Allogeneic Islet Transplantation Research Articles

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

Islet Allotransplantation in the Bone Marrow of Patients With Type 1 Diabetes: A Pilot Randomized Trial.

Results in murine and nonhuman primate suggested that the bone marrow (BM) might be an alternative site for pancreatic islet transplantation. We report the results of 2 clinical studies in patients with type 1 diabetes receiving an intra-BM allogeneic islet transplantation: a feasibility study in patients with hepatic contraindications for liver islet allotransplantation receiving a single intra-BM islet infusion (n = 4) and a pilot randomized trial (1:1 allocation using blocks of size 6) in which patients were randomized to receive islets into either the liver (n = 6) or BM (n = 3) to evaluate islet transplant function and survival. We observed no adverse events related to the intrabone injection procedure or the presence of islets in the BM. None of the recipient of an intra-BM allogeneic islet transplantation had a primary nonfunction, as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples collected during follow-up. All patients receiving islets in the BM except 1 lost islet function during the first 4 months after infusion (2 with an early graft loss). Based on biopsies and immunomonitoring, we concluded that the islet loss was primarily caused by the recurrence of autoimmunity. Bone marrow is not a suitable alternative site for pancreatic islet allotransplantation in patients with type 1 diabetes.

Australia and New Zealand Islet and Pancreas Transplant Registry Annual Report 2018-Islet Donations, Islet Isolations, and Islet Transplants.

BackgroundThis is an excerpt from chapter 4 of the annual registry report from the Australia and New Zealand islet and pancreas transplant registry. The full report is available at http://anziptr.org/reports/.MethodsWe report data for all allogeneic islet isolation and transplant activity from 2002 to end 2017. Solid organ pancreas transplantation activity is reported separately. New Zealand does not have an islet transplant program. Data analysis was performed using Stata software version 14 (StataCorp, College Station, TX).ResultsFrom 2002 to 2017, a total of 104 allogeneic islet transplants were performed in 62 recipients.ConclusionsThe number of islet transplants performed in Australia was slightly lower in 2017 but continues to increase over time.

Oxygenation strategies for encapsulated islet and beta cell transplants.

Human allogeneic islet transplantation (ITx) is emerging as a promising treatment option for qualified patients with type 1 diabetes. However, widespread clinical application of allogeneic ITx is hindered by two critical barriers: the need for systemic immunosuppression and the limited supply of human islet tissue. Biocompatible, retrievable immunoisolation devices containing glucose-responsive insulin-secreting tissue may address both critical barriers by enabling the more effective and efficient use of allogeneic islets without immunosuppression in the near-term, and ultimately the use of a cell source with a virtually unlimited supply, such as human stem cell-derived β-cells or xenogeneic (porcine) islets with minimal or no immunosuppression. However, even though encapsulation methods have been developed and immunoprotection has been successfully tested in small and large animal models and to a limited extent in proof-of-concept clinical studies, the effective use of encapsulation approaches to convincingly and consistently treat diabetes in humans has yet to be demonstrated. There is increasing consensus that inadequate oxygen supply is a major factor limiting their clinical translation and routine implementation. Poor oxygenation negatively affects cell viability and β-cell function, and the problem is exacerbated with the high-density seeding required for reasonably-sized clinical encapsulation devices. Approaches for enhanced oxygen delivery to encapsulated tissues in implantable devices are therefore being actively developed and tested. This review summarizes fundamental aspects of islet microarchitecture and β-cell physiology as well as encapsulation approaches highlighting the need for adequate oxygenation; it also evaluates existing and emerging approaches for enhanced oxygen delivery to encapsulation devices, particularly with the advent of β-cell sources from stem cells that may enable the large-scale application of this approach.

Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions.

