Type 1 diabetes is an autoimmune disease characterized by the destruction of insulin-secreting beta cells in the pancreatic islets. This results in a deficiency of endogenous insulin, necessitating conventional insulin treatment [1]. However, patients with type 1 diabetes who retain less than 5% of their beta cell volume require intensive insulin therapy to mitigate complications associated with the disease [2]. The administration of large doses of insulin can lead to life-threatening severe hypoglycemic events [3].

To reduce hypoglycemic events resulting from insulin overdose, allogeneic islet transplantation has been explored [4]. In 2000, successful clinical trials of allogeneic islet transplantation were conducted using the Edmonton protocol [5]. Subsequently, in 2016, phase 3 clinical trial outcomes were published, demonstrating that the procedure could prevent severe hypoglycemia and achieve more stable glycemic control [6]. In 2023, the US Food and Drug Administration approved the use of allogeneic islets as a biological product, thus establishing allogeneic islet transplantation as a standard treatment option for type 1 diabetes, comparable to intensive insulin therapy [7].

However, considering the large number of patients with unstable type 1 diabetes, the issue of donor shortage for allogeneic islet transplantation must be addressed. To overcome this challenge, researchers have investigated the use of porcine islets [8]. The primary benefit of porcine islet xenotransplantation is the potential for an unlimited supply of healthy islets. However, porcine islets elicit a stronger immune response than allogeneic islet transplants. Additionally, xenogeneic islets are classified as medicinal products, meaning that both the production of designated pathogen-free pigs [9] and the isolation of their islets must adhere to pharmaceutical production standards [10]. While the use of designated pathogen-free pigs reduces the risk of zoonosis, concerns regarding porcine endogenous retroviruses (PERVs) remain [11]. Moreover, such studies are subject to ethical considerations and legal regulations [12]. Despite these challenges, porcine islet xenotransplantation is considered a potential solution to address the donor shortage in allogeneic islet transplantation for patients with unstable type 1 diabetes. Clinical trials have been conducted in various countries, including Sweden, China, New Zealand, Russia, and Mexico [13–19].

In 1994, Groth et al. [13] transplanted fetal porcine islet-like cell clusters (ICCs) into 10 patients with type 1 diabetes mellitus. The porcine ICCs were delivered to the liver via the portal vein in eight patients and implanted beneath the kidney capsule in two patients. As the patients were undergoing concurrent kidney transplantation, they were induced with antithymocyte globulin (ATG) or 15-deoxyspergualin and maintained on a regimen of cyclosporine, prednisolone, and azathioprine. Of the patients who received transplants through the portal vein, four exhibited porcine C-peptide positivity in their urine for 200–400 days after transplantation. Biopsies confirmed the presence of insulin- and glucagon-positive cells, although no change in insulin requirements was noted relative to pretransplantation levels. Although no clinical benefits were observed following the transplantation of porcine ICCs, the study was the first to demonstrate that porcine islets could survive in the human body.

In 2005, a study conducted in Mexico involved transplanting macroencapsulated neonatal porcine islets with Sertoli cells into 12 adolescent patients with diabetes [14]. The macrodevice, constructed from surgical-grade stainless steel mesh tubes, was implanted subcutaneously. Over a period of 2 months, vascularized collagen tissue was allowed to infiltrate the device. Subsequently, a polytetrafluoroethylene rod was removed from the macrodevice, and 250,000 neonatal islets along with 30 to 100 Sertoli cells were implanted in its place. Eleven patients received a second transplant 6 to 9 months after the initial procedure. Of the total group, six patients experienced a reduction in insulin requirements, while the other six saw an increase. Notably, two patients achieved insulin independence. However, the clinical trial was met with criticism for being conducted on adolescent patients without a clear regulatory framework.

In 2013, a Chinese research team transplanted neonatal porcine islets into the livers of 10 adult patients with type 1 diabetes through the portal vein [15]. This clinical trial was conducted under the supervision of a Chinese governmental agency. To prevent the instant blood-mediated inflammatory reaction (IBMIR), patients received anticoagulant therapy for 7 days following transplantation. Clinically available immunosuppressants and autologous regulatory T (Treg) cells were employed to control immune responses. Although only a slight reduction in insulin requirements was noted, the procedure did not result in any zoonotic infections, including those from PERVs.

