Syngeneic Islet Research Articles

Intracranial Transplantation of Pancreatic Islets Attenuates Cognitive and Peripheral Metabolic Dysfunctions in a Rat Model of Sporadic Alzheimer's Disease.

Alzheimer's disease (AD) is often associated with brain insulin resistance and peripheral metabolic dysfunctions. Recently, we developed a model of sporadic AD associated with obesity-related peripheral metabolic abnormalities in Lewis rats using intracerebroventricular administration of streptozotocin (icv-STZ). We aimed to assess the effect of intracranially grafted pancreatic islets on cognitive and peripheral metabolic dysfunctions in the icv-STZ Lewis rats. AD-like dementia associated with obesity was induced in inbred Lewis rats using a single icv-STZ. Two months after icv-STZ, syngeneic islets (100 islets per recipient) were implanted in the cranial subarachnoid cavity of icv-STZ rats. Morris water maze and marble burying tests were used for studying cognitive and behavioral functions. Central and peripheral metabolic alterations were assessed by histological and biochemical assays. The icv-STZ induced increases in food intake, body weight, and blood levels of insulin and leptin without alteration of glucose homeostasis. Grafted islets reduced body weight gain, food consumption, peripheral insulin resistance, and hyperleptinemia. Biochemical and histological analysis of the brain revealed viable grafted islets expressing insulin and glucagon. The grafted islets did not affect expression of brain insulin receptors and peripheral glucose homeostasis. Two months after islet transplantation, cognitive and behavioral functioning in transplanted rats were significantly better than the sham-operated icv-STZ rats. No significant differences in the locomotor activity between transplanted and non-transplanted icv-STZ rats were found. Intracranial islet transplantation attenuates cognitive decline and peripheral metabolic dysfunctions providing a novel therapeutic approach for sporadic AD associated with peripheral metabolic dysfunctions.

Optimization of a Polycaprolactone (PCL) Scaffold for Islet and Cellular Transplantation

Introduction Islet transplantation is a proven effective therapy for patients with type 1 diabetes; however, donor shortage limits this therapy to very few patients with brittle diabetes. Stem cell therapies for beta cell replacement are quickly moving toward clinical application, but the optimal transplant site and delivery vehicle remains unknown. Previous studies have shown that three dimensional support and structure improves islet survival and function. To this end, we have optimized a polycaprolactone (PCL) polymer scaffold as a biocompatible delivery system for beta cell replacement. In these experiments, we used mouse islets as a proof-of-concept system, prior to application of the device for stem cell delivery. Methods PCL scaffolds of varying thickness were evaluated for maximal loading capacity in vitro with 150 mm PLGA fluorescent microspheres and mouse islets. Insulin secretion was tested in vitro using B6 mouse islets in a perifusion chamber (BioRep, Inc., Miami Lakes, FL). For in vivo testing of islet function and survival within the PCL scaffold, 200 syngeneic islets were transplanted to the epididymal fat pad of STZ-induced diabetic B6 mice, either within a PCL scaffold or directly to the epididymal fat pad. Blood glucose was measured in transplant recipient mice three times weekly for 30 days, at which point islet grafts were explanted and mice were monitored for reversion to hyperglycemia. Normal blood glucose was defined as 250mg/dL for all in vivo experiments. Results/Discussion Thicker PCL scaffolds fabricated to 1.2mm thickness showed higher loading capacity compared to 1mm thick devices (409 versus 305 microspheres, n=6 per device; Figure 1A). slets showed a similar loading pattern, confirming ample carrying capacity of the PCL scaffold. Microspheres distributed within the three-dimensional porosity of the PCL scaffold, as seen by confocal microscopy (Figure 1B). Mouse islets show no limitation or delay in insulin secretion as a result of loading within the PCL scaffold on perifusion testing (data not shown). When PCL scaffolds were loaded with syngeneic islets and transplanted to the epididymal fat pad B6 mice, 8 out of 9 (89%) mice returned to normoglycemia, while 3 out of 9 (33%) control mice receiving free islets to the epididymal fat pad returned to normoglycemia (p=0.041; Figure 2). Upon explant of islet grafts from normoglycemic mice at day 30, all mice subsequently returned to hyperglycemia, confirming function of the transplanted islets. Conclusions Our optimized PCL scaffold supports islet survival and function in a syngeneic B6 mouse model, and may provide a delivery system for future stem cell-based beta cell replacement. Research reported in this publication was supported in part by an NIAID T32 training grant from the National Institutes of Health under an award to the University of California, San Francisco (T32AI125222).

