Beta-cell Grafts 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.

Magnetic Retrieval of Encapsulated Beta Cell Transplants from Diabetic Mice Using Dual-Function MRI Visible and Retrievable Microcapsules.

Encapsulated beta cell transplantation offers a potential cure for a subset of diabetic patients. Once transplanted, beta cell grafts can help to restore glycemic control; however, locating and retrieving cells in the event of graft failure may pose a surgical challenge. Here, a dual-function nanoparticle-loaded hydrogel microcapsule is developed that enables graft retrieval under an applied magnetic field. Additionally, this system facilitates graft localization via magnetic resonance imaging (MRI), and graft isolation from the immune system. Iron oxide nanoparticles encapsulated within alginate hydrogel capsules containing viable islets are transplanted and the in vitro and in vivo retrieval of capsules containing nanoparticles functionalized with various ligands are compared. Capsules containing islets co-encapsulated with COOH-coated nanoparticles restore normal glycemia in immunocompetent diabetic mice for at least 6 weeks, can be visualized using MRI, and are retrievable in a magnetic field. Application of a magnetic field for 90 s via a magnetically assisted retrieval device facilitates rapid retrieval of up to 94% (±3.1%) of the transplant volume 24 h after surgical implantation. This strategy aids monitoring of cell-capsule locations in vivo, facilitates graft removal at the end of the transplant lifetime, and may be applicable to many encapsulated cell transplant systems.

Co-Transplant of Parathyroid Gland and Stem Cell-Derived Insulin-Producing Cells Enhances Graft Survival through Release of Pro-Angiogenic and Pro-Survival Factors

Introduction Ischemia dramatically impairs survival and function of islets after transplantation. Human Stem Cell-derived Beta Cells are currently being tested in clinical trials for replacement therapy. Although stem cell technology represents an unlimited source of beta cells, it is paramount to reduce the effects of ischemia to optimize outcome. Parathyroid glands (PTG) are known to engraft in the subcutaneous and intra-muscular sites with high success rate. The objective of this study is to investigate if PTG can protect beta-cell grafts. We hypothesize that PTG protect beta cells by both accelerating neo-vasculogenesis and by producing tropic factors that sustain beta cell viability before revascularization. Methods Stem Cell-derived Beta Cells were produced from a human embryonic stem cell line with a GFP transgene knocked into the insulin locus and a constitutively expressed luciferase transgene. Insulin-producing GFP+ cells were enriched by FACS and reaggregated to form enhanced beta clusters (eBCs). eBC were transplanted under the subcutaneous space (SQ) of NSG mice and graft mass was monitored using bioluminescence imaging. Human PTG were finely cut and co-transplanted with eBC. PTG-derived CD34+ and CD34- cells were FACS purified, cultured for 24 hours and supernatants were assessed for cytokines using Luminex. Effects of PTG-derived factors on eBCs viability after challenges of hypoxia and nutrient deprivation were assessed in vitro. Results and Discussion eBC graft mass decreased by 60% in two weeks after transplant under the SQ. However, when eBC grafts were co-transplanted with PTG, bioluminescent signal increased up to 150% (Figure 1 A and B). We further investigated the mechanisms of PTG protection. We found PTG-derived CD34+ cells produced CCL2, CXCL12, VEGF-A, and PDGF-AA (Figure 2), which have all been previously implicated in neo-vasculogenesis. Consistently, PTG co-transplant led to a dramatic increase in the ingrowth of CD31+ cells. Additionally, PTG secreted parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and γ-aminobutyric acid (GABA). These factors improved eBC survival during in vitro-simulated ischemia challenge using nutrient-deprivation and/or hypoxia (Figure 3). Conclusions Our results show that PTG co-transplantation preserves beta cell graft survival. PTG-derived CD34+ cells promote neovascularization of eBC grafts by producing angiogenic factors. PTG may also have direct cytoprotective effects on eBCs by producing PTH, PTHrP, and GABA. These properties of PTG may be harnessed to minimize ischemic injury to eBC after transplant to realize the therapeutic potential of stem cell-based therapy for diabetes.

