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BackgroundThe shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment).MethodIE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function.

Background: Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety., Methods: Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability., Results: Differentiation was capable of generating 90.4% PDX1+/NKX6.1+ pancreatic progenitors and 100% C-peptide+/NKX6.1+ iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19+ ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 104 cells/cm2 (total 8.3 × 106 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 106 cells/mL (total 105.0 × 106 cells), reducing costs by 88.8%., Conclusions: This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies., Competing Interests: A.M.J.S. serves as a consultant to ViaCyte Inc, Vertex Inc, Betalin Ltd, Hemostemix Inc, and Aspect Biosystems Ltd. A.M.J.S. is supported through a Canada Research Chair (tier 1) in Regenerative Medicine and Transplant Surgery and through grant support from the Juvenile Diabetes Research Foundation, Diabetes Canada, the Canadian Donation and Transplant Research Program, the Diabetes Research Institute Foundation of Canada, the Alberta Diabetes Foundation, and the Canadian Stem Cell Network. The other authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Generation of pure pancreatic progenitor (PP) cells is critical for clinical translation of stem cell-derived islets. Herein, we performed PP differentiation with and without AKT/P70 inhibitor AT7867 and characterized the resulting cells at protein and transcript level in vitro and in vivo upon transplantation into diabetic mice. AT7867 treatment increased the percentage of PDX1 + NKX6.1 + (-AT7867: 50.9% [IQR 48.9%-53.8%]; +AT7867: 90.8% [IQR 88.9%-93.7%]; p = 0.0021) and PDX1 + GP2 + PP cells (-AT7867: 39.22% [IQR 36.7%-44.1%]; +AT7867: 90.0% [IQR 88.2%-93.6%]; p = 0.0021). Transcriptionally, AT7867 treatment significantly upregulated PDX1 (p = 0.0001), NKX6.1 (p = 0.0005), and GP2 (p = 0.002) expression compared with controls, while off-target markers PODXL (p < 0.0001) and TBX2 (p < 0.0001) were significantly downregulated. Transplantation of AT7867-treated PPs resulted in faster hyperglycemia reversal in diabetic mice compared with controls (time and group: p < 0.0001). Overall, our data show that AT7867 enhances PP cell differentiation leading to accelerated diabetes reversal., Competing Interests: Declaration of interests A.M.J.S. serves as a consultant to ViaCyte Inc., Vertex Pharmaceuticals Inc., Betalin Therapeutics Ltd., and Aspect Biosystems Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Type 1 Diabetes (T1D) is an autoimmune destruction of pancreatic beta cells. The development of the Edmonton Protocol for islet transplantation in 2000 revolutionized T1D treatment and offered a glimpse at a cure for the disease. In 2022, the 20-year follow-up findings of islet cell transplantation demonstrated the long-term safety of islet cell transplantation despite chronic immunosuppression. The Edmonton Protocol, however, remains limited by two obstacles: scarce organ donor availability and risks associated with chronic immunosuppression. To overcome these challenges, the search has begun for an alternative cell source. In 2006, pluripotency genomic factors, coined "Yamanaka Factors," were discovered, which reprogram mature somatic cells back to their embryonic, pluripotent form (iPSC). iPSCs can then be differentiated into specialized cell types, including islet cells. This discovery has opened a gateway to a personalized medicine approach to treating diabetes, circumventing the issues of donor supply and immunosuppression. In this review, we present a brief history of allogenic islet cell transplantation from the early days of pancreatic remnant transplantation to present work on encapsulating stem cell-derived cells. We review data on long-term outcomes and the ongoing challenges of allogenic islet cell and stem cell-derived islet cell transplant.

Background: Induced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol., Methods: Sendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs., Results: Expansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67 + expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4 + Nanog + Sox2 + ) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67 + expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype., Conclusion: This study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation., (© 2023. The Author(s).)