Pancreatic islet transplantation has become an established approach to β-cell replacement therapy for the treatment of insulin-deficient diabetes. Recent progress in techniques for islet isolation, islet culture, and peritransplant management of the islet transplant recipient has resulted in substantial improvements in metabolic and safety outcomes for patients. For patients requiring total or subtotal pancreatectomy for benign disease of the pancreas, isolation of islets from the diseased pancreas with intrahepatic transplantation of autologous islets can prevent or ameliorate postsurgical diabetes, and for patients previously experiencing painful recurrent acute or chronic pancreatitis, quality of life is substantially improved. For patients with type 1 diabetes or insulin-deficient forms of pancreatogenic (type 3c) diabetes, isolation of islets from a deceased donor pancreas with intrahepatic transplantation of allogeneic islets can ameliorate problematic hypoglycemia, stabilize glycemic lability, and maintain on-target glycemic control, consequently with improved quality of life, and often without the requirement for insulin therapy. Because the metabolic benefits are dependent on the numbers of islets transplanted that survive engraftment, recipients of autoislets are limited to receive the number of islets isolated from their own pancreas, whereas recipients of alloislets may receive islets isolated from more than one donor pancreas. The development of alternative sources of islet cells for transplantation, whether from autologous, allogeneic, or xenogeneic tissues, is an active area of investigation that promises to expand access and indications for islet transplantation in the future treatment of diabetes.

Localized immune tolerance from FasL-functionalized PLG scaffolds

Intraportal allogeneic islet transplantation has been demonstrated as a potential therapy for type 1 diabetes (T1D). The placement of islets into the liver and chronic immunosuppression to control rejection are two major limitations of islet transplantation. We hypothesize that localized immunomodulation with a novel form of FasL chimeric with streptavidin, SA-FasL, can provide protection and long-term function of islets at an extrahepatic site in the absence of chronic immunosuppression. Allogeneic islets modified with biotin and engineered to transiently display SA-FasL on their surface showed sustained survival following transplantation on microporous scaffolds into the peritoneal fat in combination with a short course (15 days) of rapamycin treatment. The challenges with modifying islets for clinical translation motivated the modification of scaffolds with SA-FasL as an off-the-shelf product. Poly (lactide-co-glycolide) (PLG) was conjugated with biotin and fabricated into particles and subsequently formed into microporous scaffolds to allow for rapid and efficient conjugation with SA-FasL. Biotinylated particles and scaffolds efficiently bound SA-FasL and induced apoptosis in cells expressing Fas receptor (FasR). Scaffolds functionalized with SA-FasL were subsequently seeded with allogeneic islets and transplanted into the peritoneal fat under the short-course of rapamycin treatment. Scaffolds modified with SA-FasL had robust engraftment of the transplanted islets that restored normoglycemia for 200 days. Transplantation without rapamycin or without SA-FasL did not support long-term survival and function. This work demonstrates that scaffolds functionalized with SA-FasL support allogeneic islet engraftment and long-term survival and function in an extrahepatic site in the absence of chronic immunosuppression with significant potential for clinical translation.

PO474 Screening For Cardiovascular Risk Factors and Self Awareness Among Female Medical Students

Adipose tissue–derived mesenchymal stromal cells (AT-MSCs), widely known as multipotent progenitors, release several cytokines that support cell survival and repair. There are in vitro and in vivo studies reporting the regenerative role of AT-MSCs possibly mediated by their protective effects on functional islet cells as well as their capacity to differentiate into insulin-producing cells (IPCs).On such a basis, our goal in the present study was to use three different models including direct and indirect co-cultures and islet-derived conditioned medium (CM) to differentiate AT-MSCs into IPCs and to illuminate the molecular mechanisms of the beneficial impact of AT-MSCs on pancreatic islet functionality. Furthermore, we combined in vitro co-culture of islets and AT-MSCs with in vivo assessment of islet graft function to assess whether co-transplantation of islets with AT-MSCs can reduce marginal mass required for successful islet transplantation and prolong graft function in a diabetic rat model.Our findings demonstrated that AT-MSCs are suitable for creating a microenvironment favorable for the repair and longevity of the pancreas β cells through the improvement of islet survival and maintenance of cell morphology and insulin secretion due to their potent properties in differentiation. Most importantly, hybrid transplantation of islets with AT-MSCs significantly promoted survival, engraftment and insulin-producing function of the graft and reduced the islet mass required for reversal of diabetes.This strategy might be of therapeutic potential solving the problem of donor islet material loss that currently limits the application of allogeneic islet transplantation as a more widespread therapy for type 1 diabetes.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Patient and family expectations of beta-cell replacement therapies in type 1 diabetes

