Production of Transgenic Tilapia with Brockmann Bodies Secreting [desThrB30] Human Insulin

BACKGROUND: Tilapia are commercially important tropical fish which, like many teleosts, have anatomically discrete islet organs called Brockmann bodies. When transplanted into diabetic nude mice, tilapia islets provide long-term normoglycemia and mammalian-like glucose tolerance profiles. METHODS: Using site-directed mutagenesis and linker ligation we have "humanized" the tilapia insulin gene so that it codes for [desThrB30] human insulin while maintaining the tilapia regulatory sequences. Following microinjection into fertilized eggs, we screened DNA isolated from whole fry shortly after hatching by PCR. Positive fish were grown to sexual maturity and mated to wild-types and positive Fl's were further characterized. RESULTS: Human insulin was detected in both serum and in the clusters of beta cells scattered throughout the Brockmann bodies. Surrounding non-beta cells as well as other tissues were negative indicating beta cell specific expression. Purification and sequencing of both A-and B-chains verified that the insulin was properly processed and humanized. CONCLUSIONS: After extensive characterization, transgenic tilapia could become a suitable, inexpensive source of islet tissue that can be easily mass-produced for clinical islet xenotransplantation. Because tilapia islets are exceedingly resistant to hypoxia by mammalian standards, transgenic tilapia islets should be ideal for xenotransplantation using immunoisolation techniques.     

Glucose Homeostasis in the Teleost Fish Tilapia: Insights from Brockmann Body Xenotransplantation Studies

Certain teleost fish have large anatomically discrete isletorgans called Brockmann bodies (BBs). When transplanted intostreptozotocin diabetic athymic nude mice, tilapia BBs providelong-term normoglycemia. This has afforded us the opportunityto examine tilapia islet in vivo function in a non-piscine environmentand compare this with in vivo function in the donor species.As expected, fasting and non-fasting glycemic levels in long-termmurine recipients of tilapia BBs were analogous to correspondingvalues in donor tilapia, but, surprisingly, tilapia BB graftsprovided mammalian-like glucose tolerance profiles. Teleostfish, in general, are severely glucose intolerant. When glucosetolerance tests were performed in tilapia, the mean glucosedisappearance rates were very low; however, diabetic nude micebearing long-term tilapia BB grafts were extremely glucose responsive.This suggested a severe or absolute peripheral resistance tothe glucostatic effects of insulin. Using Western blotting withpolyclonal antibodies and then confirmed by Northern analysis,tilapia peripheral tissues appear to be devoid of GLUT-4, theinsulin-sensitive glucose transporter responsible for the hypoglycemiceffect of insulin in mammals, but not GLUT-1, the insulin independentglucose transporter. This may explain why tilapia, and possiblyother teleost fish, are severely glucose intolerant after pharmacologicglucose-loading. Because tilapia do not tend to consume largequantities of glucose in the wild, it is not surprising thatthey have evolved without a mechanism to move glucose rapidlyfrom the bloodstream into muscle and fat. Nevertheless, insulinstill appears to play an important role in maintaining normoglycemiain tilapia; however, this is mostly likely a result of its effecton glucose uptake in the liver. We also present comparativedata on tilapia beta cell function, quantification of isletcell numbers and types, islet products, insulin gene structureand expression, and beta cell sensitivity to the diabetogenicdrug streptozotocin.     

Integration of nano‐ and biotechnology for beta‐cell and islet transplantation in type‐1 diabetes treatment

Regenerative medicine using human or porcine β‐cells or islets has an excellent potential to become a clinically relevant method for the treatment of type‐1 diabetes. High‐resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell–derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre‐clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology‐based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole‐body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single‐photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin‐deficient pig model to observe transplanted porcine xeno‐islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human‐induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high‐resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.     

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.     

Islet cell engraftment and control of diabetes in rats after transplantation of pig pancreatic anlagen

The insufficient supply of tissue, loss posttransplantation, and limited potential for expansion of beta-cells restrict the use of islet allotransplantation for diabetes. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. We have shown that whole rat pancreatic anlagen isotransplanted into the omentum of rats, or xenotransplanted into costimulatory blocked mice, undergo growth and differentiate into islets surrounded by stoma without exocrine tissue. Isotransplants normalize glucose tolerance in diabetic hosts. Here, we show that embryonic day 29 porcine pancreas transplanted into the omentum of adult diabetic rats undergoes endocrine tissue differentiation over 20 wk and normalizes body weights and glucose tolerance. Unlike rat-to-rodent transplants, individual alpha- and beta-cells engraft without a stromal component, and no immunosuppression is required for pig-to-rat transplants. Herein is described a novel means to effect the xenotransplantation of individual islet cells across a highly disparate barrier.     