Background: ABO-incompatible transplantation has improved accessibility of kidney, heart, and liver transplantation. Pancreatic islet transplantation continues to be ABO-matched, yet ABH antigen expression within isolated human islets or novel human embryonic stem cell (hESC)-derived islets remain uncharacterized., Methods: We evaluated ABH glycans within human pancreata, isolated islets, hESC-derived pancreatic progenitors, and the ensuing in vivo mature islets following kidney subcapsular transplantation in rats. Analyses include fluorescence immunohistochemistry and single-cell analysis using flow cytometry., Results: Within the pancreas, endocrine and ductal cells do not express ABH antigens. Conversely, pancreatic acinar tissues strongly express these antigens. Acinar tissues are present in a substantial portion of cells within islet preparations obtained for clinical transplantation. The hESC-derived pancreatic progenitors and their ensuing in vivo-matured islet-like clusters do not express ABH antigens., Conclusions: Clinical pancreatic islet transplantation should remain ABO-matched because of contaminant acinar tissue within islet preparations that express ABH glycans. Alternatively, hESC-derived pancreatic progenitors and the resulting in vivo-matured hESC-derived islets do not express ABH antigens. These findings introduce the potential for ABO-incompatible cell replacement treatment and offer evidence to support scalability of hESC-derived cell therapies in type 1 diabetes., Competing Interests: A.M.J.S. is supported through a Canada Research Chair (Tier 1) in Regenerative Medicine and Transplant Surgery, and through grant support from the Juvenile Diabetes Research Foundation, Diabetes Canada, the Canadian Donation and Transplant Research Program, the Diabetes Research Institute Foundation of Canada, the Alberta Diabetes Foundation, and the Canadian Stem Cell Network. B.A.M.-G. is supported by the Patronato del Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (INCMNSZ), the Fundacion para la Salud y la Educacion Dr. Salvador Zubirán (FunSaEd), and the CHRISTUS Excellence and Innovation Center. A.M.J.S. serves as a consultant to ViaCyte Inc. The other authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Recapitulation of embryonic pancreatic development has enabled development of methods for in vitro islet cell differentiation using human pluripotent stem cells (hPSCs), which have the potential to cure diabetes. Advanced methods for optimal generation of stem-cell-derived islets (SC-islets) has enabled successful diabetes reversal in rodents and shown promising early clinical trial outcomes. The main impediment for use of SC-islets is concern about safety because of off-target growth resulting from contaminated residual cells. In this review, we summarize the different endocrine and non-endocrine cell populations that have been described to emerge throughout β cell differentiation and after transplantation. We discuss the most recent approaches to enrich endocrine populations and remove off-target cells. Finally, we discuss the critical quality control and release criteria testing that we anticipate will be required prior to transplantation to ensure product safety., Competing Interests: Declaration of interests A.M.J.S. serves as a consultant to ViaCyte Inc., Diagon Inc., Hemostemix Inc., Protokinetix Inc., and Pelican Therapeutics, Inc., (Copyright © 2022. Published by Elsevier Inc.)

Regulatory T cells (Tregs) have become highly relevant in the pathophysiology and treatment of autoimmune diseases, such as type 1 diabetes (T1D). As these cells are known to be defective in T1D, recent efforts have explored ex vivo and in vivo Treg expansion and enhancement as a means for restoring self-tolerance in this disease. Given their capacity to also modulate alloimmune responses, studies using Treg-based therapies have recently been undertaken in transplantation. Islet transplantation provides a unique opportunity to study the critical immunological crossroads between auto- and alloimmunity. This procedure has advanced greatly in recent years, and reports of complete abrogation of severe hypoglycemia and long-term insulin independence have become increasingly reported. It is clear that cellular transplantation has the potential to be a true cure in T1D, provided the remaining barriers of cell supply and abrogated need for immune suppression can be overcome. However, the role that Tregs play in islet transplantation remains to be defined. Herein, we synthesize the progress and current state of Treg-based therapies in T1D and islet transplantation. We provide an extensive, but concise, background to understand the physiology and function of these cells and discuss the clinical evidence supporting potency and potential Treg-based therapies in the context of T1D and islet transplantation. Finally, we discuss some areas of opportunity and potential research avenues to guide effective future clinical application. This review provides a basic framework of knowledge for clinicians and researchers involved in the care of patients with T1D and islet transplantation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Liver ischemia reperfusion injury (IRI) remains a challenge in liver transplantation. A number of compounds have previously demonstrated efficacy in mitigating IRI. Herein, we applied three specific additive strategies to a mouse IRI screening model to determine their relative potencies in reducing such injury, with a view to future testing in a large animal and clinical ex situ normothermic perfusion setting: 1) F573, a pan-caspase inhibitor, 2) anti-inflammatory anakinra and etanrecept and 3) BMX-001, a mimetic of superoxide dismutase., Methods: A non-lethal liver ischemia model in mice was used. Additives in the treatment groups were given at fixed time points before induction of injury, compared to a vehicle group that received no therapeutic treatment. Mice were recovered for 6 hours following the ischemic insult, at which point blood and tissue samples were obtained. Plasma was processed for transaminase levels. Whole liver tissue samples were processed for histology, markers of apoptosis, oxidative stress, and cytokine levels., Results: In an in vivo murine IRI model, the F573 treatment group demonstrated statistically lower alanine aminotransferase (ALT) levels (p = 0.01), less evidence of apoptosis (p = 0.03), and lower cytokine levels compared to vehicle. The etanercept with anakinra treatment group demonstrated significantly lower cytokine levels. The BMX-001 group demonstrated significantly decreased apoptosis (p = 0.01) evident on TUNEL staining., Conclusions: The administration of pan-caspase inhibitor F573 in a murine in vivo model likely mitigates liver IRI based on decreased markers of cellular injury, decreased evidence of apoptosis, and improved cytokine profiles. Anakinra with etanercept, and BMX-001 did not demonstrate convincing efficacy at reducing IRI in this model, and likely need further optimization. The positive findings set rational groundwork for future translational studies of applying F573 during normothermic ex situ liver perfusion, with the aim of improving the quality of marginal grafts., Competing Interests: The authors have declared that no competing interests exist.

