Porcine Islets Research Articles

Clinically available immunosuppression averts rejection but not systemic inflammation after porcine islet xenotransplant in cynomolgus macaques

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147days (p<.0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.

Multipotent Mesenchymal Stromal Cells Interact and Support Islet of Langerhans Viability and Function.

Type 1 diabetes (T1D) is a widespread disease, affecting approximately 41.5 million people worldwide. It is generally treated with exogenous insulin, maintaining physiological blood glucose levels but also leading to long-term therapeutic complications. Pancreatic islet cell transplantation offers a potential alternative treatment to insulin injections. Shortage of human organ donors has raised the interest for porcine islet xenotransplantation. Neonatal porcine islets are highly available, can proliferate and mature in vitro as well as after transplantation in vivo. Despite promising preclinical results, delayed insulin secretion caused by immaturity and immunogenicity of the neonatal porcine islets remains a challenge for their clinical application. Multipotent mesenchymal stromal cells (MSCs) are known to have pro-angiogenic, anti-inflammatory and immunomodulatory effects. The current state of research emphasizes the great potential of co-culture and co-transplantation of islet cells with MSCs. Studies have shown enhanced islet proliferation and maturation, insulin secretion and graft survival, resulting in an improved graft outcome. This review summarizes the immunomodulatory and anti-inflammatory properties of MSC in the context of islet transplantation.

Effects of different routes of heparin on instant blood-mediated inflammatory reaction after portal vein islet transplantation.

Heparin is mainly used as an anticoagulant in clinic, and it also has a certain anti-inflammatory effect. At present, after portal vein islet transplantation in diabetic patients, heparin is mainly infused through the peripheral veins of the limbs to achieve the purpose of anticoagulation and protection of the graft, rather than through the portal vein. In this study, animal experiments were conducted to investigate the effect of heparin infusion via the portal vein and marginal ear vein on the instant blood-mediated inflammatory reaction (IBMIR) after portal vein islet transplantation, which is the choice of anticoagulation methods for clinical islet transplantation to provide a basis for decision-making. A total of 50 neonatal pigs (Xeno-1 type, 3-5 days) were selected. Islets were isolated and purified from the pancreas of neonatal pigs. Ten non-diabetic Landrace pigs (1.5-2.0 months) served as recipients, and 12 000 IEQ/kg neonatal porcine islets were transplanted into the liver through the portal vein. All recipients received bolus injection of 50 U/kg of heparin 10 minutes before transplantation. After the bolus injection of heparin, the experimental group received heparin via the portal vein [10 U/(kg·h), 5 recipients], and the control group received heparin via the marginal ear vein [10 U/(kg·h), 5 recipients]. The superior vena cava blood was collected from the 2 groups pre-operation at 1, 3, 24 h post-operation of the transplantation. The portal vein blood was collected from the experimental group at 1 and 3 h after the transplantation as well. The levels of complement C3a, C5a, thrombin-antithrombin complex (TAT), β-thromboglobulin (β-TG), and D-dimer as well as activated partial thromboplastin time (APTT) in superior vena cava blood from 1 and 3 h post-transplantation were detected in the 2 groups, and the levels of anti-Xa and anti-IIa in the portal vein and superior vena cava blood from 1 and 3 h post-transplantation in the experimental group were detected. Twenty four hours after the transplantation, the liver tissues in the 2 groups were collected for pathological examination to observe the inflammatory cell infiltration and peripheral thrombosis around the islets graft in liver. Before transplantation, there was no statistically significant difference in C3a, C5a, TAT, β-TG, D-dimer levels and APTT between the 2 groups (all P>0.05). At 1 and 3 h after transplantation, the C3a, TAT, and D-dimer levels in the experimental group were significant decreased than those in the control groups (all P<0.05), and at 3 h after transplantation the C5a was significant decreased than that in the control group (P<0.05). At 1 and 3 h after transplantation, the anti-Xa and anti-IIa levels in the portal vein blood were significantly increased than those in the superior vena cava blood in the experimental group (all P<0.05). Pathological results showed the presence of islet cell clusters in the liver blood vessels. The thrombus formation and neutrophil infiltration around islet graft was not obvious in the experimental group, while massive thrombus formation and neutrophil infiltration in the control group. Compared with marginal ear vein infusion of heparin, the direct infusion of heparin in the portal vein has a certain inhibitory effect on complement system, coagulation system activation and inflammatory cell infiltration in portal vein islet transplantation, which may attenuate the occurrence of IBMIR.