Toward beta cell replacement for diabetes.

The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes.

The role of exendin-4-conjugated superparamagnetic iron oxide nanoparticles in beta-cell-targeted MRI

Noninvasive targeted visualization of pancreatic beta cells or islets is becoming the focus of molecular imaging application in diabetes and islet transplantation studies, but it is currently unsuccessful due to the lack of specific beta cell biomarkers. Glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in beta cells and considered as a promising target. We here developed a targeted superparamagnetic iron oxide (SPIO) nanoparticle using GLP-1 analog-exendin-4 which is conjugated to polyethylene glycol coated SPIO (PEG-SPIO). The results demonstrated that exendin-4 functionalized SPIO was able to specifically bind to and internalized by GLP-1R-expressing INS-1 cells, with the higher labeling efficiency than non-targeted nanoparticles. Notably, SPIO-exendin4 could differentially label islets in pancreatic slices or beta cell grafts in vitro. Systemic delivery of SPIO-exendin4 into nude mice bearing s.c. insulinomas (derived from INS-1 cells) leads to the accumulation of the nanoparticles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical MRI scanner at field strength of 3.0 T, and the iron deposition in tumors was further confirmed by Prussian blue staining. Furthermore, preliminary biodistribution study indicated that SPIO-exendin4 had a tendency to accumulate in pancreas. Toxicity assessments demonstrated good biocompatibility in vivo. These results suggest that SPIO-exendin4 has potential as molecularly targeted imaging agents for in vivo imaging of insulinoma, and possibly for future beta cell imaging.

Comparison of Sirolimus Alone With Sirolimus Plus Tacrolimus in Type 1 Diabetic Recipients of Cultured Islet Cell Grafts

One year survival of islet cell grafts has been reproducibly achieved under combination immune therapy including tacrolimus (TAC). However, the use of TAC causes beta-cell and renal toxicity. Because sirolimus (SIR) monotherapy was successful in kidney transplantation under antithymocyte globulin (ATG), we undertook a pilot study comparing SIR monotherapy with SIR-TAC combination therapy. Nonuremic type 1 diabetics received a cultured beta-cell graft under ATG and were randomly assigned to SIR or SIR-TAC-maintenance therapy; a second graft was implanted during posttransplantation month 3 without ATG. The planned number of patients per group (n=10) was reduced to five in view of the observed side effects. At posttransplant month 6, three SIR-patients had lost graft function and two presented marginal function; among SIR-TAC-patients, there were two early graft failures but three became insulin-independent. These three patients maintained metabolically relevant function (C-peptide >1 ng/ml and coefficient of variation fasting glycemia <25%) for more than 2 years but low-dose insulin therapy was needed from 8, 18, and 26 months posttransplant; this was still the case in two of them after reducing and stopping TAC dose. In both groups, incapacitating adverse events were attributed to sirolimus requiring its discontinuation in 4 of 10 patients; in the 3 patients with pretransplant microalbuminuria, macroalbuminuria developed which resolved when sirolimus was stopped. SIR monotherapy is not sufficient to suppress rejection after transplantation under ATG, but it can maintain survival of established beta-cell grafts. However, the risk for a SIR-induced proteinuria remains a concern.