The paucity of human donors limits broadened application of β-cell replacement therapy. Insulin-producing cells derived from human embryonic stem cells (hESCs) have recently been investigated clinically as a feasible surrogate to primary tissue. Herein, we examine the long-term efficacy of hESC-derived pancreatic endoderm cells (PECs) to maintain normoglycemia posttransplant and characterize the phenotype of the PEC grafts. Mice with chemically induced diabetes were transplanted with PECs into the subcutaneous device-less site. Transplant function was assessed through nonfasting blood glucose measurements, intraperitoneal glucose tolerance testing (IPGTT), and human C-peptide secretion for 517 days. Explanted grafts were assessed for ex vivo function and immunohistochemically. All PEC recipients ( n = 8) maintained normoglycemia until graft retrieval. IPGTTs at 365 and 517 days posttransplant did not differ ( P > 0.05), however, both demonstrated superior glucose clearance compared with nondiabetic and transplant controls ( P < 0.001). Serum C-peptide levels demonstrated significant glucose responsiveness (fasted vs. stimulated) ( P < 0.01). Small intragraft cysts were palpable in all mice, which resolved but recurred after aspiration. Cysts showed monomorphic neuroendocrine proliferation and lined by ductal epithelium. Explanted grafts demonstrated similar insulin secretory capacity as human islets and stained positively for endocrine cells. Our results demonstrate the ability of PECs to differentiate in vivo and restore glycemic control while confirming minimal proliferation and absence of neoplastic change within the grafts during the time evaluated., (© 2019 by the American Diabetes Association.)

Islet transplantation is an established clinical procedure for select patients with type 1 diabetes and severe hypoglycemia to stabilize glycemic control. Post-transplant, substantial beta cell mass is lost, necessitating multiple donors to maintain euglycemia. A potential strategy to augment islet engraftment is the co-transplantation of islets with multipotent mesenchymal stem cells to capitalize upon their pro-angiogenic and anti-inflammatory properties. Herein, we examine the in vitro and in vivo effect of co-culturing murine islets with human adipose-derived mesenchymal stem cells (Ad-MSCs). Islets co-cultured with Ad-MSCs for 48 hours had decreased cell death, superior viability as measured by membrane integrity, improved glucose stimulated insulin secretion and reduced apoptosis compared to control islets. These observations were recapitulated with human islets, albeit tested in a limited capacity. Recipients of marginal mouse islet mass grafts, co-transplanted with Ad-MSCs without a co-culture period, did not reverse to normoglycemia as efficiently as islets alone. However, utilizing a 48-hour co-culture period, marginal mouse islets grafts with Ad-MSCs achieved a superior percent euglycemia rate when compared to islets cultured and transplanted alone. A co-culture period of human islets with human Ad-MSCs may have a clinical benefit improving engraftment outcomes., Competing Interests: The authors have declared that no competing interests exist.

Aims/hypothesis: Islet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process., Methods: Human islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1 -/- mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining., Results: CFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min)., Conclusions/interpretation: The ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.