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.     

The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.     

Isolation,transplantation, and functional studies of adult porcine islets of Langerhans

Transplantation of islets of Langerhans is a possible treatment for type-I diabetes mellitus. However, there is a shortage of donors for such transplantations and the pig may be an alternative source of donor organs. The aims of the study reported here were to establish a method for adult porcine islet isolation that was based on enzymatic digestion using Liberase PI in a semiautomatic set-up, and to evaluate the in vitro and in vivo function of isolated islets. After overnight culture, isolated islets, from five of seven batches, had poor insulin response to an in vitro glucose challenge that was only partially increased by additional challenge with arginine. More than 50% of DNA and 90% of the insulin content was lost during a one-week culture period. With some batch-to-batch variation, in 15 of 25 cases, 4,000 to 7,000 porcine islets cured streptozotocin diabetic nude mice within three weeks following transplantation. In conclusion, it is possible to isolate viable islets from adult pigs, using a semiautomatic set-up. With batch-to-batch variation, the islets are able to revert diabetes mellitus when transplanted to diabetic nude mice.     

MHC-mismatched islet allografts are vulnerable to autoimmune recognition in vivo

When transplanted into type 1a diabetic recipients, islet allografts are subject both to conventional allograft immunity and, presumably, to recurrent autoimmune (islet-specific) pathogenesis. Importantly, CD4 T cells play a central role both in islet allograft rejection and in autoimmune disease recurrence leading to the destruction of syngeneic islet transplants in diabetic NOD mice. However, it is unclear how NOD host MHC class II (I-A(g7))-restricted, autoreactive CD4 T cells may also contribute to the recognition of allogeneic islet grafts that express disparate MHC class II molecules. We hypothesized that islet-specific CD4 T cells can target MHC-mismatched islet allografts for destruction via the "indirect" (host APC-dependent) pathway of Ag recognition. To test this hypothesis, we determined whether NOD-derived, islet-specific CD4 T cells (BDC-2.5 TCR transgenic cells) could damage MHC-mismatched islets in vivo independent of conventional allograft immunity. Results demonstrate that BDC-2.5 CD4 T cells can vigorously destroy MHC class II-disparate islet allografts established in NOD.scid recipients. Tissue injury is tissue-specific in that BDC-2.5 T cells destroy donor-type islet, but not thyroid allografts established in the same NOD.scid recipient. Furthermore, BDC-2.5 CD4 T cells acutely destroy MHC class II-deficient islet allografts in vivo, indicating that autoimmune pathogenesis can be completely independent of donor MHC class II expression. Taken together, these findings indicate that MHC-mismatched islet allografts can be vulnerable to autoimmune pathogenesis triggered by autoreactive CD4 T cells, presumably through indirect autoantigen recognition in vivo.     

Transcriptional profiling of stress response in cultured porcine islets

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1β, TNF-α, and IFN-γ), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and β cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.     

Islet cell transplantation for insulin-dependant diabetes mellitus: perspectives from the present and prospects for the future

The long-term complications of insulin-dependant diabetes mellitus have become a major health care problem, and it is now clear that they arise from inadequate homeostatic control of blood glucose by injected replacement insulin. Transplantation of pancreatic islets is arguably the most logical approach to restoring metabolic homeostasis in people with diabetes. This review looks at the current status of human islet transplantation and the problems that remain. These include: (1) the limited supply of human islet tissue available for transplantation; (2) the adverse effects of current immunosuppressive protocols on diabetic patients; (3) the problems of primary nonfunction of the transplanted islets; (4) the rejection of islets; and (5) the recurrence of autoimmune diabetic disease. Some of the approaches that might solve these problems are then examined: (1) immune modulation to reduce or prevent immune attack by the recipient's immune system; (2) immunoisolation to prevent recognition of the islet graft; (3) induction of tolerance; (4) xenotransplantation using islets derived from animals; and (5) gene therapy.     