Prevascularization of the Subcutaneous Space Improves Survival of Transplanted Mouse Islets

Introduction Beta cell replacement for the treatment of Type I diabetes has previously been limited by donor availability, invasive surgical techniques, and islet survival post-transplant. Transplantation into the subcutaneous space is ideal for graft accessibility and explant if necessary; however, prior investigations have shown that in comparison to other gold-standard sites (i.e., the kidney capsule), this location lacks the rich blood supply required to meet the high metabolic demands of islets. The aim of this study was to identify and demonstrate the efficacy of a novel prevascularization strategy to improve free islet graft survival in the subcutaneous space. Methods To investigate the ideal prevascularization device, four different materials were implanted into the subcutaneous space of B6 albino mice: polypropylene (PP) mesh, smooth PP, texturized polycaprolactone (PCL), and smooth PCL. Vascularity was assessed using indocyanine green fluorescence measurement at 1-week time points up to 6 weeks post implantation. At selected time points, mice were euthanized and specimens were examined using immunofluorescence histology. B6 mice that had been prevascularized for 2 weeks with textured PCL discs were made diabetic with intraperitoneal streptozosin injection (200 mg/kg). The prevascularization discs were removed, and the mice were then transplanted with 200, 400, 600, and 800 syngeneic mouse islets into the disc capsule. Blood glucose was measured for three weeks to monitor for diabetes cure. Intraperitoneal glucose tolerance test (IPGTT) was performed on transplant recipients to compare their function to diabetic and normal controls. Results When normalized to baseline fluorescence, the peak vascularity as measured by ICG fluorescence was determined for texturized PCL at the 2-week time point (Fig. 1). Histological analysis at 2 weeks (Fig. 2a) and 6 weeks (Fig. 2b) post-implantation demonstrated vascular ingrowth (vWF) and minimal foreign body reaction (macrophages, or F4/80). Diabetes reversal was not observed for 200- or 400-islet transplant recipients. 100% diabetes cure was seen with 800-islet transplants, and partial cure (50%) was seen with 600-islet transplants (Fig. 3). When subjected to IPGTT at 2 weeks post transplant, the 600-islet group showed similar blood glucose kinetics to non-diabetic control in cured mice and impaired glucose tolerance in partially cured and failed mice (Fig. 4).Conclusion Texturized PCL induces improved vascularity in the subcutaneous space of mice while inciting minimal foreign body reaction. Free islets transplanted in the subcutaneous space of mice have improved survival in the prevascularized implant capsule, and allow for comparable function to non-diabetic controls. This strategy may represent a safe and effective translational pathway for the application of subcutaneous islet transplant in human subjects in the near future. Research reported in this publication was supported in part by an NIAID T32 training grant from the National Institutes of Health under an award to the University of California, San Francisco (T32AI125222).