Background: 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., Methods: 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., Results: 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)., Conclusions: The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of "healthy" human donor pancreata restricts the broader application of this effective curative therapy. "β-Like" cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Islet transplantation is a highly effective treatment for stabilizing glycemic control for select patients with type-1 diabetes. Despite improvements to clinical transplantation, single-donor transplant success has been hard to achieve routinely, necessitating increasing demands on viable organ availability. Donation after circulatory death (DCD) may be an alternative option to increase organ availability however, these organs tend to be more compromised. The use of metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds previously demonstrated improved in vivo islet function in preclinical islet transplantation. However, the administration of MnP (BMX-001) in a DCD islet isolation and transplantation model has yet to be established. In this study, murine donors were subjected to a 15-min warm ischemic (WI) period prior to isolation and culture with or without MnP. Subsequent to one-hour culture, islets were assessed for in vitro viability and in vivo function. A 15-minute WI period significantly reduced islet yield, regardless of MnP-treatment relative to yields from standard isolation. MnP-treated islets did not improve islet viability compared to DCD islets alone. MnP-treatment did significantly reduce the presence of extracellular reactive oxygen species (ROS) (p < 0 .05). Marginal, syngeneic islets (200 islets) transplanted under the renal capsule exhibited similar in vivo outcomes regardless of WI or MnP-treatment. DCD islet grafts harvested 7 d post-transplant exhibited sustained TNF-α and IL-10, while MnP-treated islet-bearing grafts demonstrated reduced IL-10 levels. Taken together, 15-minute WI in murine islet isolation significantly impairs islet yield. DCD islets do indeed demonstrate in vivo function, though MnP therapy was unable to improve viability and engraftment outcomes.

Background: Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. However, despite early insulin independence, long-term graft attrition gradually reverts recipients to exogenous insulin dependency. Undoubtedly, as insulin producing stem cell therapies progress, a transplant site that is retrievable is desirable. This prerequisite is currently incompatible with intrahepatic islet transplantation. Herein, we evaluate the functional capacity of a prevascularized subcutaneous site to accommodate marginal islet mass transplantation in mice., Methods: Syngeneic mouse islets (150) were transplanted either under the kidney capsule (KC), into a prevascularized subcutaneous device-less (DL) site, or into the unmodified subcutaneous (SC) tissue. The DL site was created 4 weeks before diabetes induction and islet transplantation through the transient placement of a 5-Fr vascular catheter. Recipient mice were monitored for glycemic control and intraperitoneal glucose tolerance., Results: A marginal islet mass transplanted into the DL site routinely reversed diabetes (n = 13 of 18) whereas all SC islet recipients failed to restore glycemic control (n = 0 of 10, P < 0.01, log-rank). As anticipated, nearly all islet-KC mice (n = 15 of 16) became euglycemic posttransplant. The DL recipients' glucose profiles were comparable to KC islet grafts, postintrapertioneal glucose tolerance testing, whereas SC recipients remained hyperglycemic postglucose challenge. All normoglycemic mice maintained graft function for 100 days until graft retrieval. DL and KC islet grafts stained positively for insulin, microvessels, and a collagen scaffold., Conclusions: The device-less prevascularized approach supports marginal mass islet engraftment in mice.

Islet transplantation (IT) is an established clinical therapy for select patients with type-1 diabetes. Clinically, the hepatic portal vein serves as the site for IT. Despite numerous advances in clinical IT, limitations remain, including early islet cell loss posttransplant, procedural complications, and the inability to effectively monitor islet grafts. Hence, alternative sites for IT are currently being explored, with the subcutaneous space as one potential option. When left unmodified, the subcutaneous space routinely fails to promote successful islet engraftment. However, when employing the previously developed subcutaneous “deviceless” technique, a favorable microenvironment for islet survival and function is established. In this technique, an angiocatheter was temporarily implanted subcutaneously, which facilitated angiogenesis to promote subsequent islet engraftment. This technique has been employed in preclinical animal models, providing a sufficient means to develop techniques to monitor functional aspects of the graft such as angiogenesis. Here, we utilize photoacoustic imaging to track angiogenesis during the priming of the subcutaneous site by the implanted catheter at 1 to 4 weeks postcatheter. Quantitative analysis on vessel densities shows gradual growth of vasculature in the implant position. These results demonstrate the ability to track angiogenesis, thus facilitating a means to optimize and assess the pretransplant microenvironment.

Background: Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation., Methods: Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance., Results: Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels., Conclusions: The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies.

Transplantation of donor-derived islets into the liver is a successful cellular replacement therapy for individuals with diabetes. However, the hepatic vasculature is not an optimal transplant site for several reasons, including graft attrition and the inability to retrieve or image the islets. Here we describe islet transplantation into a prevascularized, subcutaneous site created by temporary placement of a medically approved vascular access catheter. In mice with streptozotocin (STZ)-induced diabetes, transplantation of ∼500 syngeneic islets into the resulting 'device-less' space reversed diabetes in 91% of mice and maintained normoglycemia for >100 days. The approach was also effective in mice with pre-existing diabetes, in another mouse strain that mounts a more vigorous inflammatory response, and across an allogeneic barrier. These results demonstrate that transient priming of a subcutaneous site supports diabetes-reversing islet transplantation in mouse models without the need for a permanent cell-encapsulation device.