The cytoprotection of chitosan based hydrogels in xenogeneic islet transplantation: An in vivo study in streptozotocin-induced diabetic mouse

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487 ± 46 to 148 ± 32 at one day postoperation and maintained in the range of 201 ± 36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29 ± 0.06 to 1.69 ± 0.65 μg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.     

Differential susceptibility of allogeneic targets to indirect CD4 immunity generates split tolerance

CD4 T cells frequently help to activate CD8 T and B cells that effect transplant rejection. However, CD4 T cells alone can reject transplants, either directly or indirectly. The relative effectiveness of indirect CD4 immunity in rejecting different types of allogeneic grafts is unknown. To address this, we used a TCR transgenic mouse model in which indirect CD4 alloimmunity alone can be studied. We challenged transgenic recipients with hematopoietic cells and shortly thereafter skin transplants that could only be rejected indirectly, and observed Ag-specific indirect donor B cell and skin rejection, but not T cell elimination, reflecting a state of split tolerance. Deficiency of indirect CD4 alloimmunity in donor T cell rejection was also apparent when acute indirect rejection of donor islets occurred despite generation and maintenance of mixed T cell chimerism, due to migration of the few passenger T cells into recipient circulation. Although passenger lymphocytes delayed indirect islet rejection, they enhanced rejection by a full repertoire capable of both direct and indirect reactivity. Interestingly, the persistence of chimerism was associated with the eventual development of tolerance, as demonstrated by acceptance of donor skin grafts given late to hematopoietic cell recipients, and hyporesponsiveness of transgenic T cells from islet recipients in vitro. Mechanistically, tolerance was recessive and associated with progressive down-regulation of CD4. Collectively, our data indicate that indirect CD4 immunity is not equally destructive toward different types of allogeneic grafts, the deficiency of which generates split tolerance. The futility of these responses can convert immunity into tolerance.     

In vivo monitoring of pancreatic beta-cells in a transgenic mouse model

We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.     

Glycemic Control Promotes Pancreatic Beta-Cell Regeneration in Streptozotocin-Induced Diabetic Mice

Background

Pancreatic beta-cells proliferate following administration of the beta-cell toxin streptozotocin. Defining the conditions that promote beta-cell proliferation could benefit patients with diabetes. We have investigated the effect of insulin treatment on pancreatic beta-cell regeneration in streptozotocin-induced diabetic mice, and, in addition, report on a new approach to quantify beta-cell regeneration in vivo.

Methodology/Principal Findings

Streptozotocin-induced diabetic were treated with either syngeneic islets transplanted under the kidney capsule or subcutaneous insulin implants. After either 60 or 120 days of insulin treatment, the islet transplant or insulin implant were removed and blood glucose levels monitored for 30 days. The results showed that both islet transplants and insulin implants restored normoglycemia in the 60 and 120 day treated animals. However, only the 120-day islet and insulin implant groups maintained euglycemia (<200 mg/dl) following discontinuation of insulin treatment. The beta-cell was significantly increased in all the 120 day insulin-treated groups (insulin implant, 0.69±0.23 mg; and islet transplant, 0.91±0.23 mg) compared non-diabetic control mice (1.54±0.25 mg). We also show that we can use bioluminescent imaging to monitor beta-cell regeneration in living MIP-luc transgenic mice.

Conclusions/Significance

The results show that insulin treatment can promote beta-cell regeneration. Moreover, the extent of restoration of beta-cell function and mass depend on the length of treatment period and overall level of glycemic control with better control being associated with improved recovery. Finally, real-time bioluminescent imaging can be used to monitor beta-cell recovery in living MIP-luc transgenic mice.     