ABSTRACTBackground: New methods of beta-cell replacement have been developed to maintain excellent glycemic control, improve quality of life, and even eliminate insulin injections in patients with type 1 diabetes mellitus (T1DM). Previously, we demonstrated that being insulin-free is the strongest motivation for accepting a newly developed therapy. Multiple allogeneic islet transplantations with immunosuppression using a human donor is the best option to be insulin-free, but the necessity for immunosuppression and donor shortage are major issues. However, these issues have been improved with scientific progress. The aim of this study was to investigate the opinions of patients and their families about the current progress. Methods: We conducted a questionnaire survey of T1DM patients (n = 47) and their family members (n = 49) about newly developed therapies: single and multiple allogeneic islet transplantation, single and multiple encapsulated allogeneic islet transplantation, single and multiple xenogeneic islet transplantation, and induced pluripotent stem cell therapy. Results: More than 90% of respondents wished to be insulin-free and have stable glycemic control. More than 90% of respondents accepted at least one of the new therapies. The current standard treatment multiple allogeneic islet transplantation was not well accepted or favored. Conclusions: The next generation of treatments, including xenotransplantation and induced pluripotent stem cell therapy, were more acceptable and favorable. Even though the majority of patients wish to become insulin-free, it is not sufficiently strong motivation for accepting newly developed treatments.

Clinical islet transplantation: is the future finally now?

Clinical pancreatic islet transplantation has evolved into a routine means to restore glycemic control in patients with type 1 diabetes mellitus (T1DM) suffering from life-threatening hypoglycemia and severe glucose liability. This chapter examines the current progress in islet transplantation while outlining the remaining limitations preventing this life-altering therapy's application to the broader T1DM population. Islet transplantation has recently been demonstrated to provide superior glycemic control with reduced glucose lability and hypoglycemic events compared with standard insulin therapy. Transplant outcomes have steadily improved, in part, reflective of refinements, including more optimal islet donors and isolations, safer transplant techniques and more effective anti-inflammatory and immunomodulatory intervention. Furthermore, latest insulin independence rates 5-years posttransplant have reached parity with pancreas transplantation. Successful completion of a recent National Institutes of Health-sponsored Phase III multicenter clinical allogeneic islet transplantation trial confirmed the safety and efficacy of this therapeutic modality and will be used in the Biological Licensure Application by the United States Food and Drug Administration. Implementation of novel immunosuppression, antiinflammatories, first-in-human stem cell and extrahepatic transplant site trials into clinical investigation has positioned β-cell replacement to become the mainstay treatment for all T1DM patients in the near future.

Report of the Key Opinion Leaders Meeting on Stem Cell-derived Beta Cells.

Beta cell replacement has the potential to restore euglycemia in patients with insulin-dependent diabetes. Although great progress has been made in establishing allogeneic islet transplantation from deceased donors as the standard of care for those with the most labile diabetes, it is also clear that the deceased donor organ supply cannot possibly treat all those who could benefit from restoration of a normal beta cell mass, especially if immunosuppression were not required. Against this background, the International Pancreas and Islet Transplant Association in collaboration with the Harvard Stem Cell Institute, the Juvenile Diabetes Research Foundation (JDRF), and the Helmsley Foundation held a 2-day Key Opinion Leaders Meeting in Boston in 2016 to bring together experts in generating and transplanting beta cells derived from stem cells. The following summary highlights current technology, recent significant breakthroughs, unmet needs and roadblocks to stem cell-derived beta cell therapies, with the aim of spurring future preclinical collaborative investigations and progress toward the clinical application of stem cell-derived beta cells.

