideally become standardized and automated. Additional efforts to develop a safe and effective β‐cell replacement strategy to treat diabetes are warranted.     

Extracellular matrix molecules and their potential contribution to the function of transplanted pancreatic islets

Extracellular matrix (ECM) molecules are responsible for structural and biochemical support, as well as for regulation of molecular signalling and tissue repair in many organ structures, including the pancreas. In pancreatic islets, collagen type IV and VI, and laminins are the most abundant molecules, but other ECM molecules are also present. The ECM interacts with specific combinations of integrin α/β heterodimers on islet cells and guides many cellular processes. More specifically, some ECM molecules are involved in beta cell survival, function and insulin production, while others can fine tune the susceptibility of islet cells to cytokines. Further, some ECM induce release of growth factors to facilitate tissue repair. During enzymatic isolation of islets for transplantation, the ECM is damaged, impacting islet function. However, restoration of the ECM in human islets (for example by adding ECM to the interior of immunoprotective capsules) has been shown to enhance islet function. Here, we provide current insight into the role of ECM molecules in islet function and discuss the clinical potential of ECM manipulation to enhance pancreatic islet function and survival.     

The bioartificial pancreas: progress and challenges

Diabetes remains a devastating disease, with tremendous cost in terms of human suffering and healthcare expenditures. A bioartificial pancreas has the potential as a promising approach to preventing or reversing complications associated with this disease. Bioartificial pancreatic constructs are based on encapsulation of islet cells with a semipermeable membrane so that cells can be protected from the host's immune system. Encapsulation of islet cells eliminates the requirement of immunosuppressive drugs, and offers a possible solution to the shortage of donors as it may allow the use of animal islets or insulin-producing cells engineered from stem cells. During the past 2 decades, several major approaches for immunoprotection of islets have been studied. The microencapsulation approach is quite promising because of its improved diffusion capacity, and technical ease of transplantation. It has the potential for providing an effective long-term treatment or cure of Type 1 diabetes.     

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

AIMS/HYPOTHESIS: Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. METHODS: Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. RESULTS: Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. CONCLUSIONS/INTERPRETATION: The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.     

Leptin modulates beta cell expression of IL-1 receptor antagonist and release of IL-1beta in human islets

High concentrations of glucose induce beta cell production of IL-1beta, leading to impaired beta cell function and apoptosis in human pancreatic islets. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of IL-1beta and protects cultured human islets from glucotoxicity. Therefore, the balance of IL-1beta and IL-1Ra may play a crucial role in the pathogenesis of diabetes. In the present study, we observed expression of IL-1Ra in human pancreatic beta cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro, chronic exposure of human islets to leptin, a hormone secreted by adipocytes, decreased beta cell production of IL-1Ra and induced IL-1beta release from the islet preparation, leading to impaired beta cell function, caspase-3 activation, and apoptosis. Exogenous addition of IL-1Ra protected cultured human islets from the deleterious effects of leptin. Antagonizing IL-1Ra by introduction of small interfering RNA to IL-1Ra into human islets led to caspase-3 activation, DNA fragmentation, and impaired beta cell function. Moreover, siIL-1Ra enhanced glucose-induced beta cell apoptosis. These findings demonstrate expression of IL-1Ra in the human beta cell, providing localized protection against leptin- and glucose-induced islet IL-1beta.     

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Aims/hypothesis

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process.

Methods

Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays.

Results

The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3.

Conclusions/interpretation

Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

     

Islet transplantation at the Diabetes Research Institute Japan

Since the Edmonton Protocol was announced, more than 600 patients with type 1 diabetes at more than 50 institutions have received islet transplantation to treat their disease. We recently established a new islet isolation protocol, called the Kyoto Islet Isolation Method, based on the Ricordi method. It includes an in-situ cooling system for pancreas procurement, pancreatic ductal protection, a modified two-layer (M-Kyoto /perfluorochemical [PFC]) method of pancreas preservation, and a new islet purification solution (Iodixanol-based solution). Using this islet isolation method, we isolated islets from 19 human pancreata of non-heart-beating donors and transplanted 16 preparations into seven patients with type 1 diabetes between April 7, 2004 and November 18, 2005. The percentage of those meeting the release criteria of the Edmonton Protocol was more than 80%. We also performed living-donor transplantation of islets for unstable diabetes on January 19, 2005. Establishment of this method enables us to make diabetic patients insulin-independent, using islets not only from two or three pancreata of non-heart-beating donors but also using islets from half a pancreas from a living donor.     

In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells

Aims/hypothesis The neogenesis of islets from cultured human adult pancreatic tissue has been reported. The islet progenitors have been thought to be ductal cells. Since previous experiments have been contaminated by a number of pre-existing islet cells, we examined their involvement in islet cell neogenesis.Methods Fresh human pancreatic cells with different purities of islet cells were grown in monolayer culture and labelled with bromodeoxyuridine. Transitional cells were analysed by double immunofluorescence staining. For purified ductal cell culture, pre-existing islets were eliminated on a magnetic cell separation system.Results We confirmed that less than 1% of the endocrine cells proliferated, mainly during the first 48 h of culture. However, a 10-fold larger proportion of the cells acquired a transitional phenotype by starting to coexpress the ductal marker cytokeratin 19 (CK19). These cells represented more than 10% of all endocrine cells after 1 day in culture, and 6% at 5 days of culture. Using magnetic cell sorting, we eliminated cells expressing neural cell adhesion molecule (N-CAM), after which we obtained 99.7% pure non-endocrine CK19-rich cell populations. These cell populations could be expanded in vitro. However, their endocrine differentiation capacity was severely reduced as compared with the original mixed cell cultures.Conclusions/interpretation These results suggest that islet neogenesis in this culture system at least partly represents the de-differentiation of islet cells into a duct-cell-like phenotype, with further re-differentiation in appropriate conditions. The plasticity of differentiated human pancreatic cell types may thus be an important mechanism of human pancreas regeneration.     

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes

Aims/hypothesis

Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets.

Methods

Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS.

Results

Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45⁺ leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c⁺ cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets.

Conclusions/interpretation

Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.     

The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells

Aims/hypothesis

The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue.

Methods

HDCs (n=4), NPIs (n=9) and human islets (n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days.

Results

HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control (p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups.

Conclusions/interpretation

Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance.