Site For Islet Transplantation Research Articles

Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

Pancreatic Islet Transplantation into the Submandibular Gland: Our Experimental Experience and a Review of the Relevant Literature.

Pancreatic islet transplantation is a promising therapy for type 1 diabetes. Islet transplantation is clinically performed through intra-portal infusion, which is associated with several drawbacks, including poor engraftment. The histological resemblance between the submandibular gland and the pancreas renders it an attractive alternative site for islet transplantation. In this study, we refined the technique of islet transplantation into the submandibular gland to achieve good morphological features. Then, we transplanted 2600 islet equivalents into the submandibular glands of diabetic Lewis rats. Intra-portal islet transplantation was performed in diabetic rats as a control. Blood glucose levels were followed for 31 days, and an intravenous glucose tolerance test was performed. Immunohistochemistry was used to demonstrate the morphology of transplanted islets. Follow-up after transplantation showed that diabetes was cured in 2/12 rats in the submandibular group in comparison to 4/6 in the control group. The intravenous glucose tolerance test results of the submandibular and intra-portal groups were comparable. Immunohistochemistry showed large islet masses in the submandibular gland in all examined specimens with positive insulin staining. Our results show that submandibular gland tissue can support the islet function and engraftment but with considerable variability. Good morphological features were achieved using our refined technique. However, islet transplantation into rat submandibular glands did not demonstrate a clear advantage over conventional intra-portal transplantation.

Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Bioengineered omental transplant site promotes pancreatic islet allografts survival in non-human primates

The transplanting islets to the liver approach suffers from an immediate posttransplant loss of islets of more than 50%, progressive graft dysfunction over time, and precludes recovery of grafts should there be serious complications such as the development of teratomas with grafts that are stem cell-derived islets (SC-islets). The omentum features an attractive extrahepatic alternative site for clinical islet transplantation. We explore an approach in which allogeneic islets are transplanted onto the omentum, which is bioengineered with a plasma-thrombin biodegradable matrix in three diabetic non-human primates (NHPs). Within 1week posttransplant, each transplanted NHP achieves normoglycemia and insulin independence and remains stable until termination of the experiment. Success was achieved in each case with islets recovered from a single NHP donor. Histology demonstrates robust revascularization and reinnervation of the graft. This preclinical study can inform the development of strategies for β cell replacement including the use of SC-islets or other types of novel cells in clinical settings.

Successful Islet Transplantation Into a Subcutaneous Polycaprolactone Scaffold in Mice and Pigs.

In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice' and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans. In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs. To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Advantages of the retroperitoneal retrocolic space as the transplant site for encapsulated xenogeneic islets.

Islet allotransplantation has demonstrated improved clinical outcomes using the hepatic portal vein as the standard infusion method. However, the current implantation site is not ideal due to the short-term thrombotic and long-term immune destruction. Meanwhile, the shortage of human organ donors further limits its application. To find a new strategy, we tested a new polymer combination for islet encapsulation and transplantation. Meanwhile, we explored a new site for xenogeneic islet transplantation in mice. We synthesized a hydrogel combining alginate plus poly-ethylene-imine (Alg/PEI) for the encapsulation of rat, neonatal porcine, and human islets. Transplantation was performed into the retroperitoneal retro-colic space of diabetic mice. Control mice received free islets under the kidney capsule or encapsulated islets into the peritoneum. The biochemical indexes were measured, and the transplanted islets were harvested for immunohistochemical staining of insulin and glucagon. Mice receiving encapsulated rat, porcine and human islets transplanted into the retroperitoneal space maintained normoglycemia for a median of 275, 145.5, and 146 days, respectively. In contrast, encapsulated xenogeneic islets transplanted into the peritoneum, maintained function for a median of 61, 95.5, and 82 days, respectively. Meanwhile, xenogeneic islets transplanted free into the kidney capsule lost their function within 3 days after transplantation. Immunohistochemical staining of encapsulated rat, porcine and human islets, retrieved from the retroperitoneal space, allowed to distinguish morphological normal insulin expressing β- and glucagon expressing α-cells at 70, 60, and 100 days post-transplant, respectively. Transplantation of Alg/PEI encapsulated xenogeneic islets into the retroperitoneal space provides a valuable new implantation strategy for the treatment of type 1 diabetes.