Reduction of marginal mass required for successful islet transplantation in a diabetic rat model using adipose tissue–derived mesenchymal stromal cells

Background aimsAdipose tissue–derived mesenchymal stromal cells (AT-MSCs), widely known as multipotent progenitors, release several cytokines that support cell survival and repair. There are in vitro and in vivo studies reporting the regenerative role of AT-MSCs possibly mediated by their protective effects on functional islet cells as well as their capacity to differentiate into insulin-producing cells (IPCs). MethodsOn such a basis, our goal in the present study was to use three different models including direct and indirect co-cultures and islet-derived conditioned medium (CM) to differentiate AT-MSCs into IPCs and to illuminate the molecular mechanisms of the beneficial impact of AT-MSCs on pancreatic islet functionality. Furthermore, we combined in vitro co-culture of islets and AT-MSCs with in vivo assessment of islet graft function to assess whether co-transplantation of islets with AT-MSCs can reduce marginal mass required for successful islet transplantation and prolong graft function in a diabetic rat model. ResultsOur findings demonstrated that AT-MSCs are suitable for creating a microenvironment favorable for the repair and longevity of the pancreas β cells through the improvement of islet survival and maintenance of cell morphology and insulin secretion due to their potent properties in differentiation. Most importantly, hybrid transplantation of islets with AT-MSCs significantly promoted survival, engraftment and insulin-producing function of the graft and reduced the islet mass required for reversal of diabetes. ConclusionsThis strategy might be of therapeutic potential solving the problem of donor islet material loss that currently limits the application of allogeneic islet transplantation as a more widespread therapy for type 1 diabetes.

A model for determining an effective in vivo dose of transplanted islets based on in vitro insulin secretion.

Allogeneic islet transplantation for the treatment of type 1 diabetes often requires multiple implant procedures, from as many as several human pancreas donors, to achieve lasting clinical benefit. Given the limited availability of human pancreases for islet isolation, porcine islets have long been considered a potential option for clinical use. Agarose-encapsulated porcine islets (macrobeads) permit long-term culture and thus a thorough evaluation of microbiological safety and daily insulin secretory capacity, prior to implantation. The goal of this study was the development of a method for determining an effective dose of encapsulated islets based on their measured in vitro insulin secretion in a preclinical model of type 1 diabetes. Spontaneously diabetic BioBreeding diabetes-prone rats were implanted with osmotic insulin pumps in combination with continuous glucose monitoring to establish the daily insulin dose required to achieve continuous euglycaemia in individual animals. Rats were then implanted with a 1×, 2× or 3× dose (defined as the ratio of macrobead in vitro insulin secretion per 24hours to the recipient animal's total daily insulin requirement) of porcine islet macrobeads, in the absence of immunosuppression. In vivo macrobead function was assessed by recipient non-fasted morning blood glucose values, continuous glucose monitoring and the presence of peritoneal porcine C-peptide. At the end of the study, the implanted macrobeads were removed and returned to in vitro culture for the evaluation of insulin secretion. Diabetic rats receiving a 2× macrobead implant exhibited significantly improved blood glucose regulation compared to that of rats receiving a 1× dose during a 30-day pilot study. In a 3-month follow-up study, 2× and 3× macrobead doses initially controlled blood glucose levels equally well, although several animals receiving a 3× dose maintained euglycaemia throughout the study, compared to none of the 2× animals. The presence of porcine C-peptide in rat peritoneal fluid 3months post-implant and the recurrence of hyperglycaemia following macrobead removal, along with the finding of persistent in vitro insulin secretion from retrieved macrobeads, confirmed long-term graft function. Increasing dosages of islet macrobeads transplanted into diabetic rats, based on multiples of in vitro insulin secretion matched to the recipient's exogenous insulin requirements, correlated with improved blood glucose regulation and increased duration of graft function. These results demonstrate the usefulness of a standardized model for the evaluation of the functional effectiveness of islets intended for transplantation, in this case using intraperitoneally implanted agarose macrobeads, in diabetic rats. The results suggest that some features of this islet-dosing methodology may be applicable, and indeed necessary, to clinical allogeneic and xenogeneic islet transplantation.

