The strategy for in vitro differentiation of ESCs/iPSCs into pancreatic cells mimics the molecular regulatory mechanisms of pancreatic development in vivo. It involves the use of a combination of growth factors and small molecules to activate developmental signaling pathways and transcription factor networks. Through staged induction, pancreatic progenitor cells and endocrine progenitor cells eventually differentiate into mature endocrine cells (α, β, δ) cells (36–39). D’Amour et al. first attempted to establish a protocol for generating hormone-expressing cells that can synthesize and release multiple hormones (40). Rezania et al. reported a seven-step differentiation protocol, in which the resulting cells expressed key markers of mature β-cells, such as MAFA, PDX1, NKX6.1, and INS, and exhibited similar functionality to human islets ( Figure 2 ) (41). Subsequently, Pagliuca et al. utilized human ESCs and employed a stepwise induction method with the addition of various factors in basal medium to successfully cultivate insulin-secreting β-cells (SC-β-cells), which functioned as fully functional pancreatic β-cells. Upon transplantation into mice, SC-β-cells showed detectable insulin secretion within two weeks, with secretion levels changing in response to blood glucose levels (42, 43). Directed differentiation protocols have also been reported for iPSCs, enabling the generation of cells expressing insulin and other mature β-cell markers (44, 45).

Because ESCs/iPSCs have good proliferation and differentiation ability and can produce large numbers of cells, they are ideal candidates for differentiation into islet β-cells and have broad application prospects for treating T1D. Therefore, how to produce SC-β-cells in vitro in large quantities has become the focus of research. The key transcription factors for differentiating ESCs/iPSCs into SC-β-cells in vitro are PDX1 and NKX6.1, both of which are highly expressed in pancreatic progenitor cells and required for producing monohormone, glucose-reactive β-cells (46, 47). Several research teams have reported differentiating ESCs/iPSCs into PDX1 and NKX6.1 co-expressing pancreatic progenitor cells (48, 49) in monolayer culture, and with improved experimental conditions, up to 90% of PDX1+/NKX6.1+ co-positive pancreatic progenitor cells were produced. Simultaneously, differentiating pancreatic progenitor cells into pancreatic β-cells has also made substantial progress, and the efficiency of pancreatic progenitor cells producing β-cells in vitro increased to about 40% (21, 42), although these β-cells are still different from human β-cells in other functions despite being responsive to glucose. To further improve the function of SC-beta cells, multiple research teams provided insights, such as Juan et al., who found that co-culturing with factors regulating circadian rhythm could enhance SC-beta cell function (50), Leonardo by altering the signaling pathway of SC-β-cells differentiation process (51), while Aharon et al. modified the nutrients in the medium used for in vitro differentiation to further enhance generating functional SC-β-cells in vitro (52), and could also induce SC-β-cells by simulating the 3D culture system of human pancreatic development (53). In addition, Isaura (54) and Mariana (55) et al. recently updated and detailed the recent progress in using ESCs/iPSCs derived islet β-cells in vitro. Some of the above protocols, although not reaching the level of the original human islet β-cells, promoted the development of stem cells differentiating into islet β-cells in vitro.

With the continuous development of stem cell technology, pancreatic β-cells products derived from ESCs/iPSCs are gradually used in clinical trials. A clinical trial conducted in 2014 (NCT02239354) used a stem cell-derived pancreatic endoderm cell population (PEC-01) developed by ViaCyte, Inc., which matured into insulin-producing endocrine cells in vivo over several months in animal models (56–58)., and in the clinical trials they have developed an immune protective device (PEC - Encap, VC - 01) is used to encapsulate PEC - 01, the device is a kind of biological membranes in order to eliminate the need for immunosuppression. The results of the trial showed that PEC-01 cell population could differentiate into β-cells and other islet cells after implantation under the patient’s skin, but excessive fibrosis around the device resulted in the end of the trial due to immune rejection (29, 30). To address this problem, the new device was modified with an opening in the biofilm that allowed vascularization, enhanced nutrient exchange so that host cells could also penetrate the device, and immunosuppressive therapy was administered after transplantation, while a more mature and functional cell population (PEC-02) was used. The results of a subsequent clinical trial (NCT03163511), published in 2021, showed that transplanted cells matured from pancreatic progenitor cells to pancreatic endocrine cells six months after transplantation, producing glucose-reactive C-peptide (59, 60) in six of the 17 patients who underwent the trial. Although the circulating C-peptide levels observed in these studies are still low, all demonstrate the potential of ESCs/iPSCs to differentiate into renewable islet β-cells. Most importantly, both studies, although in early stage clinical studies, did not identify any serious safety issues related to the transplanted cells, including tumor formation. ViaCyte was later acquired by Vertex. Another direction of clinical trials is transplanting fully differentiated SC-β-cells, which have been successful in non-human primates (61, 62). The most promising clinical trial to date is Vertex’s Phase I/II trial in 2021 (NCT04786262), which uses cells made of fully differentiated islet cells derived from pluripotent stem cells (VX-880) injected into the liver via a traditional portal route. Immunosuppressive therapy was also used to protect the transplanted islet cells from immune rejection. Some early results from the trial were recently published, with significant circulating C-peptide levels detected three months after transplantation and patients’ blood sugar significantly controlled, And well tolerated treatment (63). VX-880 is a novel stem cell derived product for the treatment of T1D, and the trial is continuing in the United States and Canada to further evaluate the safety and efficacy of the product. As the technology develops, more clinical trials are expected.