A Novel Subcutaneous Pancreatic Islet Transplantation Method Using the New Device of Recombinant Peptide Containing Human Type I Collagen Material with RGD motif (Arg-Gly-Asp)

Introduction Subcutaneous islet transplantation has many advantages including its minimal invasiveness, thus it is considered to be one of the ideal transplant sites for pancreatic islets, compared with the intraportal islet transplantation. However, it is well known that subcutaneous site has poor vascularization. Although the effectiveness of basic fibroblast growth factor (bFGF) on this issue was previously reported, it often causes bleeding, making it difficult to use for diabetic patients. In order to overcome these drawbacks, we herein investigated a new material device, Recombinant Peptide(RCP) containing human type I collagen together with RGD motif (Arg-Gly-Asp), as alternative for bFGF pretreatment. Materials and Methods The RCP (10 days before islet transplantation:RCP 10D group, 4 weeks before islet transplantation:RCP 4W group) or vehicles containing 10 ug of bFGF togetehr with 10 U of heparin (bFGF 10 group) were implanted into the subcutaneous space of streptozotocin-induced diabetic C57BL/6 mice, and marginal amount of syngeneic islets(660 IEQs) were transplanted into the prevascularized space after removal of the devices. The portal vein transplantation group (Ipo group), the group with only vehicle not containing bFGF or heparin (bFGF 0 group), the Sham group, and the group without any prevascularization procedures (None group) were set up and examined as control groups. Results and Discussion The cure rates of the Ipo and bFGF 10 group were significantly higher than those of the Sham and None groups (P <0.01, 0.05). In the RCP groups, the efficiency in the RCP 4W group was better than that in the bFGF 10 group, furthermore, comparable to that in the Ipo group (100% vs. 75% vs. 87.5%). In contrast, the cure rate of the RCP 10D group was significantly lower than that of the RCP 4W and Ipo groups (P <0.01, 0.05). Unlike bFGF, no bleeding and effusion were observed in the RCP groups. The area under the curve of the RCP 4W group in the glucose tolerance test was even better than that of the Ipo group (23,866.5±3372 vs. 31,581.7±3310 min*mg/d), and it was also significantly lower than that of the None, Sham, and RCP 10D groups (P<0.01,resectively). The number of vWF-positive vessels in the bFGF 10 group was significantly higher than that in the None and bFGF 0 groups (P<0.01,resectively), whereas the efficiency of neovascularization in the RCP groups appeared to be lower than the bFGF 10 group, suggesting that the other factors including the compensation of the lost extracellular matrices of the isolated islets, as well as neovascularization, might be crucial for successful subcutaneous islet transplantation. Conclusions The present study showed that subcutaneous islet transplantation could be performed as efficiently as intraportal islet transplantation with the establishment of an optimized protocol by introducing RCP device. In this regard, the use of materials that induce efficient prevascularization would be a novel therapeutic approach for improving subcutaneous islet engraftment. Megumi Goto. Maya Imura.

Evaluation of encapsulating and microporous nondegradable hydrogel scaffold designs on islet engraftment in rodent models of diabetes.

Islet transplantation is a promising therapeutic option for type 1 diabetes mellitus, yet the current delivery into the hepatic portal vasculature is limited by poor engraftment. Biomaterials have been used as a means to promote engraftment and function at extrahepatic sites, with strategies being categorized as encapsulation or microporous scaffolds that can either isolate or integrate islets with the host tissue, respectively. Although these approaches are typically studied separately using distinct material platforms, herein, we developed nondegradable polyethylene glycol (PEG)-based hydrogels for islet encapsulation or as microporous scaffolds for islet seeding to compare the initial engraftment and function of islets in syngeneic diabetic mice. Normoglycemia was restored with transplantation of islets within either encapsulating or microporous hydrogels containing 700 islet equivalents (IEQ), with transplantation on microporous hydrogels producing lower blood glucose levels at earlier times. A glucose challenge test at 1 month after transplant indicated that encapsulated islets had a delay in glucose-stimulated insulin secretion, whereas microporous hydrogels restored normoglycemia in times consistent with native pancreata. Encapsulated islets remained isolated from the host tissue, whereas the microporous scaffolds allowed for revascularization of the islets after transplant. Finally, we compared the inflammatory response after transplantation for the two systems and noted that microporous hydrogels had a substantially increased presence of neutrophils. Collectively, these findings suggest that both encapsulation and microporous PEG scaffold designs allow for stable engraftment of syngeneic islets and the ability to restore normoglycemia, yet the architecture influences islet function and responsiveness after transplantation.