Background: Pancreas dissociation is a critical initial component of the islet isolation procedure and introduces high variability based on factors including the enzyme type, specificity and potency. Product refinement and alterations to the application strategies have improved isolation outcomes over time; however, islet utilization from donor organs remains low. In this study we evaluate a low endotoxin-high activity grade neutral protease in clinical islet isolation., Materials and Methods: The use of a non-collagenolytic enzyme, either thermolysin or high active neutral protease, was randomized in clinical islet isolations to evaluate efficacy. Additionally a retrospective comparison to neutral protease NB was conducted., Results: The thermolysin group had lower trapped islet population and increased purity and post-culture islet mass in comparison to high active grade neutral protease. Comparison of neutral protease NB GMP grade to high active neutral protease displayed no measurable difference in islet mass or viability and transplantation outcomes at 1 mo post-transplant were favorable for both groups., Conclusions: High activity neutral protease can generate clinical grade islets and may prove beneficial to islet function and viability based on a reduced endotoxin load but dosing of neutral protease requires ongoing optimization.

Background: Large numbers of islets are lost in the early phase after clinical islet transplantation, through apoptosis, necrosis, or innate inflammatory injury. We previously demonstrated the efficacy of a series of caspase inhibitors in mouse models on islet engraftment through reduction in early posttransplant apoptosis. We studied IDN6556, a caspase inhibitor with a first-pass effect, in a large animal (pig) intraportal marginal mass islet autotransplant model., Methods: Total pancreatectomy and marginal mass islet autotransplantation were carried out in Yucatan miniature swine to explore the effects of IDN6556 on islet engraftment. Pigs were treated with IDN6556 at a dose of 20 mg/kg orally twice daily (n=7) or phosphate-buffered saline control (n=6) orally for 7 days, and blood glucose was monitored for 1 month. Glucose tolerance and acute insulin release were determined at 1 month., Results: There were no differences in islet procurement, isolation, or islet functional parameters between the two groups. Pigs receiving IDN6556 had lower fasting blood glucose level after transplantation and a higher percentage (100% vs. 33.3%) showed fasting blood glucose levels less than 11 mM. This translated into an enhanced metabolic reserve and acute insulin release for pigs in the treatment group., Conclusions: IDN6556 led to enhanced islet engraftment in this large animal islet transplant model. Although this study has limitations including a short interval of study (1 month) and the use of unpurified islets, the results justify early clinical trials of IDN6556 in islet transplantation.

One limitation of current islet transplantation protocols is the loss of up to 70% of the transplanted islet mass. Inflammatory events play a major role in islet loss including the cytokines TNFα and IL-1. Resveratrol, a compound with anti-inflammatory and anti-oxidant properties, has the potential to mitigate islet loss. Using a syngeneic marginal  after mouse islet transplantation model we tested the ability of resveratrol to enhance islet engraftment. We failed to show a difference in diabetes reversal between mice treated with vehicle and those treated with either 10 mg/kg (47.1% for resveratrol vs. 35.3% for control) or 50 mg/kg (20% for resveratrol vs. 22.2% for control) of resveratrol daily for three weeks. In addition, at one month there was no difference in glucose tolerance or graft survival (10 mg/kg: 552.6 ng/ml resveratrol group vs. 576.6 ng/ml control group; 50 mg/kg: 463 ng/ml resveratrol group vs. 444.1 ng/ml control group). In summary, over a wide range of doses, resveratrol did not exert a benefit on mouse islet engraftment. Further studies should be conducted with human islets before deeming resveratrol ineffective in islet engraftment and survival.

