Porcine Islet Xenotransplantation Research Articles

The porcine islet-derived organoid showed the characteristics as pancreatic duct

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)—cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)—cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19000IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Porcine embryonic stem cells: An alternative solution for the shortage of human islets to treat type 1 diabetes?

AbstractTransplantation of human embryonic stem cell (ESC) derived beta‐like cells and xenotransplantation of porcine islets are two potential cures for type 1 diabetes (T1D). We have previously reported the isolation of porcine ESCs and have also separately shown that islets from genetically modified pigs can effectively cure diabetes in a preclinical baboon model. Here we discuss the possibility of combining these technologies to produce gene‐edited porcine ESC‐derived islets as an alternative for treating T1D, which may offer several advantages compared with these approaches.

Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.

106.2: 10-year follow-up of type 1 diabetic patients following an encapsulated porcine islet xenotransplantation without immunosuppression

106.2: 10-year follow-up of type 1 diabetic patients following an encapsulated porcine islet xenotransplantation without immunosuppression

316.5: Clinical trial protocol for porcine islet xenotransplantation in South Korea

316.5: Clinical trial protocol for porcine islet xenotransplantation in South Korea

Neonatal islets from human PD-L1 transgenic pigs reduce immune cell activation and cellular rejection in humanized nonobese diabetic-scid IL2rγnull mice

Strong xenorejection limits the clinical application of porcine islet transplantation in type 1 diabetes. Targeting T cell-mediated rejection is one of the main approaches to improve long-term graft survival. Here we study engraftment and survival of porcine islet cells expressing human programmed cell death ligand-1 (hPD-L1) in a humanized mouse model. Neonatal islet-like clusters (NPICCs) from transgenic hPD-L1 (hPD-L1-Tg) and wild-type (Wt) pigs were transplanted into nonobese diabetic-scid IL2rγnull mice stably reconstituted with human immune cells (hPD-L1 n = 10; Wt n = 6). Primary endpoint was development of normoglycemia during a 16-week observation period after transplantation. Secondary endpoints were porcine C-peptide levels and immune cell infiltration. Animals transplanted with hPD-L1-Tg neonatal islet-like clusters achieved a superior normoglycemic rate (50% versus 0%) and significantly higher plasma C-peptide levels as compared to the Wt group, indicating long-term beta cell function. Intracytoplasmic fluorescence-activated cell sorting analysis and immunohistochemistry revealed significantly decreased frequencies of interferonγ-expressing splenic hCD8-positive T cells and reduced intragraft-infiltrating immune cells. We here demonstrate that expression of hPD-L1 provides strong islet xenograft protection without administration of immunosuppressive drugs. These findings support the hypothesis that hPD-L1 has the capacity to control cellular rejection and therefore represents a very promising transgene candidate for clinical porcine islet xenotransplantation.

Acceptance of encapsulated porcine islet xenotransplantation by patients and doctors in the US

AbstractIntroductionPreviously, we performed encapsulated porcine islet xenotransplantation for the treatment of unstable type 1 diabetic patients and demonstrated the clinical benefit and safety in New Zealand and Argentina. Conversely, the treatment of type 1 diabetes differs from country to country; therefore, understanding the acceptance of the new treatment by patients and medical doctors in each country is important. In this study, a survey study of the acceptance of the encapsulated porcine islet xenotransplantation by the type 1 diabetic patients and medical doctors in the US was conducted.MethodsThe questionnaires consisted of the acceptance of the encapsulated porcine islet xenotransplantation without immunosuppression, and the reasons for selecting and not selecting. Moreover, we conducted a sub‐analysis among patient groups regarding severe hypoglycemia and HbA1c levels.ResultsThe majority of patients and doctors (patients, 63.8%; doctors, 70.0%) had a positive opinion to accept this treatment. A significantly high number of doctors selected no immunosuppression for selecting this treatment (patients, 35.5%; doctors, 53.6%; p < 0.001), and a significantly high number of patients selected not insulin free for not selecting this treatment (patients 20.4%, doctors 12.4%, p < 0.05). The high HbA1c group had the highest ‘definitely’ accept rate, which was significantly higher than those of the other HbA1c groups (High HbA1c group, 50.0%; other HbA1c groups, 22.2%; p < 0.05).ConclusionIn conclusion, the majority of US patients and doctors had positive opinions to accept the encapsulated porcine islet xenotransplantation. Type 1 diabetic patients with high HbA1c levels had the highest ‘definitely’ acceptance rates.