Establishing the Controlled Delivery of VEGF Using a Hydrogel Loaded Soft Robotic Drug Delivery System With the Aim to Prevascularise Implant Site for Islet Transplantation: Erratum.

Establishing the Controlled Delivery of VEGF Using a Hydrogel Loaded Soft Robotic Drug Delivery System With the Aim to Prevascularise Implant Site for Islet Transplantation: Erratum.

Development of a novel method for measuring tissue oxygen pressure to improve the hypoxic condition in subcutaneous islet transplantation

Subcutaneous tissue is a promising site for islet transplantation, but poor engraftment, due to hypoxia and low vascularity, hinders its prevalence. However, oxygen partial pressure (pO2) of the subcutaneous space (SC) and other sites were reported to be equivalent in several previous reports. This contradiction may be based on accidental puncture to the indwelling micro-vessels in target tissues. We therefore developed a novel optical sensor system, instead of a conventional Clark-type needle probe, for measuring tissue pO2 and found that pO2 of the SC was extremely low in comparison to other sites. To verify the utility of this method, we transplanted syngeneic rat islets subcutaneously into diabetic recipients under several oxygenation conditions using an oxygen delivery device, then performed pO2 measurement, glucose tolerance, and immunohistochemistry. The optical sensor system was validated by correlating the pO2 values with the transplanted islet function. Interestingly, this novel technique revealed that islet viability estimated by ATP/DNA assay reduced to less than 75% by hypoxic condition at the SC, indicating that islet engraftment may substantially improve if the pO2 levels reach those of the renal subcapsular space. Further refinements for a hypoxic condition using the present technique may contribute to improving the efficiency of subcutaneous islet transplantation.

A Prevascularized Sinus Tract on the Liver Surface for Islet Transplantation.

The lack of a suitable transplantation site has become a bottleneck restricting the development of islet transplantation. In this study, for the first time, a prevascularized sinus tract (PST) for islet transplantation was constructed in a mouse model by temporarily embedding a 4× silk thread between the liver surface and the attached decellularized human amniotic membrane. After which, the characteristics of the PST and the function of the islet graft within the PST were evaluated. The results showed that PST was lined with granulation tissue, the blood vessel density of the local tissue increased, and proangiogenic proteins were upregulated, which mimics the microenvironment of the islets in the pancreas to a certain extent. Transplantation of ~200 syngeneic islets into the PST routinely reversed the hyperglycemia of the recipient mice and maintained euglycemia for >100 d until the islet grafts were retrieved. The islet grafts within the PST achieved better results to those in the nonprevascularized control groups and comparable results to those under the kidney capsule with respect to glycemic control and glucose tolerance. By attaching a decellularized human amniotic membrane to the surface of mouse liver and temporarily embedding a 4× silk thread, the PST formed on the liver surface has a favorable local microenvironment and is a potential clinical islet transplantation site.

Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

SummaryAdoptive transfer of chimeric antigen receptor regulatory T cells (CAR Tregs) is a promising way to prevent allograft loss without the morbidity associated with current therapies. Non-human primates (NHPs) are a clinically relevant model to develop transplant regimens, but manufacturing and engraftment of NHP CAR Tregs have not been demonstrated yet. Here, we describe a culture system that massively expands CAR Tregs specific for the Bw6 alloantigen. In vitro, these Tregs suppress in an antigen-specific manner without pro-inflammatory cytokine secretion or cytotoxicity. In vivo, Bw6-specific CAR Tregs preferentially traffic to and persist in bone marrow for at least 1 month. Following transplant of allogeneic Bw6+ islets and autologous CAR Tregs into the bone marrow of diabetic recipients, CAR Tregs traffic to the site of islet transplantation and maintain a phenotype of suppressive Tregs. Our results establish a framework for the optimization of CAR Treg therapy in NHP disease models.