Background: Islet transplantation has become a viable option for selected type 1 diabetic patients; however, a significant portion need to return to exogenous insulin. The predominant factors include impaired islet engraftment and early islet loss. Caspase inhibition is a potent way to improve islet engraftment, but all tested compounds so far have not been clinically relevant. IDN-6556 (PF3491390) has already been used clinically and can be delivered orally with high portal vein concentrations., Methods: Mice were given a marginal mass islet graft of either mouse or human islets and treated with either IDN-6556 (10 or 20 mg/kg ip bid) or vehicle and followed for diabetes reversal. At 1 month post-transplant, mice were subjected to a glucose tolerance test and an assessment of graft mass. In separate experiments, human islets were cultured with IDN-6556 or vehicle to assess for islet survival and viability., Results: In both syngeneic mouse islets and human islets transplanted into immunodeficient mice, IDN-6556 (20 mg/kg) given for 7 days post-transplant led to a significantly enhanced rate of diabetes reversal as compared to vehicle. In addition, mice receiving caspase inhibitor displayed improved glucose tolerance and graft survival at the 1-month point. We also found protective effects in vitro for islet viability and marked reduction in apoptosis in vivo., Conclusion: Taken together, these results demonstrate the effectiveness of caspase inhibition with IDN-6556 on islet transplantation and in particular islet engraftment and survival., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Islet transplantation has become a very promising treatment for type 1 diabetes. To facilitate further clinical improvements in this exciting field, rodent islets are used to evaluate new strategies and modifications. One method to purify islets is on a density gradient, although the optimal gradient component can be debated. N=6 separate mouse islet isolations were used and the resulting islets were separated and purified on either a Ficoll, Histopaque, Dextran or Iodixanol gradient. Islets were assessed for recovery, viability, purity and in vitro functionality. Aliquots were transplanted into diabetic mice to assess in vivo functionality and survival. There was no difference in the number of islets recovered across groups nor in the size of recovered islets. Use of a Ficoll or Histopaque gradient led to the most pure and viable islets in comparison to Dextran and Iodixanol. Functionally, islets isolated on a Ficoll gradient had the highest glucose-stimulated insulin release in vitro while performing equally to Histopaque and Dextran gradients in vivo. Using a Ficoll gradient, however, comes at a higher monetary cost. We recommend using a Histopaque gradient, which led to the isolation of viable and functional islets with a reduced cost as compared to a Ficoll gradient.

Improved posttransplant monitoring and on-time detection of rejection could improve islet transplantation outcome. The present study explored the possibility of detecting harmful events after mouse islet transplantation measuring the immune responsiveness against islet extracts. Mouse islet transplantations were performed using various donor/recipient combinations, exploring autoimmune (NOD/SCID to NOD, n = 6) and alloimmune events (C57BL/6 to BALB/c, n = 20), a combination of both (C57BL/6 to NOD, n = 8), the absence of both (BALB/c to BALB/c, n = 21), or naive, nontransplanted control mice (n = 14). The immune reactivity was measured by ELISPOT, looking at the ex vivo release of IFN-γ from splenocytes stimulated by islet donor extracts (sonicated islets). The immune reactivity was not altered in the syngeneic and autoimmune models, demonstrating similar levels as nontransplanted controls (p = 0.46 and p = 0.6). Conversely, the occurrence of an allogeneic rejection alone or in combination to autoimmunity was associated to an increase in the level of immune reactivity (p = 0.023 and p = 0.003 vs. respective controls). The observed increase was transient and lost in the postrejection period or after treatment with CTLA4-Ig. Overall, allogeneic rejection was associated to a transient increase in the reactivity of splenocytes against islet proteins. Such a strategy has the potential to improve islet graft monitoring in human and should be further explored.

Background: After the discontinuation of the manufacturing Liberase HI because of a small potential for prion disease transmission, Roche Diagnostics (Indianapolis, IN) developed a new enzyme product (Liberase MTF [mammalian tissue free]), which is similar to Liberase HI with the exception that no mammalian tissue is used in the manufacture of the collagenase component. We report our experience using the MTF enzyme in clinical islet isolations compared with Serva NB-1 with modified enzyme delivery method., Methods: Islets were isolated from 41 pancreata using MTF enzyme (n=17) or NB-1 enzyme (n=24). NB-1 enzymes were delivered using a modified (nonsimultaneous) enzyme delivery method whereas isolations using MTF used the standard method of simultaneous collagenase and thermolysin perfusion. Islets were purified on a COBE 2991 Cell Blood Processor and subsequently cultured., Results: The average islet mass after purification was 392+/-36 x 10 islet equivalent (IE) for MTF versus 371+/-40 x 10 IE for Serva NB-1 (P=0.63). Post-IE/cm of tissue was 110+/-9 x 10 IE/cm and 91+/-11 x 10 IE/cm for MTF and NB-1, respectively (P=0.07). The isolation success rate (>400,000 IE) for MTF was 53% compared with 33% for Serva (P=0.33)., Conclusion: We conclude that MTF may be successfully used for high-yield human islet isolation and clinical transplantation and provides similar quality islets to those derived using NB-1.