Synergistic Effect of Islet Magnetic Resonance Imaging Using Direct Linking of Ferumoxytol to Surface of PEGylated Islet

Purpose: We previously reported the feasibility of islet magnetic resonance imaging (MRI) using ferumoxytol, which is the only clinically-available ultrasmall superparamagnetic iron oxide. However, the labeling efficacy was not sufficient for its clinical use in islet transplantation. We aimed to evaluate the feasibility of islet MRI using direct linking of ferumoxytol to islet surface through PEGylation. Method: Islets were labeled by surface modification with up to 4% PEG-heparin-ferumoxytol. We compared islet function and viability of control islets and ferumoxytol-heparin-PEGylated islets. Efficacy of labeling with ferumoxytol-heparin-PEGylation and ferumoxytol alone was assessed in both ex vivo and in vivo models. Results: Labeling of islets with up to 2% PEG-heparin-ferumoxytol did not derange ex vivo islet viability and function. The T2* relaxation time was optimal when islets were labeled with 1 to 4% PEG-heparin-ferumoxytol. The labeling intensity in the ex vivo MRI of ferumoxytol-heparin-PEGylated islets was stronger than islets labeled with ferumoxytol alone. In syngeneic renal subcapsular islet transplantation, labeling with ferumoxytol-heparin-PEGylation showed better in vivo labeling efficacy than that of islets labeled with ferumoxytol alone. After labeling with ferumoxytol-heparin-PEGylation, there was a correlation between the total area of visualized islets and the transplanted islet mass in syngeneic mouse intraportal islet transplantation, and the visibility was also confirmed in the preliminary analysis of non-human primate intraportal islet transplantation model. Conclusion: Direct linking of ferumoxytol to islet surface through PEGylation improved the labeling efficacy and it could be used for the islet MRI in clinical islet transplantation. Disclosure H. Lee: None. Y. Kwon: None. H. Kim: None. M.R. Haque: None. G. Kim: None. S. Jin: None. M. Lee: None. Y. Byun: None. J. Kim: None.

Are Liver Cystic Lesions after Islet Transplantation Caused by Streptozotocine?

Introduction: Pancreatic islets (PI) transplanted (Tx) into the portal vein cause a permanent exposure of surrounding liver tissue to insulin in high concentration. There were published several papers pronouncing the hypothesis of a direct triggering effect of Tx PI on a development of focal morphological changes in liver tissue (tumors, cysts). At the same time, streptozotocine (STZ) was supposed to play a minor but not negligible role in this phenomenon. Methods: Syngeneic pancreatic islets were transplanted into the healthy or diabetic (50mg/kg STZ) Brown Norway rats. Group A (n=5)-healthy, Tx of 450 PI into the portal vein; Group B (n=17)-STZ diabetes, Tx of 450 PI into the portal vein; Group C (n=8)-STZ diabetes, Tx of 100 PI under the left kidney capsule; Group D (n=7)-STZ diabetes, human insulin pellet s.c. (changed each 6th week); All recipients were monitored for 10 months including MRI of liver. At the end of study each liver was examined by histology. The ultrastructure of liver was examined by electron microscopy (TEM) 3 hours after injection of STZ. Results: Tx of PI in suboptimal number normalized fasting blood glucose levels but did not normalize glucose assimilation during IVGTT in all diabetic animals. While liver of Group A recipients remained intact, in animals of Groups B and C multiple voluminous cystic complexes with cholangiocyte cubic epithelium lining were detected after 10 months irrespective of Tx site. In animals of Group D, blood glucose levels were nearly normalized six months after STZ injection and there was no need to replace pellets later on. There were simple, individual cystic lesions within liver in this group after 10 months. There were not any typical lesions in TEM examined samples. Conclusions: The injection of STZ and subsequent Tx of PI can cause cholagiogenic cystic lesions development in rats. The therapy by human insulin did not stimulate the development of cystic lesions by the equal efficiency. STZ itself cannot cause the cystic transformation of BN rat liver. Disclosure L. Kosinová: None. A. Patikova: None. E. Sticova: None. A. Vojtíšková: None. L. Bartonova: None. M. Cahova: None. J. Kriz: None.