Cryoprecipitated plasma perfusion preservation and cold storage preservation of duct-ligated pancreatic allografts

It has been shown previously that, at least in dogs, a vascularized pancreatic allograft with ligation of the pancreatic duct can maintain normal beta-cell function for at least 5 years. In this study common organ preservation techniques, as used in human cadaveric kidney transplantation, were applied to canine pancreatic allografts to determine the influence of 24-hour preservation on beta-cell function, graft survival and histological appearances. Three groups of dogs were compared: group A consisted of 9 dogs with fresh grafts; group B consisted of 5 dogs who received grafts preserved for 24 hours by pulsatile hypothermic cryoprecipitated plasma perfusion; and group C was composed of 5 animals who received grafts that had been flushed with Collins' solution followed by hypothermic storage for 24 hours. It appeared that both preservation methods were equally effective and that preservation did not alter either the graft's function or its histological appearance. No significant differences after transplantation were observed in the endocrine function tests of the three groups when compared with the preoperative values; neither was there a significant difference in the mean graft survival time between the groups.

PERCUTANEOUS TRANSHEPATIC BETA CELL TRANSPLANTATION IN TYPE 1 DIABETIC PATIENTS USING A COMBINED ULTRASOUND- AND FLUOROSCOPY-GUIDED TECHNIQUE.

P447 Aims: To evaluate the safety and feasibility of percutaneous transhepatic beta cell transplantation in patients with type 1 diabetes mellitus using a combined ultrasound- and fluoroscopy-guided puncture technique. Methods: Between December 2001 and November 2003, 15 C-peptide negative type 1 diabetic patients underwent 31 percutaneous beta cell transplantation procedures in our JDRF center. In 13 cases the transplant procedure was done under sedation, while in 18 cases general anesthesia was given, in 12 cases using a laryngeal mask and in 6 an endotracheal tube. In all procedures percutaneous access to the right portal vein was made under ultrasound guidance and subsequent catherisation of the main portal vein was performed under fluoroscopic control. The volume of beta cell grafts injected for each procedure was recorded as well as the total procedure time. Clinical, biochemical and radiological evaluation was performed before and after the procedure. Results: In all cases it was possible to achieve access to the portal vein (median number of seeker needle passes = 1, range 1 – 6). The volume of cultured beta cell grafts injected for each transplantation was 0.58 ml (range 0.26 – 1.60 ml) and the recorded procedure time (from puncture to catheter withdrawal) was 19 min (range 10 – 80 min). Three patients presented with transient abdominal pain immediately after the procedure; postprocedural duplex ultrasound of the liver revealed a patent portal vein and endbranches in all cases and a minor perihepatic fluid collection in 3 patients. From the end of week 1 up to week 3, an increase in liver transaminases was measured in all recipients (3.0 fold, range 1.8 – 11.3 fold). Conclusions: The combined ultrasound and fluoroscopic monitoring of percutaneous transhepatic injection is a new, safe and reproducible radiological procedure for transplantation of beta cell grafts in type 1 diabetic patients.

Follow-up study of the revascularization process of purified rat islet beta-cell grafts

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3–5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10–14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

Beta cells are important for islet innervation: evidence from purified rat islet-cell grafts

Pancreatic islets receive an extensive and complex innervation that includes sympathetic, noradrenergic nerve fibres also storing neuropeptide Y. Islets transplanted to the kidney capsule become progressively reinnervated, mainly by sympathetic fibres and to a lesser extent by parasympathetic and sensory fibres. The density of nerve fibres in the islet grafts is often higher than in the graft-bearing organ, suggesting that the grafted islets contain factors that promote ingrowth of nerve fibres. To find out if beta cells are of any importance for attracting nerve fibres, purified preparations of rat islet beta and non-beta cells were transplanted to the kidneys of nude mice. Some of the mice were rendered diabetic by alloxan injection before transplantation. Immunocytochemical analysis revealed that the beta-cell grafts became richly reinnervated by noradrenergic (tyrosine hydroxylase-containing) nerve fibres, also storing neuropeptide Y. Non-beta islet-cell grafts were virtually devoid of demonstrable nerve fibres. There was no discernible difference in the reinnervation pattern between diabetic and non-diabetic mice. The findings indicate that factors mediating islet neurotrophism are produced by the beta cells.

Transplantation of purified islet cells in diabetic BB rats

The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.