Patients' opinions 10 years after receiving encapsulated porcine islet xenotransplantation without immunosuppression.

Previously we performed clinical encapsulated neonatal porcine islet transplantation under comprehensive regulation, and demonstrated the efficacy and safety. To analyze the patients' quality of life (QOL), we assessed patients' opinions 10 years after islet xenotransplantation. Twenty-one type 1 diabetic patients received microencapsulated neonatal porcine islet transplants in Argentina were enrolled. Seven patients were enrolled in efficacy and safety study and 14 patients were enrolled in safety studies. Patients' opinions related to the current and pre-transplant status of diabetes control, blood glucose levels, severe hypoglycemia, and hyperglycemia required hospitalization were analyzed. In addition, opinions related to islet xenotransplantation were assessed. At the time of this survey, average HbA1c was still significantly lower compared to pre-transplantation (8.5±0.9 (%) at pre-transplant and 7.4±0.5(%) at the survey, p<.05) and average insulin dose were also lower (0.95±0.32 (IU/kg) at pre-transplant and 0.73±0.27 (IU) at the survey). The majority of patients improved diabetes control (71%), blood glucose levels (76%), severe hypoglycemia (86%) and hyperglycemia required hospitalization (76%), and no patients deteriorated in all of the categories when compared with pre-transplantation. No patients had cancer, or psychological problem, and one patient had a serious adverse event. The majority of patients wanted to recommend this treatment to other patients (76%) and receive booster transplantation (85.7%). The majority of patients had positive opinions related to the encapsulated porcine islet xenotransplantation 10 years after transplantation.

Establishment of a donor pig for xenotransplantation clinical trials based on the principle of Changsha Communiqué

AbstractBackgroundXenotransplantation is a potential way to reduce the shortage of the needed organ grafts for the end‐stage disease. Immune rejection, physiological incompatibility and bio‐safety are the most critical issues.MethodsTo ensure the safety and efficacy of gene editing, second‐ and third‐generation sequencing technologies have allowed us to obtain a clearer genetic background of donor pigs for xenotransplantation. Based on the Changsha Communiqué, the local DPF‐ excluded lists and DPF donor facility were established in Changsha, China. A pig‐to‐human islet clinical trial was conducted and overseen by the respective Chinese governmental agency.ResultsThe DPF standards for pig husbandry eliminated specific pathogens in donor pigs. We have established a PERV‐C free, genetic information clean, DPF donor for xenotransplantation. A clinical trial of ten adult patients (9M:1F) with type 1 diabetes who received DPF porcine islet xenotransplantation via the portal vein were performed. Clinical accepted immunosuppressant drugs and autologous Treg were used for controlling immune rejection. No cross‐species infection events occurred in this trial, and importantly, no cross‐species transmission of PERV was found.ConclusionsXenotransplantation is a pioneer study and safety is the most important issue. The fundamental principles for establishing xenotransplantation donor pigs should follow the Changsha Communiqué (2008), the second WHO consultation,and the 2018 Changsha Communiqué which would finally help reducing the risks of xenotransplantation.

Procedure of Adult Porcine Islet Isolation.