A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver

Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice. In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice. Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Display of CD47 protein on pancreatic islet grafts improves engraftment following intraportal transplantation

Abstract Intraportal islet transplantation is a curative therapy for type 1 diabetes. However, early islet engraftment is hampered by a thrombotic and inflammatory reaction defined as an instant blood-mediated inflammatory reaction (IBMIR). The CD47 molecule is an important regulator of IBMIR, by blocking phagocytosis. We hypothesized that the transient display of the CD47 protein on the surface of islet grafts may prevent early islet destruction by IBMIR following intraportal transplantation. We generated a chimeric CD47 fused with a modified streptavidin (SA-CD47). SA-CD47 was transiently displayed on the surface of rat splenocytes that had been modified with biotin taking advantage of the strong affinity between biotin and streptavidin (SA). SA-CD47 protein was also effectively displayed on the surface of biotinylated islets. Importantly, in a syngeneic marginal mass intraportal islet transplantation model, 7/8 streptozotocin-diabetic C57BL/6 mice transplanted with 125 SA-CD47 engineered islets established euglycemia long term (&amp;gt;80 days). In marked contrast, only 1/7 mice achieved euglycemia when transplanted with SA-engineered islet grafts. The engrafted islets in the SA-CD47 group had a comparable response to naïve mice in the intraperitoneal glucose tolerance test. Flow cytometric and qRT-PCR analyses of liver tissues did not reveal significant changes in cellular and molecular immune responses between SA-CD47 and SA-engineered control groups, suggesting that inhibition of islet phagocytosis as underlying mechanism of islet engraftment and survival. Collectively, these results demonstrate the utility of SA-CD47 as a regulator of IBMIR for prevention of early islet graft loss following intraportal transplantation

Expansion of transplanted islets in mice by co-transplantation with adipose tissue-derived mesenchymal stem cells

The shortage of donor islets is a significant obstacle for widespread clinical application of pancreatic islet transplantation. To investigate whether adipose tissue-derived mesenchymal stem cells (ADSCs) induce expansion of transplanted islets, we performed co-transplantation experiments in a mouse model. Streptozotosin (STZ)-induced diabetic mice transplanted with 50 syngeneic islets remained hyperglycemic. However, hyperglycemia was ameliorated gradually when 50 islets were co-transplanted with ADSCs but not separately grafted into the contralateral kidney. Insulin and proinsulin contents of 120-day grafts containing 50 islets co-transplanted with ADSCs were significantly increased compared with those of 50 isolated islets. The Ki67-positive ratios in islets of the naïve pancreas, at 30 and 120 days grafts were 0.23%, 2.12%, and 1.52%, respectively. Ki67-positive cells were predominantly Pdx1+ and insulin+ cells. These results demonstrate that co-transplantation with ADSCs induces proliferation of transplanted islets in mice, suggesting a potential solution for the low efficiency of islet transplantation.

Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats

A decrease in mass of isografts and a decline in islet function are major challenges in islet transplantations. Despite this, transplantation of 84 h harvested pancreatic duct ligation (PDL) tissues have been shown to have the same functional ability to foetal pancreata, but there was only 40% success in reverting hyperglycaemia. We tested the potential of early islets with mesenchymal stromal cells (MSCs) to promote isogeneic grafts survival and to restore normoglycemia in diabetic rats, in comparison with late islets. Islets were isolated from injured adult pancreata of donor rats at 24 h post ligation either with MSCs (24 h islet/MSC+) or without MSCs (24 h islet/MSC-), and at 84 h without MSCs (84 h islet/MSC-). These cells were transplanted under the renal capsule of syngeneic STZ-diabetic recipient rats. The islet grafts were monitored using the BGLs of recipients and the immunohistomorphology of the grafts were analysed using anti-insulin and anti-Ki67 antibodies. The mean BGL in 24 h islet/MSC+ recipients was reduced over time toward the control value. The curves of the mean BGLs in the control islet/MSC- and the 24 h islet/MSC- recipients dropped significantly below the control normal glucose group’s levels to reach their nadirs on weeks 4 and 6, respectively. Both curves had a peak overshoot on week 9, with no statistical significant difference between them. Engrafted islets were evident in these recipients, lasted for 5 and 6 weeks and correspondingly survived failure. However, insulin+ cells were present in the isografts of all recipients; but, only isografts in the 24 h islet/MSC+ presented with a homogenous subcapsular beta cell mass. In addition, the tendency of 24 h islet/MSC- to restore normoglycaemia with its survival capacity was statistically highly significant compared to the 84 islet/MSC- recipients (80%; 20%; p = 0.001). Transplantation of early islets with MSCs from injured adult pancreata prolongs islet graft survival and improves isograft function in diabetic rats. This novel observation requires much further exploration for its clinical application, but this model already provides hope for new sources of donor islets for transplantation.

BMX-001, a novel redox-active metalloporphyrin, improves islet function and engraftment in a murine transplant model.

Islet transplantation has become a well-established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX-001 (MnTnBuOE-2-PyP5+ [Mn(III) meso-tetrakis-(N-b-butoxyethylpyridinium-2-yl)porphyrin]) and its earlier derivative, BMX-010 (MnTE-2-PyP [Mn(III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin]) could improve islet function and engraftment outcomes. Long-term culture of human islets with BMX-001, but not BMX-010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24hours with 34μmol/L BMX-001 exhibited improved insulin secretion (n=3 isolations, P<.05) in response to glucose relative to control islets. In addition, 34μmol/L BMX-001-supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P<.05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34μmol/L BMX-001-treated islet recipients became euglycemic (n=11 of 19) compared with 19% of nontreated control islet recipients (n=3 of 19, P<.05). Of murine recipients receiving a marginal dose of human islets cultured with 34μmol/L BMX-001, 92% (n=12 of 13) achieved euglycemia compared with 57% of control recipients (n=8 of 14, P=.11). These results demonstrate that the administration of BMX-001 enhances in vitro viability and augments murine marginal islet mass engraftment.

The Optimization of the Prevascularization Procedures for Improving Subcutaneous Islet Engraftment.

Subcutaneous islet transplantation is associated with minimal invasiveness, but poor vascularization. Thus, the optimization of the prevascularization procedures is crucial for improving the outcomes. Although the effectiveness of basic fibroblast growth factor (bFGF) was reported, the optimal procedures remain unclear. We sought to optimize the prevascularization procedures including the use of a novel scaffold, recombinant peptide (RCP). Devices containing various amount of bFGF with/without heparin or RCP were implanted into the subcutaneous space of diabetic C57BL/6 mice. Syngeneic islets were transplanted into the prevascularized space. Blood glucose, intraperitoneal glucose tolerance, and immunohistochemistry were evaluated. The cure rates in all the device groups irrespective of bFGF doses were considerably higher than in the nondevice group. The cure rate in the bFGF0 group was unexpectedly higher than that in the subcutaneous islet transplant alone group (the None group) (57.1% vs 28.6%). Glucose tolerance was ameliorated in the bFGF10(-), 10(+) and 15(-) groups. The number of von Willebrand factor-positive vessels in the bFGF10(+) group was significantly higher than that in the None and bFGF0 groups (P < 0.01). Taken together, the bFGF10(+) group was considered to have received optimized procedures. In a marginal graft model, the efficiency in the RCP group was better than that in the bFGF10(+) group, furthermore, comparable to that in the intraportal transplantation group. Unlike bFGF, no bleeding and effusion were observed in the RCP group. These results suggest that optimizing biomaterials to induce efficient prevascularization could be a novel strategy for improving subcutaneous islet transplantation.