Mesenchymal Stem Cells Secretions Enhanced ATP Generation on Isolated Islets during Transplantation

ABSTRACT The success of islet transplantation in both basic research and clinical settings has proven that cell therapy has the potential to cure diabetes. Islets intended for transplantation are inevitably subjected to damage from a number of sources, including ischemic injury during removal and delivery of the donor pancreas, enzymatic digestion during islet isolation, and reperfusion injury after transplantation in the recipient. Here, we found that protein factors secreted by porcine adipose-tissue mesenchymal stem cells (AT-MSCs) were capable of activating preserved porcine islets. A conditioned medium was prepared from the supernatant obtained by culturing porcine AT-MSCs for 2 days in serum-free medium. Islets were preserved at 4°C in University of Wisconsin solution during transportation and then incubated at 37°C in RPMI-1620 medium with fractions of various molecular weights prepared from the conditioned medium. After treatment with certain fractions of the AT-MSC secretions, the intracellular ATP levels of the activated islets had increased to over 160% of their initial values after 4 days of incubation. Our novel system may be able to restore the condition of isolated islets after transportation or preservation and may help to improve the long-term outcome of islet transplantation. Abbreviations: AT-MSC, adipose-tissue mesenchymal stem cell; Cas-3, caspase-3; DAPI, 4,6-diamidino-2-phenylindole; DTZ, dithizone; ES cell, embryonic stem cell; FITC, fluorescein isothiocyanate; IEQ, islet equivalent; INS, insulin; iPS cell, induced pluripotent stem cell; Luc-Tg rat, luciferase-transgenic rat; PCNA, proliferating cell nuclear antigen; PDX1, pancreatic and duodenal homeobox protein-1; UW, University of Wisconsin; ZO1, zona occludens 1.

MRI monitoring of transplanted neonatal porcine islets labeled with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles in a mouse model.

Islet transplantation is a potential treatment option for certain patients with type 1 diabetes; however, it still faces barriers to widespread use, including the lack of tools to monitor islet grafts post-transplantation. This study investigates whether labeling neonatal porcine islets (NPI) with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) affects their function, and whether this nanoparticle can be utilized to monitor NPI xenografts with magnetic resonance imaging (MRI) in a mouse model. In vitro, PVP-SPIO-labeled NPI in an agarose gel was visualized clearly by MRI. PVP-SPIO-labeled islets were then transplanted under the kidney capsules of immunodeficient nondiabetic and diabetic mice. All diabetic mice that received transplantation of PVP-SPIO-labeled islets reached normoglycemia. Grafts appeared as hypo-intense areas on MRI and were distinguishable from the surrounding tissues. Following injection of spleen cells from immunocompetent mice, normoglycemic recipient mice became diabetic and islet grafts showed an increase in volume, accompanied by a mixed signal on MRI. Overall, this study demonstrates that PVP-SPIO did not affect the function of NPI that PVP-SPIO-labeled islets were easily seen on MRI, and changes in MRI signals following rejection suggest a potential use of PVP-SPIO-labeled islets to monitor graft viability.

307.1: Bionic Pancreas - The First Results of Functionality Bionic Tissue Model With Pancreatic Islets