Intrapleural transplantation of allogeneic pancreatic islets achieves glycemic control in a diabetic non-human primate

Clinical islet transplantation has relied almost exclusively on intraportal administration of pancreatic islets, as it has been the only consistent approach to achieve robust graft function in human recipients. However, this approach suffers from significant loss of islet mass from a potent immediate blood-mediated inflammatory response (IBMIR) and a hypoxic environment. To avoid these negative aspects of the portal site, we explored an alternative approach in which allogeneic islets were transplanted into the intrapleural space of a non-human primate (NHP), treated with an immunosuppression regimen previously reported to secure routine survival and tolerance to allogeneic islets in NHP. Robust glycemic control and graft survival were achieved for the planned study period of >90days. Our observations suggest the intrapleural space provides an attractive locale for islet transplantation due to its higher oxygen tension, ability to accommodate large transplant tissue volumes, and a lack of IBMIR-mediated islet damage. Our preliminary results reveal the promise of the intrapleural space as an alternative site for clinical islet transplantation in the treatment of type 1 diabetes.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing β cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

Local Dexamethasone Administration Delays Allogeneic Islet Graft Rejection in the Anterior Chamber of the Eye of Non-Human Primates

Pancreatic islet transplantation into the anterior chamber of the eye (ACE) has been shown to improve glycemic control and metabolic parameters of diabetes in both murine and primate models. This novel transplantation site also allows the delivery of therapeutic agents, such as immunosuppressive drugs, locally to prevent islet graft rejection and circumvent unwanted systemic side effects. Local intravitreal administration of micronized dexamethasone implant was performed prior to allogeneic islet transplantation into the ACEs of non-human primates. Two study groups were observed namely allogeneic graft without immunosuppression (n = 4 eyes) and allogeneic graft with local immunosuppression (n = 8 eyes). Survival of islet grafts and dexamethasone concentration in the ACE were assessed in parallel for 24 weeks. Allogeneic islet grafts with local dexamethasone treatment showed significantly better survival than those with no immunosuppression (median survival time- 15 weeks vs 3 weeks, log-rank test p<0.0001). Around 73% of the grafts still survived at week 10 with a single local dexamethasone implant, where the control group showed no graft survival. Dexamethasone treated islet grafts revealed a good functional response to high glucose stimulation despite there was a transient suppression of insulin secretion from week 8 to 12. Our findings show a significant improvement of allografts survival in the ACE with local dexamethasone treatment. These results highlight the feasibility of local administration of pharmacological compounds in the ACE to improve islet graft survival and function. By eliminating the need for systemic immunosuppression, these findings may impact clinical islet transplantation in the treatment of diabetes, and the ACE may serve as a novel therapeutic islet transplantation site with high potential for local pharmacological intervention.

P.123: Establishing the Controlled Delivery of VEGF Using a Hydrogel Loaded Soft Robotic Drug Delivery System With the Aim to Prevascularise Implant Site for Islet Transplantation

P.123: Establishing the Controlled Delivery of VEGF Using a Hydrogel Loaded Soft Robotic Drug Delivery System With the Aim to Prevascularise Implant Site for Islet Transplantation