Local, Controlled Release In Vivo of Vascular Endothelial Growth Factor Within a Subcutaneous Scaffolded Islet Implant Reduces Early Islet Necrosis and Improves Performance of the Graft

Islet transplantation remains the only alternative to daily insulin therapy for control of type 1 diabetes (T1D) in humans. To avoid the drawbacks of intrahepatic islet transplantation, we are developing a scaffolded islet implant to transplant islets into nonhepatic sites. The implant test bed, sized for mice, consists of a limited (2-mm) thickness, large-pore polymeric sponge scaffold perforated with peripheral cavities that contain islets suspended in a collagen hydrogel. A central cavity in the scaffold holds a 2-mm diameter alginate sphere for controlled release of the angiogenic cytokine vascular endothelial growth factor (VEGF). Host microvessels readily penetrate the scaffold and collagen gel to vascularize the islets. Here, we evaluate the performance of the implant in a subcutaneous (SC) graft site. Implants incorporating 500 syngeneic islets reversed streptozotocin-induced diabetes in mice approximately 30 d after SC placement. Controlled release of a modest quantity (20 ng) of VEGF within the implant significantly reduced the time to normoglycemia compared to control implants lacking VEGF. Investigation of underlying causes for this effect revealed that inclusion of 20 ng of VEGF in the implants significantly reduced central necrosis of islets 24 h after grafting and increased implant vascularization (measured 12 d after grafting). Collectively, our results demonstrate (1) that the scaffolded islet implant design can reverse diabetes in SC sites in the absence of prevascularization of the graft site and (2) that relatively low quantities of VEGF, delivered by controlled release within the implant, can be a useful approach to limit islet stress after grafting.

Immunoisolation of murine islet allografts in vascularized sites through conformal coating with polyethylene glycol.

Islet encapsulation may allow transplantation without immunosuppression, but thus far islets in large microcapsules transplanted in the peritoneal cavity have failed to reverse diabetes in humans. We showed that islet transplantation in confined well-vascularized sites like the epididymal fat pad (EFP) improved graft outcomes, but only conformal coated (CC) islets can be implanted in these sites in curative doses. Here, we showed that CC using polyethylene glycol (PEG) and alginate (ALG) was not immunoisolating because of its high permselectivity and strong allogeneic T cell responses. We refined the CC composition and explored PEG and islet-like extracellular matrix (Matrigel; MG) islet encapsulation (PEG MG) to improve capsule immunoisolation by decreasing its permselectivity and immunogenicity while allowing physiological islet function. Although the efficiency of diabetes reversal of allogeneic but not syngeneic CC islets was lower than that of naked islets, we showed that CC (PEG MG) islets from fully MHC-mismatched Balb/c mice supported long-term (>100days) survival after transplantation into diabetic C57BL/6 recipients in the EFP site (750-1000 islet equivalents/mouse) in the absence of immunosuppression. Lack of immune cell penetration and T cell allogeneic priming was observed. These studies support the use of CC (PEG MG) for islet encapsulation and transplantation in clinically relevant sites without chronic immunosuppression.