Introduction: 3D bioprinting using living cells is the latest technique in the field of biomedical engineering. One of the most important points of the procedure is to preserve the cells and leave them fully functional in the resulting bioconstruction. In the case of bioprinting of bionic tissue, it is important to provide cells with access to nutrients and gas exchange. These are the first results of printed bionic petals and the first studies on a mouse model. Material: The research was carried out on 60 mice (SCID). Diabetes induction was not undertaken in small animal studies because T1D is difficult to achieve in a mouse model. The mice were divided into 3 groups: a control; the IsletTx in which porcine pancreatic islets were transplanted under the renal capsule; 3D Bioprint in which the 3D bioink petals consisted of bioink A and porcine islets. The petals were transplanted into the dorsal part of the muscles under the skin. Daily glucose measurement was performed and the level of C-peptide was tested every 7 days. Results: The results obtained in mice initially showed no differences in the concentration of peptide-C and glucose between the groups. However, as early as 7 days after transplantation, both parameters analyzed in the fasting state were significantly lower in the IsletsTx and 3Dbioprinted groups compared to the control group. On day 14, decreased values of C-peptide and glucose were observed only in the group with petals transplants. Conclusion: Transplantation of bionic petals in mice resulted in a decrease in mean glucose levels. The mice showed a reduced concentration of their own C-peptide, which can indicate relief in mice’s owns islets function. None of the animals died due to postoperative complications or the lack of biocompatibility with the bionic structure. The positive effect of transplantation was maintained throughout the experiment, which proves the optimal selection of the composition of the bioink and the bioprinting parameters. This study was financially supported by The National Centre for Research and Development STRATEGMED3/305813/2/NCBR/2017.

402.3: Long-term Preservation of Isolated Human, Mouse, Porcine Islets and Human Stem Cell Derived Beta Cells (HUES-8 Cell Lines) Using a High Throughput Vitrification-Rewarming Modified Cryomesh Technique to Successfully Cure Diabetes in a Mouse With Transplantation

402.3: Long-term Preservation of Isolated Human, Mouse, Porcine Islets and Human Stem Cell Derived Beta Cells (HUES-8 Cell Lines) Using a High Throughput Vitrification-Rewarming Modified Cryomesh Technique to Successfully Cure Diabetes in a Mouse With Transplantation

P.127: The Influence of Temperature Conditions and Reconstruction of the Extracellular Matrix Environment on the Viability of Pancreatic Islets

Introduction: Pancreatic islet transplantation is one of the methods of treating patients with T1D. However, for the procedure to be successful, the high quality of the implanted islets is of key importance for a successful islet transplant. Following islet isolation, it has been shown that mainly β-cells undergo apoptosis due to vascular loss and hypoxia. The cellular stresses to which islets are subjected during isolation (for example disturbance of the cell-matrix relationship). Appropriate storage conditions for insulated islands are of key importance for their functionality. The most common method of islet storage is the introduction into a suspension culture. However, because these are micro organs, the storage of pancreatic islets is a more accurate term. Therefore, this study aimed to demonstrate whether for pancreatic islets it is better to cultivate them in standard conditions (37oC; 5% CO2), or maybe stored them at 4oC. Material: The research material was porcine pancreatic islets. Islets were isolated from the native organ using collagenase and a Ricordi chamber. After isolation, the material was divided into 4 research groups: 1. control (islands suspended in CMRL 1066 medium) - traditional breeding conditions (temperature 37oC; 5% CO2); 2. CMRL 1066 suspended islets stored at 4oC; 3. Islets in CMRL 1066 medium with the addition of dECM (decellularized extracellular matrix) - in traditional culture conditions; 4. Islets in CMRL 1066 supplemented with dECM stored at 4oC. Each variant was tested in 3 replicates and each trial was 3000iEq. Additionally, each tested variant was tested on pancreatic islets that were cleaned or not immediately after isolation. Results: The research showed that the storage temperature, reduced to 4oC, and the addition of ECM significantly influenced the functionality of the islets. This variant tested (uncleaned islands) showed functionality even 5 days after isolation. On the other hand, islets incubated at 37°C without the addition of dECM showed no functionality after 2 days of the experiment. The remaining variants showed a gradual loss of functionality. However, the 4oC variant was better in each case. Conclusion: Pancreatic islets, as micro organs with their vascularization, need appropriate storage conditions after isolation to ensure their proper functioning. Based on these studies, it was shown that the islands should be stored and not cultivated as single-cell lines. In addition, the addition of dECM allows for the recreation of the matrix-cell environment, which is appropriate storage conditions - corresponding to the native environment. This study was financially supported by The National Centre for Research and Development STRATEGMED3/305813/2/NCBR/2017.