Islet transplantation is a useful therapeutic choice for severe diabetes mellitus; however, limited donor supplies have interfered with the use of this treatment. Therefore, the establishment of alternative donor sources and engineered tissue, which enables to produce appropriate insulin for controlling blood glucose, is an important challenge. The adult pig is a promising and feasible donor source and materials for the engineered tissue for the clinical setting among various candidates. The recent progress of gene-editing technology contributes to possible clinical porcine xenotransplantation, including porcine islet xenotransplantation. For the success of future clinical porcine islet xenotransplantation, establishing an islet isolation technique for acquiring adequate, good-quality porcine islets is equally important to use a gene-edited pig. However, the characteristics of porcine islets are different from other species; therefore, establishing a suitable technique for porcine islets is challenging. Impact statement Recent technological progress promotes the feasibility of xenotransplantation, including islet xenotransplantation, for clinical setting. Adult pig is a promising and feasible donor source for islet xenotransplantation and engineered tissue, which enables to control blood glucose in recipients. It is important to acquire porcine islets in good qualities for the promotion, however, establishing a technique for adult porcine islet isolation is important but challenging because of the vulnerability of adult porcine islets. Deciding the proper timing of stopping pancreatic digestion is one of the important factors for obtaining adult porcine islets in good quality.

Heterogenous expression of endocrine and progenitor cells within the neonatal porcine pancreatic lobes-Implications for neonatal porcine islet xenotransplantation.

Neonatal porcine islets (NPIs) are a source of islets for xenotransplantation. In the pig, the pancreatic lobes remain separate, thus, when optimizing NPI isolation, the pancreatic lobes included in the pancreatic digest should be specified. These lobes are the duodenal (DL), splenic (SL) and connecting (CL) lobe that correspond to the head, body-tail, and uncinate process of the human pancreas. In this study we are the first to evaluate all three neonatal porcine pancreatic lobes and NPIs isolated from these lobes. We report, a significant difference in endocrine and progenitor cell composition between lobes, and observed pancreatic duct glands (PDG) within the mesenchyme surrounding exocrine ducts in the DL and CL. Following in vitro differentiation, NPIs isolated from each lobe differed significantly in the percent increase of endocrine cells and final cell composition. Compared to other recipients, diabetic immunodeficient mice transplanted with NPIs isolated from the SL demonstrated euglycemic control as early as 4 weeks (p < 0.05) and achieved normoglycemia by 6 weeks post-transplant (p < 0.01). For the first time we report significant differences between the neonatal porcine pancreatic lobes and demonstrate that NPIs from these lobes differ in xenograft function.

The Effects of Photobiomodulation Therapy on Porcine Islet Insulin Secretion.

Background and objective: Xenotransplantation of porcine islets to human recipients has been investigated as a potential cure for type 1 diabetes. However, the porcine islets have poor insulin secretion capacity compared with human islets. The objective of this study was to evaluate the effect of photobiomodulation therapy (PBMT) in insulin secretion on isolated porcine islets. Methods: Eight pancreata were harvested from crossbred market porcine and the islets were isolated from the pancreas. The isolated islets were treated with PBMT (wavelength: 633 nm and dosages: 0.0, 15.6, and 31.3 J/cm2) followed by 30-min incubation in low (3.0 mM) or high (16.7 mM) glucose. The relative percentage differences on insulin secretion between three dosages were compared in low and high glucose, respectively. Results: Insulin secretion was higher in samples exposed to 15.6 J/cm2 PBMT in low glucose (p < 0.05), but not in high glucose. When evaluating sex differences, male islets had higher insulin secretion by 15.6 J/cm2 PBMT in low glucose compared with females (p < 0.05). No significant differences were seen in high glucose. When compared within the control groups (0.0 J/cm2 PBMT), the relative changes on insulin secretion in high glucose was significantly higher on male islets (p < 0.05), but not on female islets. Conclusions: PBMT may increase insulin secretion on isolated porcine islets in basal condition, but it may not improve islets' glucose responsiveness to secrete insulin. Male porcine islets may respond to PBMT and glucose stimuli better than female islets on insulin secretion.

CTLA4Ig/VISTAIg combination therapy selectively induces CD4+ T cell-mediated immune tolerance by targeting the SOCS1 signaling pathway in porcine islet xenotransplantation.