Development of the Total Pancreatectomy and Autologous Islet Transplantation Models as the Step for Allogenic Islet Transplantation Experiments in the Swine

Development of the Total Pancreatectomy and Autologous Islet Transplantation Models as the Step for Allogenic Islet Transplantation Experiments in the Swine

Improvement of Metabolic Function after Intra-Portal Allogeneic Islet Transplantation Using Induction Treatment of the Non-Hematopoetic Erythropoietin Analogue Cibinetide in a Mouse Model

Improvement of Metabolic Function after Intra-Portal Allogeneic Islet Transplantation Using Induction Treatment of the Non-Hematopoetic Erythropoietin Analogue Cibinetide in a Mouse Model

Immunomodulation with SA-FasL-Engineered Microgels Achieves Long-Term Survival of Allogeneic Islet Grafts

Introduction and Objective Allogenic islet transplantation has shown efficacy in the clinic for the treatment of type 1 diabetes. However, sustained survival of allogeneic islet grafts requires chronic immunosuppression that has significant adverse effects. T effector (Teff) cells recognizing and responding to allograft antigens are the major culprit of graft rejection. Upon activation, Teff cells upregulate Fas death receptor on their surface and become sensitive to FasL-mediated apoptosis. Fas pathway, therefore, presents an important target for immunomodulation to block alloreactive responses with significant therapeutic potential. Herein, we assessed the efficacy of PEG microgels engineered with a novel form of FasL chimeric with a modified form of core streptavidin, SA-FasL, in achieving sustained survival of allogeneic islet grafts in the absence of chronic immunosuppression. Materials and Methods Microgels presenting biotin on their surface were produced by reacting biotin-PEG-thiol with a maleimide-terminated 4-arm poly(ethylene) glycol macromer, and generating 200 μm diameter microgels crosslinked with dithiothreitol. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) taking the advantage of the high affinity interaction between biotin and streptavidin. The apoptotic activity of SA-FasL-engineered microgels was tested on mouse A20 B cell lymphocytes in vitro. The immunomodulatory function of microgels for the prevention of graft rejection was tested by mixing ~500 BALB/c naïve islets with 1000 SA-FasL-engineered microgels and transplanting under the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. A group of recipients were also treated transiently with rapamycin (0.2 mg/kg daily for 15 days starting the day of transplantation). Results and Discussion Biotin-PEG microgels were efficiently coupled with SA-FasL, and showed a dose-dependent apoptotic activity in A20 cells. Co-transplantation of SA-FasL-engineered microgels with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified microgels) with 20% surviving for a 200-day observation period. Transient use of low dose rapamycin, enhanced survival to > 90% of the grafts. In marked contrast, only 20% of islets co-transplanted with PEG microgels, and short-course rapamycin, survived long-term. Importantly, CD4+CD25+FoxP3+ Treg cells were required for graft survival as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. Conclusion These results provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential. Funded in part by NIH (grants R21EB020107, R21AI113348, 185 R56AI121281, and F30AR069472) and the Juvenile Diabetes Research Foundation (2-SRA-186 2014-287-Q-R).