307.4: Subcutaneous Bioabsorption of Nanofibrous Scaffolds Influence the Engraftment and Function of Neonatal Porcine Islets Xenografts in Mice

Dr. Korbutt and Dr. Pepper Introduction: The subcutaneous space is currently being pursued as an alternative transplant site for human pancreatic islets, stem cell and xenogeneic derived-islets, due to its retrievability, minimally invasiveness, accommodation of large transplant volumes, and potential for monitoring graft function. However, transplantation of islets into an unmodified subcutaneous niche fails to reverse diabetes due to a lack of adequate blood supply. Biomaterial nanofibrous scaffolds can be functionalized to enhance the host tissues integration, provide a platform for the delivery of pro-engraftment growth factors, and immunomodulatory agents. Method: Herein, we utilize poly (ε-caprolactone) (PCL) and poly (lactic-co-glycolic acid) (PLGA) polymers to make nanofibrous scaffolds and functionalized with bioactive peptides, to prime the subcutaneous space into a more suitable environment. We implanted the nylon catheter (2 cm length, 6 French diameter) (deviceless space, DL), and nanofibrous scaffolds (scaffold was wrapped around the nylon catheter) such as PCL, PCL+RGD+VEGF (PCL+R+V), PCL+RGD+Laminin (PCL+R+L), PLGA and PLGA+Gelatin (PLGA+G) into the subcutaneous space of immunodeficient B6.129S7-Rag1tm1Mom/J mice for four weeks to create a prevascularized space. After 4 weeks, DL and scaffolds implanted mice and those intended for kidney capsule (KC) implantation were rendered diabetic by intraperitoneal injection of 180 mg/kg streptozotocin. Subsequently, neonatal porcine islets (3000 NPI) were transplanted under the KC or within the subcutaneous DL and scaffolds. Graft function was evaluated by monitoring non-fasting blood glucose, stimulated porcine insulin measurement, intraperitoneal glucose tolerance test, and histochemical analysis. Results: Our preliminary scaffolds implantation (no cells) study demonstrates that PCL, PCL+R+V and PCL+R+L scaffolds did not absorb and partially integrated with the host tissues (PCL based scaffolds remain intact at the implanted site) whereas PLGA and PLGA+G scaffolds were completely absorbed and integrated with the host tissues (no remnants of PLGA based scaffolds at the implanted site), and we also observed that there were numerous blood vessels innervated in and around these scaffolds. Compared with peptide functionalized PCL scaffolds, PLGA and PLGA+G fibrous scaffolds with NPI resulted in 86% and 100% euglycemia (*p< 0.05, **p<0.01 respectively), superior glucose clearance (*p<0.05) and greater stimulated porcine insulin secretion (*p<0.05). Moreover, PLGA and PLGA+G scaffolds exhibited comparable graft functions with the positive controls (DL and KC)(p>0.05). Conclusion: Our study demonstrates that PLGA based fibrous scaffolds facilitates the engraftment and function of NPIs in the subcutaneous space of diabetic mice. These collective data emphasize the support of biomaterial implants on cellular graft function in the subcutaneous space for clinical islet xenotransplant applications in a near future.

P.168: Biofabrication of a Functional Vascularized Endocrine Pancreas (VEP) for Type 1 Diabetes