Objective: Costimulation blockade has emerged as a selective nontoxic maintenance therapy in transplantation. However, these drugs must be combined with other immunomodulatory agents to ensure long-term graft survival., Research Design and Methods: Recent work has demonstrated that caspase inhibitor therapy (EP1013) prevents engraftment phase islet loss and markedly reduces the islet mass required to reverse diabetes. The "danger" hypothesis suggests that reduction in graft apoptosis should reduce the threshold for immunosuppression and increase the possibility for tolerance induction. Thus, the impact of combination of EP1013 treatment with costimulation blockade (CTLA4-Ig) was investigated in this study., Results: Islet allografts were completed in fully major histocompatibility complex (MHC)-mismatched mice (Balb/C to B6). When animals received vehicle or EP1013, there was no difference in graft survival. CTLA4-Ig resulted in prolonged graft survival in 40% of the animals, whereas EP1013+CLTA4-Ig resulted in a significant increase in graft survival (91% >180 days; P = 0.01). Ex vivo analysis revealed that animals receiving EP1013 or EP1013+CTLA4-Ig had a reduced frequency of alloreactive interferon (IFN)-gamma-secreting T-cells and an increased frequency of intragraft Foxp3(+) Treg cells. Alloantibody assays indicated that treatment with EP1013 or CTLA4-Ig prevented allosensitization., Conclusions: This study suggests that addition of caspase inhibitor therapy to costimulation blockade will improve clinical transplantation by minimizing immune stimulation and thus reduce the requirement for long-term immunosuppressive therapy. The approach also prevents allosensitization, which may be an important component of chronic graft loss in clinical transplantation.

The culture of human islets is associated with approximately 10-20% islet loss, occasionally preventing transplantation. Preconditioning of the islets to improve postculture yields would be of immediate benefit, with the potential to increase both the number of transplanted patients and their metabolic reserve. In this study, the effect of liraglutide, a long-acting human glucagon-like peptide 1 analogue, on cultured human islets was examined. Culture with liraglutide (1 micromol/l) was associated with a preservation of islet mass (significantly more islets at 24 and 48 h, compared to control; P < or = 0.05 at 24 and 48 h) and with the presence of larger islets (P < or = 0.05 at 48 h). These observations were supported by reduced apoptosis rates after 24 h of treatment. We also demonstrated that human islet engraftment is improved in C57Bl/6-RAG(-/-) mice treated with liraglutide 200 microg/kg sc twice daily (P < or = 0.05), suggesting that liraglutide should be continued after transplantation. Overall, these data demonstrate the beneficial effect of liraglutide on cultured human islets, preserving islet mass. They support the design of clinical studies looking at the effect of liraglutide in clinical islet transplantation.

Background: Successful islet isolation relies heavily on enzyme products. Among them, Liberase was used in islet transplantation programs until the islet community was notified of the use of a bovine brain component during the manufacturing process. To minimize potential risk of prion disease transmission, many islet isolation facilities switched to Serva enzyme, which is considered to pose less risk. However, this conversion significantly affected the field in transplant activity. Here, we report our successful conversion from Liberase to Serva collagenase with the use of a modified digestion protocol., Methods: We compared the quality of Serva versus Liberase enzyme using chromatography and collagenase activity assay. On the basis of the findings, we developed a pancreas digestion protocol optimized for Serva enzyme, where only collagenase was injected into the pancreas through the duct, and then neutral protease was added to the circulating system during the digestion phase., Results: Class I collagenase activity of Serva was remarkably reduced compared with Liberase. Chromatography of Serva demonstrated suspected degradation of class I collagenase. When the modified protocol was applied to donor pancreata more than 35 years of age, we recovered 3119+/-147 islet equivalent/g pancreas with a success rate of 51% (35/68), whereas the standard method yielded only 1809+/-266 islet equivalent/g pancreas (P=0.02) with a success rate of 11% (1/9). This beneficial effect of the modified method was, however, diminished when applied to younger donor pancreata., Conclusions: Our study brings new insight into the role of collagenase and noncollagenolytic protease on pancreas dissociation.

Although insulin independence is maintained in most islet recipients at 1 yr after transplant, extended follow-up has revealed that many patients will eventually require insulin therapy. Previous studies have shown that islet autografts are prone to chronic failure in large animals and humans, suggesting that nonimmunological events contribute to islet graft functional decay. Early intervention with therapies that promote graft stability should provide a measurable benefit over time. In this study, the efficacy of the long-acting glucagon-like peptide-1 analog liraglutide was explored in a porcine marginal mass islet autograft transplant model. Incubation with liraglutide enhanced porcine islet survival and function after prolonged culture. Most vehicle-treated (83%) and liraglutide-treated (80%) animals became insulin independent after islet autotransplantation. Although liraglutide therapy did not improve insulin independence rates or blood glucose levels after transplant, a significant increase in insulin secretion and acute-phase insulin response was observed in treated animals. Surprisingly, no evidence for deterioration of graft function was observed in any of the transplanted animals over more than 18 months of follow-up despite significant weight gain; in fact, an enhanced response to glucose developed over time even in control animals. Histological analysis showed that intraportally transplanted islets remained highly insulin positive, retained alpha-cells, and did not form amyloid deposits. This study demonstrates that marginal mass porcine islet autografts have stable long-term function, even in the presence of an increasing metabolic demand. These results are discrepant with previous large animal studies and suggest that porcine islets may be resistant to metabolic failure.