Comparison of Xenogeneic Encapsulated Islet Transplantation in Nonhuman Primates With and Without Immunosuppression

Previously, we have shown the long-term efficacy and biocompatibility of chitosan-coated alginate capsule in allogeneic islet transplantation with canine models of diabetes. In this study, we compared the efficacy and immune response of chitosan-coated alginate capsule in xenogeneic islet transplantation with non-human primates (NHPs). Porcine islets were encapsulated alginate crosslinked with BaCl2, followed by suspension in chitosan solution. Encapsulated porcine islets were transplanted intraperitoneally in NHPs with and without immunosuppressants. Anti-αGal IgM and IgG was measured from the day of transplantation. After transplantation of encapsulated porcine islets, hyperglycemia and exogenous insulin requirements were decreased in the NHP recipients. Porcine C-peptide was detected in plasma after transplantation, but decreased with time. Anti-αGal IgM and IgG began to increase a week after transplantation in NHP without immunosuppressants, while they showed little increase in NHP with immunosuppressants. Exogenous insulin began to be administered to the NHP recipients from 3 to 5 days after transplantation regardless of immunosuppressants. However, daily insulin requirement increased earlier in NHP without immunosuppressants than those with immunosuppressants. Retrieved encapsulated porcine islets from showed fibrosis more than 70% in NHP without immunosuppressants, whereas only 5% of retrieved capsules showed fibrosis in NPH with immunosuppressants. In addition, in NPH with immunosuppressants, dithizone positive islets were observed over a month after transplantation. The use of immunosuppressants in xenogeneic encapsulated islet transplantation in NHP may help reduce fibrosis and improve islet function by modulating immune reaction. Disclosure E. Lee: None. H. Park: None. J. Kim: None. Y. Yang: None. J. Lim: None. H. Kim: None. S. Lee: None. J. Cho: None. B. Cha: None. K. Yoon: None.

Identification of the Subsets of IL-10-Producing Regulatory B Cells in the Course of Tolerance Induction and Maintenance in Islet Allotransplantation

BackgroundOur previous study demonstrated that regulatory B cells (Bregs) play an indispensable role in dual anti-CD45RB/anti-TIM-1 antibody-induced transplantation tolerance through an IL-10-dependent pathway. However, the potentially specific subsets of Bregs have not been clearly demonstrated. In this study, we aimed to detect the subsets of the Bregs possessing the ability to produce IL-10 in the early stage and tolerance maintenance stage of allogeneic islet transplantation treated by dual anti-CD45RB/anti-TIM-1. MethodsIsolated Balb/C islets were transplanted under the kidney capsule of C57BL/6J diabetic mice. The recipients were treated with anti-CD45RB and anti-TIM-1. Two major IL-10-producing Breg subsets of CD19+CD5+CD1d+ B10 cells and CD19+TIM-1+B cells, and the T cells of CD4+CD25+ (Tregs), CD4+IFN-γ+ Th1 cells, and CD4+IL-4+ Th2 cells in splenocytes of grafted recipients were detected by flow cytometry after 1 week, 2 weeks, and more than 100 days posttransplantation. ResultsOn day 14 after islet transplantation, the frequency of CD19+CD5+CD1d+ B10 cells in recipient mice increased significantly, compared with day 0 and 7 (P = .001, P < .001, respectively), while it dropped to normal level in the recipients with long-term graft survival. In contrast, there was significant increase of CD19+TIM-1+B cells on day 14 (P = .003) and day 100 after transplant (P = .009). CD4+CD25+ Treg cells have similar increasing tendency with CD19+TIM-1+B cells, as they increased from normal 10% to almost 20% both on day 14 and day 90 after transplantation. Conversely, the expression of CD4+IFN-γ+ Th1 cells decreased significantly on day 14 (P = .004) and on day 100 after transplant (P = .002). As another kind of helper T cells, CD4+IL-4+ Th2 cells increased only on day 14 after transplant (P < .001). ConclusionsCD19+CD5+CD1d+ B10 cells may play an important role only in the early stage of transplantation tolerance induction by dual anti-CD45RB/anti-TIM-1 antibody treatment, whereas CD19+TIM-1+B cells may play crucial parts in the whole process of tolerance induction and maintenance.