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140mmHg, compared to 45mmHg under ambient air. In vitro, islets cultured under 140mmHg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45mmHg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P < 0.05 and P < 0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P < 0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P < 0.05 and P < 0.05, respectively). The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

Effect of liver histopathology on islet cell engraftment in the model mimicking autologous islet cell transplantation

ABSTRACTBackground: The inflammatory milieu in the liver as determined by histopathology is different in individual patients undergoing autologous islet cell transplantation. We hypothesized that inflammation related to fatty-liver adversely impacts islet survival. To test this hypothesis, we used a mouse model of fatty-liver to determine the outcome of syngeneic islet transplantation after chemical pancreatectomy. Methods: Mice (C57BL/6) were fed a high-fat-diet from 6 weeks of age until attaining a weight of ≥28 grams (6–8 weeks) to produce a fatty liver (histologically > 30% fat);steatosis was confirmed with lipidomic profile of liver tissue. Islets were infused via the intra-portal route in fatty-liver and control mice after streptozotocin induction of diabetes. Outcomes were assessed by the rate of euglycemia, liver histopathology, evaluation of liver inflammation by measuring tissue cytokines IL-1β and TNF-α by RT-PCR and CD31 expression by immunohistochemistry. Results: The difference in the euglycemic fraction between the normal liver group (90%, 9/10) and the fatty-liver group (37.5%, 3/8) was statistically significant at the 18th day post- transplant and was maintained to the end of the study (day 28) (p = 0.019, X2 = 5.51). Levels of TNF–α and IL-1β were elevated in fatty-liver mice (p = 0.042, p = 0.037). Compared to controls cytokine levels were elevated after islet cell transplantation and in transplanted fatty-liver mice as compared to either fatty- or islet transplant group alone (p = NS). A difference in the histochemical pattern of CD31 could not be determined. Conclusion: Fatty-liver creates an inflammatory state which adversely affects the outcome of autologous islet cell transplantation.

Local delivery of fingolimod from three-dimensional scaffolds impacts islet graft efficacy and microenvironment in a murine diabetic model.

The local delivery of immunosuppressive agents could significantly promote the success of islet transplantation for the treatment of Type 1 diabetes. Fingolimod, a clinically-approved sphingosine-1-phosphate receptor agonist, has been found to dampen allograft islet rejection in rodent models when delivered systemically. Herein, we engineered a platform for the local delivery of fingolimod by incorporating it within a macroporous polydimethylsiloxane (PDMS) scaffold specifically designed for islet transplantation. In vitro drug release studies quantifying kinetics confirmed sustained release within targeted dose levels for >7 days. Fingolimod-PDMS scaffolds containing syngeneic islets were subsequently transplanted into diabetic mice for examination of the effect of local fingolimod release on engraftment. Surprisingly, either delayed or abrogated efficacy was observed when scaffolds contained a dosage of fingolimod >0.5% w/w; despite drug release rates estimated at ~80-fold less than published systemic delivery reports where no detrimental effects were noted. Histological analysis of explants indicated a dose-dependent modulation of cellular migration and phenotype at the graft site, with high doses impairing host infiltration and engraftment while lower doses promoted leucocyte migration. Mechanistic in vivo and in vitro studies observed unique host and islet responses to local fingolimod delivery, with impairment of murine islet viability and function. Overall, this study confirmed the ability to modulate local delivery of fingolimod in a sustained-release manner using a three-dimensional PDMS scaffold; however, the observed detrimental impacts at the site of islet transplantation do not support further investigation of local delivery at the graft site in murine models.

A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival

1) The silk fibroin scaffold presented in the present study provides an open platform for scaffold development in islet transplantation, with heparinisation as an example. 2) Both heparin and silk fibroin have been used clinically. The excellent in vivo therapeutic outcome reported here may therefore be clinically relevant and provide valuable insights for bench to bed translation. 3) Compared to conventional clinical islet transplantation, during which islets are injected via the hepatic portal vein, the physical/mechanical properties of silk fibroin scaffolds create a more accessible transplantation site (i.e., within fat pad), which significantly reduces discomfort. 4) Islet implantation into the fat pad also avoids an instant blood mediated inflammatory response, which occurs upon contact of islet with recipient's blood during intraportal injection, and prolongs survival and function of implanted islets.