Background: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Islet Organ (VIO, lung scaffold repopulated by murine islets and HUVEC cells), we prototyped our platform to engineer an upgrade based on human blood-derived endothelial cells (BOECs - Blood Outgrowing Endothelial Cells) and immature neonatal porcine islet clusters (NPIs). Material and Methods: NPIs and BOECs phenotype profile was assessed by flow cytometry, insulin secretion test and tubule formation assay. Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with NPIs and BOECs, generating a Vascularized Endocrine Pancreas (VEP). For ex vivo maturation VEP was cultured for 7 days in a customized bioreactor specifically designed to allow cell integration. The NPIs β cell death in mature VEPs was estimated during ex vivo organ maturation evaluating miR-375 expression by droplet digital PCR compared to batch matched NPIs in standard condition. Matured VEPs and control NPIs function were measured by dynamic glucose perifusion and insulin quantification (by ELISA/IF). Once validated in vitro, VEPs were subcutaneously transplanted in diabetic immunodeficient NSG recipients and compared for in vivo function with matched NPIs transplanted in different implantation sites: kidney capsule (KC-NPIs), deviceless (DL-NPIs) and liver (LV-NPIs). Results: Matured VEPs showed regenerated vascular network (CD31+) with NPIs (insulin+) integrated. miR-375, a marker of β cell death, was expressed in NPIs but not in BOECs, as expected. VEP was able to significantly reduce β cell death: the amount of lost NPIs were ≤ 5% during VEPs maturation, while >71% during in vitro culture (p<0.05). Matured VEPs were able to sustain NPIs engraftment, survival and significantly improve insulin secretion during the maturation process compared to batch matched NPIs cultured in standard conditions (AUC VEPs first phase: 3.765±0.90; NPIs 1.60±0.25 p<0.01). In long-term transplants in diabetic mice, VEPs demonstrated a significant NPIs engraftment with a prompt VEPs function after implantation and the reversal of the glycemia within 2 days until 60 days after implantation. Additionally, VEPs showed a significant superior function compared to all the internal controls (KC-NPIs, DL-and LV-NPIs). Conclusion: We conclude that ex vivo bio-fabrication of VEPs enables NPIs engraftment, maturation and vascularization before transplantation. VEP is the first organ to our knowledge assembled with relevant source of endocrine and endothelial cells suitable for future clinical translation. Given recent progress in gene editing, this technology may enable assembly of functional personalized endocrine organs to escape immunoreaction against the graft. This study was supported by a grant from Juvenile Diabetes Research Foundation (JDRF 1-SRA-2019-771-S-B).

Butyrate and Class I Histone Deacetylase Inhibitors Promote Differentiation of Neonatal Porcine Islet Cells into Beta Cells.

Neonatal porcine islets-like clusters (NPICCs) are a promising source for cell therapy of type 1 diabetes. Freshly isolated NPICCs are composed of progenitor cells and endocrine cells, which undergo a maturation process lasting several weeks until the normal beta cell function has developed. Here, we investigated the effects of short-chain fatty acids on the maturation of islet cells isolated from two to three day-old piglets. NPICCs were cultivated with acetate, butyrate and propionate (0–2000 µM) for one to eight days. Incubation with butyrate resulted in a significant upregulation of insulin gene expression and an increased beta cell number, whereas acetate or propionate had only marginal effects. Treatment with specific inhibitors of G-protein-coupled receptor GPR41 (β-hydroxybutyrate) and/or GPR43 (GPLG0974) did not abolish butyrate induced insulin expression. However, incubation of NPICCs with class I histone deacetylase inhibitors (HDACi) mocetinostat and MS275, but not selective class II HDACi (TMP269, MC1568) mimicked the butyrate effect on beta cell differentiation. Our study revealed that butyrate treatment has the capacity to increase the number of beta cells, which may be predominantly mediated through its HDAC inhibitory activity. Butyrate and specific class I HDAC inhibitors may represent beneficial supplements to promote differentiation of neonatal porcine islet cells towards beta cells for cell replacement therapies.

Anti-thymoglobulin induction improves neonatal porcine xenoislet engraftment and survival.

Porcine islet xenotransplantation is a viable strategy to treat diabetes. Its translation has been limited by the pre-clinical development of a clinically available immunosuppressive regimen. We tested two clinically relevant induction agents in a non-human primate (NHP) islet xenotransplantation model to compare depletional versus nondepletional induction immunosuppression. Neonatal porcine islets were isolated from GKO or hCD46/GKO transgenic piglets and transplanted via portal vein infusion in diabetic rhesus macaques. Induction therapy consisted of either basiliximab (n=6) or rhesus-specific anti-thymocyte globulin (rhATG, n=6), combined with a maintenance regimen using B7 costimulation blockade, tacrolimus with a delayed transition to sirolimus, and mycophenolate mofetil. Xenografts were monitored by blood glucose levels and porcine C-peptide measurements. Of the six receiving basiliximab induction, engraftment was achieved in 4 with median graft survival of 14 days. All six receiving rhATG induction engrafted with significantly longer xenograft survival at 40.5 days (P=0.03). These data suggest that depletional induction provides superior xenograft survival to nondepletional induction, in the setting of a costimulation blockade-based maintenance regimen.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.

From insulin injections to islet transplantation: An overview of the journey.