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells, investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4, PD-1, TIGIT and VISTA, and the frequency of CTLA4+ CD4+ , TIGIT+ CD4+ , VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets, whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling.

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.

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.

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.

Coagulation, inflammation, and CD46 transgene expression in neonatal porcine islet xenotransplantation.

Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet xenotransplantation and relate it to early inflammation, we examined porcine islets transplanted into non-human primates using a dual-transplant model to directly compare islet characteristics. α1,3-Galactosyltransferase gene-knockout (GTKO) islets with and without expression of the human complement regulatory transgene CD46 (hCD46) were studied. Biologically inert polyethylene microspheres were used to examine the generic pro-thrombotic effects of particle embolization. Immunohistochemistry was performed 1 and 24hours after transplantation. Xeno-islet transplantation activated both extrinsic and intrinsic coagulation pathways. The intrinsic pathway was also initiated by microsphere embolization, while extrinsic pathway tissue factor (TF) and platelet aggregation were more specific to engrafted islets. hCD46 expression significantly reduced TF, platelet, fibrin, and factor XIIIa accumulation in and around islets but did not alter intrinsic factor activation. Layers of TF+ cells emerged around islets within 24hours, particularly co-localized with vimentin, and identified as CD3+ and CD68+ cells inflammatory cells. These findings detail the origins of thrombosis following islet xenotransplantation, relate it to early immune activation, and suggest a role for transgenic hCD46 expression in its mitigation. Layers of TF-positive inflammatory cells and fibroblasts around islets at 24hours may have important roles in the progressive events of thrombosis, inflammatory cell recruitment, rejection, and the ultimate outcome of transplanted grafts. These suggest that the strategies targeting these elements could yield more progress toward successful xenogeneic islet engraftment and survival.

Novel Immunomodulatory Approaches for Porcine Islet Xenotransplantation.

Porcine islet xenotransplantation is a promising alternative to overcome the shortage of organ donors. For the successful application of islet xenotransplantation, robust immune/inflammatory responses against porcine islets should be thoroughly controlled. Over the last few decades, there have been numerous attempts to surmount xenogeneic immune barriers. In this review, we summarize the current progress in immunomodulatory therapy for the clinical application of porcine islet xenotransplantation. Long-term graft survival of porcine islets was achieved by using anti-CD154 Ab-based regimens in a preclinical non-human primate (NHP) model. However, owing to a serious complication of thromboembolism in clinical trials, the development of an anti-CD154 Ab-sparing immunosuppressant procedure is required. The efficacy of new immunosuppressive practices that employ anti-CD40 Abs or other immunosuppressive reagents has been tested in a NHP model to realize their utility in porcine islet xenotransplantation. The recent progress in the development of immunomodulatory approaches, including the immunosuppressive regimen, which enables long-term graft survival in a pig-to-non-human primate islet xenotransplantation model, with their potential clinical applicability was reviewed.

Current status of porcine islet xenotransplantation.

Human islet transplantation has proven to be a highly effective treatment for patients with labile type 1 diabetes mellitus, which can free patients from daily glucose monitoring and insulin injections. However, the shortage of islet donors limits its' broad application. Porcine islet xenotransplantation presents a solution to the donor shortage and recent advances in genetic modification and immunosuppressive regimens provide renewed enthusiasm for the potential of this treatment. Advances in genetic editing technology are leading to multigene modified porcine islet donors with alterations in expression of known xenoantigens, modifications of their complement and coagulation systems, and modifications to gain improved immunological compatibility. Recent NHP-based trials of costimulation blockade using CD154 blockade show promising improvements in islet survival, whereas results targeting CD40 are less consistent. Furthermore, trials using IL-6 receptor antagonism have yet to demonstrate improvement in glucose control and suffer from poor graft revascularization. This review will detail the current status of islet xenotransplantation as a potential treatment for type I diabetes mellitus, focusing on recent advances in porcine xenogeneic islet production, assessment in nonhuman primate preclinical models, the outcome of human clinical trials and review barriers to translation of xenoislets to the clinic.