164-OR: Brown Adipose Tissue as a Novel Site for Islet Transplantation

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells. While islet transplantation is a promising alternative to insulin therapy for T1D treatment, finding ideal transplant sites that are readily accessible, exhibit glucose regulation, are immune-privileged, and promote vascularization for islet survival has remained a challenge. A novel site which exhibits these traits is the brown adipose tissue (BAT). BAT functions as a thermogenic adipose tissue that maintains body temperature and regulates metabolism. We hypothesize that murine islets transplanted into BAT will display normal islet graft and BAT function while providing immunological protection. Syngeneic transplant of 250 NOD.scid islets into the BAT of streptozotocin-treated diabetic NOD.scid mice restored euglycemia for over ten weeks, which was comparable to kidney capsule transplant controls. BAT histology and mRNA analysis assessed ten weeks after transplantation showed engrafted islets maintain expression of hallmark hormones, like insulin and glucagon, and critical transcription factors. Evaluation of BAT function after transplantation revealed no impairments in glucose and insulin tolerance, cold tolerance, or whole-body energy expenditure. Flow cytometry analysis displayed an enrichment of immunomodulatory T regulatory cells and anti-inflammatory M2 macrophages in BAT compared to kidney, possibly allowing greater immunoprotection for engrafted islets. Strikingly, adoptive transfer and allogeneic transplant studies revealed that islets transplanted into BAT showed significant delays in autoimmune and allograft rejection compared to islets transplanted under the kidney capsule. Overall, our data suggest BAT as a novel and efficacious site for islet transplantation to potentially combat T1D. Disclosure J. Kepple: None. J. Barra: None. C. S. Hunter: None. H. M. Tse: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK099550 to H.M.T.), (DK111483 to C.S.H.), (DK126456 to H.M.T., C.S.H.), (F31DK121414 to J.K.); JDRF (SRA-2016-270-S-B, 2-SRA-2019-692-S-B to H.M.T.); National Institute of General Medical

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Bioluminescence Imaging In Vivo Confirms the Viability of Pancreatic Islets Transplanted into the Greater Omentum.

The liver is the most widely used site for pancreatic islet transplantation. However, several site-specific limitations impair functional success, with instant blood-mediated inflammatory reaction being the most important. The aim of this study was to develop a preclinical model for placement of the islet graft into a highly vascularized omental flap using a fibrin gel. For this purpose, we tested islet viability by bioluminescence imaging (BLI). Pancreatic islets were isolated from luciferase-positive and luciferase-negative rats, mixed at a 1:1 ratio, placed into a plasma-thrombin bioscaffold, and transplanted in standard (10 pancreatic islets/g wt; n = 10) and marginal (4 pancreatic islets/g wt; n = 7) numbers into the omentums of syngeneic diabetic animals. For the control, 4 pancreatic islets/g were transplanted into the liver using the standard procedure (n = 7). Graft viability was tested by bioluminescence at days 14, 30, 60, and 90 post transplant. Glucose levels, intravenous glucose tolerance, and serum C-peptide were assessed regularly. Nonfasting glucose levels < 10 mmol/l were restored in all animals. While islet viability in the omentum was clearly detected by stable luminescence signals throughout the whole study period, no signals were detected from islets transplanted into the liver. The bioluminescence signals were highly correlated with stimulated C-peptide levels detected at 80 days post transplant. Glucose tolerance did not differ among the 3 groups. We successfully tested a preclinical model of islet transplantation into the greater omentum using a biocompatible scaffold made from autologous plasma and human thrombin. Both standard and marginal pancreatic islet numbers in a gel-form bioscaffold placed in the omentum restored glucose homeostasis in recipients with diabetes. Bioluminescence was shown promising as a direct proof of islet viability.

The Liver Surface Is an Attractive Transplant Site for Pancreatic Islet Transplantation.