When, in 1869, Paul Langerhans detected the "islands of tissue" in the pancreas, he took the first step on a journey towards islet transplantation as a treatment for type 1 diabetes. The route has embraced developments across biosciences, surgery, gene therapy and clinical research. This review highlights major milestones along that journey involving whole pancreas transplantation, islet transplantation, the creation of surrogate insulin-secreting cells and novel islet-like structures using genetic and bio-engineering technologies. To obviate the paucity of human tissue, pluripotent stem cells and non-β-cells within the pancreas have been modified to create physiologically responsive insulin-secreting cells. Before implantation, these can be co-cultured with endothelial cells to promote vascularisation and with immune defence cells such as placental amnion cells to reduce immune rejection. Scaffolds to contain grafts and facilitate surgical placement provide further opportunities to achieve physiological insulin delivery. Alternatively, xenotransplants such as porcine islets might be reconsidered as opportunities exist to circumvent safety concerns and immune rejection. Thus, despite a long and arduous journey, the prospects for increased use of tissue transplantation to provide physiological insulin replacement are drawing ever closer.

Adult Pig Islet Isolation.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

Semi-automated digital quantification of cellular infiltrates for in vivo evaluation of transplanted islets of Langerhans encapsulated with bioactive materials.

In the field of xenotransplantation, digital image analysis (DIA) is an asset to quantify heterogeneous cell infiltrates around transplanted encapsulated islets. RGD-alginate was used to produce empty capsules or to encapsulate neonatal porcine islets (NPI) with different combinations of human pancreatic extracellular matrix (hpECM), porcine mesenchymal stem cells (pMSC) and a chitosan anti-fouling coating. Capsules were transplanted subcutaneously in rats for one month and then processed for immunohistochemistry. Immunostainings for macrophages (CD68) and lymphocytes (CD3) were quantified by DIA in two concentric regions of interest (ROI) around the capsules. DIA replicability and reproducibility were assessed by two blind operators. Repeatability was evaluated by processing the same biopsies at different time points. DIA was also compared with quantification by point counting (PC). Methodology validation: different sizes of ROIs were highly correlated. Intraclass correlation coefficients confirmed replicability and reproducibility. Repeatability showed a very strong correlation with CD3 stains and moderate/strong for CD68 stains. Group comparisons for CD68 IHC at each time point proved internal consistency. Point counting and DIA were strongly correlated with both CD3 and CD68. Capsule biocompatibility: Macrophage infiltration was higher around capsules containing biomaterials than around empty and RGD-alginate-NPI capsules. Lymphocytic infiltration was comparable among groups containing cells and higher than in empty capsules. We validated a semi-automated quantification methodology to assess cellular infiltrates and successfully applied it to investigate graft biocompatibility, showing that neonatal porcine islets encapsulated in alginate alone triggered less infiltration than capsules containing islets and bioactive materials.

Carbohydrate antigen microarray analysis of serum IgG and IgM antibodies before and after adult porcine islet xenotransplantation in cynomolgus macaques.

Understanding the anti-carbohydrate antibody response toward epitopes expressed on porcine cells, tissues, and organs is critical to advancing xenotransplantation toward clinical application. In this study, we determined IgM and IgG antibody specificities and relative concentrations in five cynomolgus monkeys at baseline and at intervals following intraportal xenotransplantation of adult porcine islets. This study utilized a carbohydrate antigen microarray that comprised more than 400 glycoconjugates, including historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications. The elicited anti-carbohydrate antibody responses were predominantly IgM compared to IgG in 4 out of 5 monkeys. Patterns of elicited antibody responses greater than 1.5 difference (log2 base units; 2.8-fold on a linear scale) from pre-serum to post-serum sampling specific for carbohydrate antigens were heterogeneous and recipient-specific. Increases in the elicited antibody response to α-Gal, Sda, GM2 antigens, or Lexis X antigen were found in individual monkeys. The novel carbohydrate structures Galβ1-4GlcNAcβ1-3Galβ1 and N-linked glycans with Manα1-6(GlcNAcβ1-2Manα1–3)Manβ1-4GlcNAcβ structure were common targets of elicited IgM antibodies. These results provide important insights into the carbohydrate epitopes that elicit antibodies following pig-to-monkey islet xenotransplantation and reveal possible targets for gene editing.

Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.