This prospective study assessed lymphocyte subsets in the peripheral blood of 42 islet allograft recipients using flow cytometry from 2 weeks and up to 2 years post-transplantation. Subjects received daclizumab (n = 16), Thymoglobulin (n = 12) or alemtuzumab (n = 14). Alemtuzumab was associated with an early (within 1 month) and transient (up to 6 months) increase in the frequency of CD3(+) CD4(+) Foxp3(+) T cells, while daclizumab induced a near complete loss of these cells (P

The aim of this study was to investigate whether the use of a medically approved biodegradable scaffold as a solid support system would enhance graft survival following transplantation into the omental pouch in a preclinical large animal model. Six beagle dogs underwent total pancreatectomy followed by islet autotransplantation into the omental pouch. Four dogs received islets seeded in a biodegradable polymer scaffold and two received free islets without a scaffold. All four animals that received islets in the scaffold became normoglycemic without exogeneous insulin injection. One dog, transplanted with the largest number of islets, maintained a normal metabolic state until the graft was removed at 5 months posttransplant. In two out of the three that received a marginal islet mass, insulin independence was sustained up to 2 months. In contrast, two dogs transplanted with a similar marginal mass without the scaffold never became normoglycemic. Histological examination of the grafts in the scaffold showed numerous well-granulated, insulin-containing cells as well as glucagon-positive cells. These results indicate that biodegradable scaffolds may enhance survival and function of islet grafts. Manipulation of the microenvironment of transplanted islets may constitute the basis for new approaches to enhance islet engraftment.

Objective: Clinical islet transplantation can provide insulin independence in patients with type 1 diabetes, but chronic graft failure has been observed. This has been attributed in part to loss of >or=60% of the transplanted islets in the peritransplant period, resulting in a marginal implant mass. Strategies designed to maximize survival of the initial islet mass are likely to have major impact in enhancing long-term clinical outcomes. EP1013 (N-benzyloxycabonyl-Val Asp-fluoromethyl ketone [zVD-FMK]), is a broad-spectrum caspase selective inhibitor with no observed toxicity in rodents., Research Design and Methods: The therapeutic benefit of EP1013 was examined in a syngeneic rodent islet transplant model using deceased donor human islets to determine whether the amount of tissue required to restore euglycemia in diabetic animals could be reduced., Results: EP1013 (combined pretransplant islet culture for 2 h and in vivo treatment for days 0-5 posttransplant) significantly improved marginal islet mass function following syngeneic islet transplantation in mice, even at lower doses, compared with previous studies using the pan-caspase inhibitor N-benzyloxycabonyl-Val Ala-Asp-fluoromethyl ketone (zVAD-FMK). EP1013 supplementation in vitro improved human islet yields following prolonged culture and reversed diabetes following implantation of a marginal human islet mass (80-90% reduction) into mice., Conclusions: Our data suggest that EP1013 therapy will markedly reduce the islet mass required in clinical islet transplantation, improving insulin independence rates following single-donor infusion.

Combinatorial chemistry has recently burst on the scene as a valuable tool for the discovery of new drug candidates. The ability to synthesize hundreds of compounds for screening is a useful complement to rational drug design. There are many similarities between the design of new therapeutic agents and the development of new asymmetric ligands, the most important of which is the limitation of a rational design strategy. For this reason a program was begun that would allow the use of combinatorial technology in the development of new ligands for transition metal catalyzed asymmetric reactions. Because of the large number of catalytic reactions they are involved in the system was based around phosphine ligands. This paper reports the synthesis of phosphine derivatives of alanine, proline, and the aromatic amino acids tyrosine and hydroxyphenylglycine. Examples of the use of these amino acids in the synthesis of peptides possessing helical and beta-turn secondary structures are presented. Metal complexes of these peptide-based ligands are used in hydrogenation and alkylation reactions., (Copyright 2005 Wiley Periodicals, Inc.)