Clinical trials of microencapsulated neonatal porcine islet transplantation have been conducted in New Zealand, Russia, and Argentina from the early 2000s to the present [16–19]. These trials have shown a reduction in insulin dose and improved hypoglycemic awareness in most recipients, with some patients achieving long-term insulin independence. Consequently, this approach to xenogeneic islet transplantation is regarded as promising. The results of these clinical trials are summarized in Table 1.

In Korea, a sponsor-initiated trial using the islets of adult Seoul National University (SNU) pigs was approved by the Ministry of Food and Drug Safety in December 2022. However, due to internal issues with the sponsor, the trial has not yet commenced. Once underway, it will be conducted under a clear regulatory framework. Clinical trials require robust regulatory oversight to ensure safety and efficacy, and this trial is designed to adhere to those standards.

The age of donor pigs significantly influences the properties of islet products (Table 2) [20]. Researchers have experimented with the isolation and transplantation of pancreatic islets from pigs of various ages [21]. The islet structure in fetal and neonatal pigs is markedly different from that in adult pigs, primarily because the matrix and connective tissue in the pancreas of younger pigs are not fully developed. Consequently, ICCs can be relatively easily isolated from these organs through enzymatic digestion [22]. ICCs are primarily composed of endocrine tissue, which survives during in vitro culture, while the exocrine tissue dies. This process enables the purification of ICCs. However, ICCs require a maturation period after transplantation before they can begin secreting insulin. Therefore, even if porcine ICCs avoid immune rejection and successfully engraft, exogenous insulin therapy is necessary until their maturation is complete.

Isolation methods used for fetal and neonatal pancreases have been adapted for use with young pig pancreases. Typically, approximately 30,000 islet equivalents (IEQ) can be harvested from the pancreas of one young pig. However, Matsumoto et al. [23] reported isolating up to 180,000 IEQ from a single young pig. Islets from young pigs offer several advantages over neonatal islets, including higher islet yield and the capacity for immediate insulin secretion, which is attributed to their maturation during the culture period.

Despite the benefit of immediate insulin secretion, islets from young adult and adult pigs are delicate, which complicates the process of islet isolation. It is widely recognized that the success and yield of islet isolation depend heavily on the breed and age of the pig. Although the SNU miniature pig is used as an adult, it has demonstrated a higher yield in comparison to other breeds, such as Prestige World Genetics miniature pigs and adult market pigs sourced from a local slaughterhouse [24]. Consequently, the choice of pig breed should be made carefully when considering the use of adult pig islets for clinical or research applications.

In collaboration with the World Health Organization, the International Xenotransplantation Association (IXA) published a consensus statement in 2009 to guide the safe conduct of clinical trials involving porcine islet cells. This guidance is detailed in “The International Xenotransplantation Association consensus statement on the conditions for undertaking clinical trials of porcine islet products in type 1 diabetes.” One obstacle to the clinical adoption of porcine islet xenotransplantation is the difficulty in meeting success criteria in preclinical studies involving nonhuman primates (NHPs) [25]. The updated IXA consensus statement from 2016 specify requirements for NHP studies. These include maintaining fasting blood glucose levels below 150 mg/dL and nonfasting levels below 200 mg/dL, either without the need for exogenous insulin or with a significant reduction in insulin requirements, in at least four of six consecutive NHPs following porcine islet transplantation with a clinically available immunosuppressive regimen [26]. Also, all cases should have a follow-up period of at least 6 months, with one or two successful cases ideally undergoing follow-up for 12 months.

Various research teams have conducted studies on porcine islet xenotransplantation in preclinical NHP models to adhere to the IXA guidelines. Among these efforts, the present article summarizes the results of groups that achieved long-term graft survival (Table 3) [27–43]. In 2015, our group was the first to demonstrate a proof of concept by successfully reversing diabetes in five diabetic NHPs. This was accomplished through the transplantation of wild-type porcine islets, combined with an immunosuppressive regimen that included an anti-CD154 monoclonal antibody (mAb; 5C8)—which is not considered clinically applicable—and low-dose sirolimus, with or without ex vivo expanded Treg cells [27]. Subsequently, we reported similar, albeit less robust, outcomes using an anti-CD40 mAb (2C10R4) [28], which has potential for clinical use. Most recently, we maintained normal glycemic control in three of seven diabetic NHPs and significantly reduced the need for exogenous insulin in five of these seven animals. This was achieved using a clinically applicable immunosuppressive regimen that included ATG, adalimumab, anakinra, tocilizumab, intravenous immunoglobulin, tacrolimus, belimumab, rapamycin, and tofacitinib [29]. While this study could substantially advance the area of islet xenotransplantation clinical trials, the intensity of the immunosuppressive protocol used limits its widespread application. Consequently, additional strategies may be necessary, such as employing genetically engineered pigs or developing novel immunosuppressive drugs with fewer adverse effects.