Novel Consistently Ex-vivo Expanded CD8+CD25+FoxP3+Treg Cells Prolongs the Allogeneic Islet Survival

Abstract In addition to CD4+ regulatory T cells(Tregs), adoptive transfer of CD8+CD25+FoxP3+T cells is emerging as an alternative adjunctive therapy to diminish current reliance on lifelong, nonspecific immunosuppression after transplantation. Here we evaluated the possible therapeutic application of consistently ex-vivo expanded mouse CD8+CD25+FoxP3+T cells to prevent the rejection of allogeneic islets. Around 80% of mouse CD8+CD25+T and CD4+CD25+T cells expressed FoxP3+T cells in spleen. Higher level of FoxP3 was expressed in CD8+Tregs compared to CD4+Tregs. Ex-vivo enriched CD8+CD25+Treg cells were successfully and consistently expanded to 600–1,000 times from the initial culture for 12 days with optimized culture conditions. High concentration of IL-2 (&amp;gt;500units) and TGF-beta together with aCD3-and aCD28 stimulation were essential to the massive ex-vivo expansion. Expanded Tregs showed 99% of FoxP3 expression and expressed high level of CD27, CD47 and Lag-3 in their surface. Very high amount of Granzyme B and minor amount of IFN-r and IL-17 were xpressed. Expanded CD8+Tregs successfully inhibits the proliferation and activation of CD4+T cells in vitro. In the allogenic islet transplantation model (Balb.Cà B6), Single injection of ex-vivo expanded CD8+Tregs prolonged islet allograft survival more than 100 days after the reconstitution with CD90.2+T cells in immunodeficient B6 rag2(−/−) mice by inhibiting antigen-specific IFN-r producing cell generation. In this study, we proved that ex-vivo expanded CD8+Treg retain regulatory activity and can protect islet allograft rejection by single administration. These implies that ex-vivo expanded CD8+Tregs has clinical relevance of an adjunctive cellular therapy.

Microporous Polymer Scaffolds for the Transplantation of Embryonic Stem Cell Derived Pancreatic Progenitors to a Clinically Translatable Site for the Treatment of Type I Diabetes.

Type I diabetes mellitus, which affects an estimated 1.5 million Americans, is caused by autoimmune destruction of the pancreatic beta cells that results in the need for life-long insulin therapy. Allogeneic islet transplantation for the treatment of type I diabetes is a therapy in which donor islets are infused intrahepatically, which has led to the transient reversal of diabetes. However, therapeutic limitations of allogeneic transplantation, which include a shortage of donor islets, long-term immunosuppression, and high risk of tissue rejection, have led to the investigation of embryonic or induced pluripotent stem cells as an unlimited source of functional beta-cells. Herein, we investigate the use of microporous scaffolds for their ability to promote the engraftment of stem cell derived pancreatic progenitors and their maturation toward mono-hormonal insulin producing β-cells at a clinically translatable, extrahepatic site. Initial studies demonstrated that microporous scaffolds supported cell engraftment, and their maturation to become insulin positive; however, the number of insulin positive cells and the levels of C-peptide secretion were substantially lower than what was observed with progenitor cell transplantation into the kidney capsule. The scaffolds were subsequently modified to provide a sustained release of exendin-4, which has previously been employed to promote maturation of pancreatic progenitors in vitro and has been employed to promote engraftment of transplanted islets in the peritoneal fat. Transplantation of stem cell derived pancreatic progenitors on scaffolds releasing exendin-4 led to significantly increased C-peptide production compared to scaffolds without exendin-4, with C-peptide and blood glucose levels comparable to the kidney capsule transplantation cohort. Image analysis of insulin and glucagon producing cells indicated that monohormonal insulin producing cells were significantly greater compared to glucagon producing and polyhormonal cells in scaffolds releasing exendin-4, whereas a significantly decreased percentage of insulin-producing cells were present among hormone producing cells in scaffolds without exendin-4. Collectively, a microporous scaffold, capable of localized and sustained delivery of exendin-4, enhanced the maturation and function of pluripotent stem cell derived pancreatic progenitors that were transplanted to a clinically translatable site.