Development of the islets, exocrine pancreas, and related ducts in the Nile tilapia, Oreochromis niloticus (Pisces: Cichlidae)

Pancreatic development and the relationship of the islets with the pancreatic, hepatic, and bile ducts were studied in the Nile tilapia, Oreochromis niloticus, from hatching to the onset of maturity at 7 months. The number of islets formed during development was counted, using either serial sections or dithizone staining of isolated islets. There was a general increase in islet number with both age and size. Tilapia housed in individual tanks grew more quickly and had more islets than siblings of the same age left in crowded conditions. The pancreas is a compact organ in early development, and at 1 day posthatch (dph) a single principal islet, positive for all hormones tested (insulin, SST-14, SST-28, glucagon, and PYY), is partially surrounded by exocrine pancreas. However, the exocrine pancreas becomes more disseminated in older fish, following blood vessels along the mesenteries and entering the liver to form a hepatopancreas. The epithelium of the pancreatic duct system from the intercalated ducts to the main duct entering the duodenum was positive for glucagon and SST-14 in 8 and 16 dph tilapia. Individual insulin-immunopositive cells were found in one specimen. At this early stage in development, therefore, the pancreatic duct epithelial cells appear to be pluripotent and may give rise to the small islets found near the pancreatic ducts in 16-37 dph tilapia. Glucagon, SST-14, and some PPY-positive enteroendocrine cells were present in the intestine of the 8 dph larva and in the first part of the intestine of the 16 dph juvenile. Glucagon and SST-14-positive inclusions were found in the apical cytoplasm of the mid-gut epithelium of the 16 dph tilapia. These hormones may have been absorbed from the gut lumen, since they are produced in both the pancreatic ducts and the enteroendocrine cells. At least three hepatic ducts join the cystic duct to form the bile duct, which runs alongside the pancreatic duct to the duodenum.     

Clusterin expression in porcine endocrine cells during islet development.

Clusterin is a well-known glycoprotein expressed by many cell types involved in multiple physiological functions. In rat pancreatic tissue it is expressed along with islet cell development and found to be involved in regeneration of pancreatic endocrine cells after various types of tissue injuries. These results led us to propose that clusterin might play a crucial role in organization and assembling processes of islet cells during pre- and postnatal development. Therefore, the aim of this study was to find out whether and in which cell type clusterin is expressed during islet cell organization in the porcine species which could play a future role in the field of xenotransplantation. For this purpose we examined the expression pattern of clusterin at different developing stages in the porcine pancreas by double-immunostaining with antibodies against chromogranin A and clusterin, and clarified whether distinct islet hormones were coexpressed with clusterin. Further, we checked by RT-PCR whether clusterin was locally expressed or possibly locally bound to the corresponding receptor. In newborn and up to 3-month-old animals clusterin was found to be expressed in a special cell type which is closely associated and intermingled with other endocrine cells. In fully developed adult islets clusterin-cells then reorganize and were found to be mainly localized in the mantle area of Langerhans islets. Double-immunostaining with antibodies against clusterin and different islet hormones such as insulin, glucagon, and somatostatin clearly demonstrate that clusterin expression was found in an own special cell type and it is also present in a subset of glucagon producing A-cells. Taken together, our results show that clusterin expression in porcine species is found in an own, as yet unidentified cell type during postnatal developmental stages, and probably labels immature precursor cells in adult animals, which finally have the potential to differentiate into glucagon-expressing cells.     

Quantitative micro positron emission tomography (PET) imaging for the in vivo determination of pancreatic islet graft survival

Islet transplantation is an attractive approach for treating type-1 diabetes, but there is a massive loss of transplanted islets. It is currently only possible to estimate islet mass indirectly, through measurement of circulating C-peptide and insulin levels. This type of estimation, however, is not sufficiently sensitive or reproducible for follow-up of individuals who have undergone islet transplantation. Here we show that islet graft survival could be assessed for 1 month in diabetic NOD mice using 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG)-positron emission tomography (PET) technology, the PET signal reflecting insulin secretory capacity of transplanted islets. Expression of the gene encoding viral interleukin-10 (vIL-10), was measurable in real time with PET scanning. Additionally, we addressed the clinical potential of this approach by visualizing transplanted islets in the liver, the preferred clinical transplantation site. We conclude that quantitative in vivo PET imaging is a valid method for facilitating the development of protocols for prolonging islet survival, with the potential for tracking human transplants.     

Adhesion of pancreatic beta cells to biopolymer films

Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 μm) as compared with large islets (>125 μm), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium‐free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 676–685, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Incorporation of Bone Marrow Cells in Pancreatic Pseudoislets Improves Posttransplant Vascularization and Endocrine Function

Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10³ cells. To create bone marrow cell-enriched pseudoislets 2×10³ islet cells were co-cultured with 2×10³ bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.