The production of galactosyltransferase knockout pigs in 2002 was a key milestone in xenotransplantation research, eliminating the α-galactosidase (αGal) antigen and leading to the first major wave of advancements in the field [44]. Since then, the advent of clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) technology has enabled the creation of various genetically engineered pigs. These include animals with multiple human gene knockins to mitigate immune responses; those with knock-outs of multiple carbohydrate antigens such as Gal, 5NeuGc, and B4GalNT2; and even PERV-free pigs. This development has prompted a second wave of progress in xenotransplantation [45]. The authors anticipate that the third wave will incorporate innovative immunological therapies. These may include chemical drugs or antibodies that regulate T cell receptor (TCR) signaling, costimulation, and cytokines, as well as cell-based treatments such as Treg cells.

Regarding potential strategies, first, interventions targeting TCR signaling (also known as signal 1) have been utilized to prevent graft rejection. Numerous immunosuppressive drugs have been developed to target TCR signaling pathways as therapeutic interventions [46]. Chemical immunosuppressive drugs, including mammalian target of rapamycin inhibitors (sirolimus and everolimus), calcineurin inhibitors (tacrolimus and cyclosporine), the Janus kinase inhibitor tofacitinib, and purine and pyrimidine synthesis inhibitors (6-mercaptopurine, mycophenolate mofetil, and methotrexate), have been widely used in preclinical NHP studies of porcine islet xenotransplantation to interfere with the signals that activate T cells and promote their proliferation [47]. In addition to methods that inhibit the TCR signaling pathway, approaches that deplete T cells (such as ATG; polyclonal immunoglobulins [Igs] from rabbits immunized with human thymocytes; or alemtuzumab, an anti-CD52 mAb) or induce T cell anergy (such as daclizumab or basiliximab, blocking and internalizing the interleukin [IL]-2 receptor to reduce IL-2–dependent T cell activation and proliferation) are being employed in combination [48]. In preclinical NHP studies of porcine islet xenotransplantation, all groups that demonstrated long-term graft survival used sirolimus/everolimus, mycophenolate mofetil, tacrolimus, or tofacitinib (Table 3). When costimulatory blockades such as anti-CD154, anti-CD40, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) Ig, or anti-LFA-1 were used, long-term graft survival was achieved even with the use of a single signal 1 blocker. However, in immunosuppressive regimens that are clinically available without costimulatory blockade, long-term graft survival was only observed when three signal 1 blockers—sirolimus, tacrolimus, and tofacitinib—were used together [29]. Thus, it appears essential to incorporate costimulatory blockade in preclinical NHP studies of porcine islet xenotransplantation to ensure long-term graft survival.

Second, a costimulatory blockade strategy, such as anti-CD40L, anti-CD40, anti-LFA-1, LFA-3 Ig, or CTLA-4 Ig, can be considered, with a primary focus on inhibiting the CD40-CD40L pathway. Preclinical studies have shown that CD40-CD40L blockade exhibits remarkable efficacy in inducing tolerance across various autoimmune disease models and in promoting the acceptance of skin and organ transplants [49]. In preclinical trials involving NHPs with porcine islet transplants, long-term islet survival was observed using anti-CD154 mAbs [27,30–36]. The success of these antibodies is attributed to their critical role in the development of autoimmune diseases and the mediation of graft rejection. These impressive preclinical outcomes have spurred considerable interest in developing anti-CD154 therapies for human diseases, leading to clinical trials of native IgG1 CD154 antibodies in the late 1990s. However, these trials were halted due to fatal thromboembolic events caused by the simultaneous binding of CD154 to Fc receptors on platelets [50]. Recent advancements in immunotherapy have focused on modifying or substituting immunoglobulins to prevent binding to FcγRIIA, thereby avoiding platelet activation. Notable examples include frexalimab and tegoprubart, which are humanized CD154-specific IgG1 antibodies with silencing mutations in the Fc domain. Additionally, dapirolizumab pegol, a polyethylene glycol-conjugated anti-CD154 Fab fragment, and dazodalibep, a nonantibody biological antagonist, are being examined. These CD154 inhibitors have shown a favorable safety profile, with no thromboembolic activity observed in hundreds of patients and healthy controls. Promising results have also emerged from phase 2 clinical trials for conditions like systemic lupus erythematosus, Sjogren syndrome, and multiple sclerosis, with several candidates advancing to pivotal phase 3 trials [51].