In the current clinical islet transplantation, intraportal transplantation is regarded as the gold-standard procedure. However, in this procedure, 50 to 70% of the transplanted islets are immediately damaged due to a strong innate immune response based on islet–blood contact. We investigated the transplant efficiency of a novel method of liver surface transplantation using a syngeneic keratinocyte sheet to avoid islet–blood contact. To examine the influence of the keratinocyte sheet, substantial amounts of syngeneic islets (8 IEQs/g) were transplanted on the liver surface of diabetic rats, while marginal amounts of islets (4 IEQs/g) were transplanted via intraportal transplantation to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and in vivo imaging findings of the cell sheet were evaluated. The study showed that islet transplantation to the liver surface immediately followed by a syngeneic keratinocyte sheet covering was effective for curing diabetic rats, while no rats were cured in the group without the cell sheet. Notably, islet grafts transplanted via this approach appeared to penetrate into the liver parenchyma. However, the transplant efficiency did not reach that of intraportal transplantation. Further refinements of this approach by introducing mesothelial or fibroblast cell sheets in combination with a preferable scaffold for islet grafts may help to improve the transplant efficiency.

Suitability of denervated muscle flaps as recipient sites for pancreatic islet cell transplantation.

BackgroundExtensive research has been conducted on islet transplantation as a possible cure for diabetes. Islet transplantation in the liver via the portal vein has shown remarkable results, but numerous other recipient sites are currently being investigated. We aimed to show the effectiveness of using a muscle flap as a recipient site for islet transplantation.MethodsIslet cells were harvested from 12 isogenic Lewis rats, and then diabetes was induced in another 12 isogenic Lewis rats by streptozotocin injection. In six rats, 3,000 islets were transplanted into gastrocnemius muscle flaps, and in the other six rats, the same number of islets were transplanted into the gastrocnemius muscle. The transplanted islet cell function between the two groups was compared by means of blood glucose tests, glucose tolerance tests, immunohistochemistry, and real-time reverse transcription polymerase chain reaction.ResultsIn the muscle flap group, blood glucose levels significantly decreased after islet transplantation. Blood glucose levels were significantly different between the two groups at 3 weeks after transplantation. The muscle flap group showed nearly normoglycemic results upon the glucose tolerance test, whereas the muscle group was hyperglycemic. Immunohistochemical evaluation showed positive results against insulin and glucagon in biopsies of both groups, and the islet cell density was higher in the muscle flap group. There were no statistically significant differences between the two groups in real-time reverse transcription polymerase chain reaction results.ConclusionsOur results suggest that muscle flaps are promising candidates for islet cell transplantation.

A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas.

Although sites for clinical or experimental islet transplantation are well established, pancreatic islet survival and function in these locations remain unsatisfactory. A possible factor that might account for this outcome is local hypoxia caused by the limited blood supply. Here, we modified a prevascularized tissue-engineered chamber (TEC) that facilitated the viability and function of the seeded islets in vivo by providing a microvascular network prior to transplantation. TECs were created, filled with Growth Factor-Matrigel™ (Matrigel™) and then implanted into the groins of mice with streptozotocin-induced diabetes. The degree of microvascularization in each TECs was analyzed by histology, real-time PCR, and Western blotting. Three hundred syngeneic islets were seeded into each chamber on days 0, 14, and 28 post-chamber implantation, and 300, 200, or 100 syngeneic islets were seeded into additional chambers on day 28 post-implantation, respectively. Furthermore, allogeneic or xenogeneic islet transplantation is a potential solution for organ shortage. The feasibility of TECs as transplantation sites for islet allografts or xenografts and treatment with anti-CD45RB and/or anti-CD40L (MR-1) was therefore explored. A highly developed microvascularized network was established in each TEC on day 28 post-implantation. Normalization of blood glucose levels in diabetic mice was negatively correlated with the duration of prevascularization and the number of seeded syngeneic islets. Combined treatment with anti-CD45RB and MR-1 resulted in long-term survival of the grafts following allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry demonstrated that the frequency of CD4+Foxp3-Treg and CD4+IL-4+-Th2 cells increased significantly after tolerogenic xenograft transplantation, while the number of CD4+IFN-γ-Th1 cells decreased. These findings demonstrate that highly developed microvascularized constructs can facilitate the survival of transplanted islets in a TECs, implying its potential application as artificial pancreas in the future.