The anti-CD40 inhibitor, a component of the CD40-CD40L blockade, has also displayed promising results in both preclinical and clinical trials. In NHP models, an anti-CD40 mAb (chi220; with B cell-depleting effects) significantly prolonged the survival of neonatal porcine islet grafts, with five of six recipients achieving insulin independence. In that study, up to 50,000 IEQ/kg of neonatal porcine islets were transplanted into six pancreatectomized diabetic macaques via the portal vein. The immunosuppression regimen included induction therapy with CD40-mAb and basiliximab, along with maintenance therapy using sirolimus and belatacept [37]. Separately, Shin et al. [28] demonstrated more than 321 days of adult porcine islet graft survival using a combination of ATG, cobra venom factor, antitumor necrosis factor (TNF), anti-CD40 (2C10R4), rapamycin, and tacrolimus. In another report, iscalimab also displayed a favorable safety profile and improvements compared to placebo in non-thymectomized patients with moderate to severe myasthenia gravis [52]. Clinical trials using anti-CD40 antibodies for various autoimmune diseases are ongoing, and the results are eagerly awaited.

Third, a strategy to inhibit cytokines should be considered to prevent the rejection of porcine islets. Both clinical islet allotransplantation and porcine islet xenotransplantation involve infusing islets through the portal vein to allow the transplanted islets to engraft in the liver. Consequently, the islets come into direct contact with the recipient’s blood, resulting in IBMIR and massive early islet loss. IBMIR is a complex phenomenon that triggers the activation of the complement system and coagulation pathway. Platelets rapidly bind to the islets, and neutrophils and monocytes infiltrate the area [53]. During IBMIR, proinflammatory cytokines such as high-mobility group box 1 protein, IL-8, TNF-α, IL-1β, and IL-6 are produced, leading to further islet destruction [54]. Various inhibitors of proinflammatory cytokines are available. For TNF-α, options include etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab [55]. Anakinra, a recombinant form of the human IL-1 receptor, can be used to block both IL-1α and IL-1β, while canakinumab, a human IgG1/κ mAb, specifically targets IL-1β [56]. For IL-6, options include tocilizumab, an antibody that blocks the IL-6 receptor, and siltuximab, an antibody that targets IL-6 itself [57]. The efficacy of TNF-α blockers (infliximab, certolizumab pegol, and golimumab), an IL-1β blocker (canakinumab), and an IL-6 blocker (siltuximab) has not yet been explored in preclinical NHP studies of porcine islet xenotransplantation, and this information is highly anticipated.

Fourth, Treg cells have been investigated in preclinical NHP studies of porcine islet xenotransplantation to induce immune tolerance. Shin et al. [35] utilized autologous Treg cells characterized by CD4(+) CD25(high) CD127(low) markers in combination with immunosuppressive agents such as ATG, cobra venom factor, anti-CD154 mAb, and sirolimus. In their study, two diabetic NHPs exhibited promising results; one achieved 500 days of graft survival and the other 1,000 days, illustrating the potential of Treg cells in promoting immune tolerance [35]. Although polyclonal Treg cells are a promising tool for inducing transplantation tolerance, their lack of specificity requires the administration of high doses. By genetically modifying these cells to express chimeric antigen receptors (CAR) that target mismatched donor human leukocyte antigen molecules, CAR Treg cells could become much more effective in preventing allograft rejection [58]. Researchers are also examining the use of swine leukocyte antigen-specific CAR Treg cells to induce tolerance in xenotransplantation. This strategy is expected to be incorporated into future porcine islet xenotransplantation protocols, offering new potential for improved outcomes [59].

The advancements in TCR signaling blockades, CD40-CD40L costimulation blockades, cytokine blockers, and Treg cell therapies offer hope for increased clinical accessibility. These developments have potential applications in the xenotransplantation of porcine organs, positioning them within what could be considered a “third wave” in this field. Xenotransplantation research is a highly active field in South Korea. As part of a national initiative, the government plans to invest approximately 38 billion Korean won into this area of study over the next 4 years. Preclinical studies involving NHPs are underway, with a focus on the transplantation of solid organs such as kidneys, hearts, and livers, as well as cellular tissues including islets, corneas, and skin. To advance xenotransplantation research in South Korea, sustained investment from both the government and the private sector, coupled with regulatory reforms, is crucial.