In addition to using ESC/iPSC-based techniques to induce the differentiation of transplantable β-cells in vitro, Zeng et al. proposed an alternative solution. Using single-cell sequencing technology, they discovered a previously unreported cell population in the mouse pancreas: protein C receptor-positive (Procr+) pancreatic cell population. These Procr+ endocrine progenitor cells can be cultured and induced to differentiate into islet-like cells. In a transplantation model of diabetic mice, transplanted islet-like organs reversed the disease (36). This finding provides a new direction for the direct extraction of target cells from the pancreas and induction of their differentiation into islet-like organs.

In addition to using stem cell-derived islet beta cells to replace donor islet cells, another potential option is xenotransplantation using porcine islets. Compared to human islets, pig reproduction is easier and pig islets are more readily available. More importantly, pig insulin is highly similar to human insulin, differing by only one amino acid. Pig insulin has been used to treat diabetes for decades. Pigs have organs similar in size to humans, enabling production of a sufficient number of islets for xenotransplantation. They are the most promising donor source for xenotransplantation. Although more porcine islet cells are required to achieve adequate insulin secretion compared to human donor islets, porcine islets appear to outperform human islets in studies. Porcine islet cell xenotransplantation has achieved insulin function in non-human primates, suggesting feasibility in clinical settings. Shin et al. reported long-term survival of adult porcine islets transplanted into five rhesus monkeys for over 20 months. These early trials suggest pig islets have great potential to address donor islet shortages for T1D patients.

However, there are still some urgent problems to be solved in the use of porcine islet xenotransplantation, the first of which is graft rejection. For example, infusion of porcine islets into the portal vein leads to activation of complement and clotting pathways, resulting in platelet aggregation and thrombosis at the transplant site and hyperacute rejection (64). This is followed by human responses to porcine islet antigens (Galactose α1,3-galactose and N-Glycolylneuraminic acid), as well as zoonotic infections caused by endogenous retroviruses.

In the meantime, solutions are being tried. One strategy is encapsulating islet cells without immunosuppression to solve the immune rejection problem in porcine islet xenotransplantation. Various natural or synthetic biomaterials are used for encapsulation, such as polyethylene glycol diacrylate (PEG-DA) (65), agarose (66), and other biological materials like alginate (67). Coating islet cells with alginate films containing polyethylene glycol acrylate has allowed survival up to 6 months without immunosuppression (68, 69). However, encapsulation risks hypoxia and nutrient deficiency in islet cells, delayed glucose and insulin diffusion affecting glucose regulation (70). One possible immunosuppression approach is co-stimulatory blocking. Studies in non-human primates showed anti-CD154 monoclonal antibodies combined with stimulus-blocking and standard immunization regimens injected through the portal vein prolonged transplanted porcine mice survival. However, no clinically available anti-CD154 monoclonal antibodies exist due to high thrombosis risk (71). We summarized relevant studies using immunosuppressive therapy to prolong porcine islet survival post-transplantation in Table 2 . Technological developments like gene editing technologies like CRISPR/Cas9 potentially eliminate endogenous viruses in pigs, improving porcine islet xenotransplantation safety to humans (31). Gene editing overexpresses or knocks out multiple genes finding the best transgenic pigs for islet transplantation, avoiding xenotransplantation rejection (72). Recent studies showed targeted controlled mutational events successfully generated in pig cells through nuclease-directed homologous recombination (32).

In general, various differentiation protocols are available to induce the transformation of ESCs/iPSCs into insulin-producing cells. Clinical trial results have shown its safety and tolerance, making it a hot topic in current research with broad application prospects. However, the approach of directly selecting cells from the pancreatic tissue to induce pancreatic-like organs should not be abandoned. Finally, although extensive data on pig islets are still required from nonhuman primates for safety validation before clinical trials, they have gained popularity among many researchers. These different sources of β-cell replacement provide abundant choices for future clinical applications, allowing personalized treatment plans based on individual patient conditions. We summarized the advantages and disadvantages of using ESCs/iPSCs derived islet β-cells and porcine islet instead of β-cells as shown in Table 3 .

Mesenchymal stem cells (MSC),also known as stromal cells or mesenchymal progenitor cells, are a kind of non-hematopoietic stem cells derived from mesoderm, with multi-directional differentiation potential and strong self-renewal ability (80). MSC is relatively easy to obtain, can be obtained from human and rodent peripheral blood, placental tissue, umbilical cord blood, bone marrow cavity tissue and adipose tissue and other tissues and organs, and can be expanded and induced to differentiate in vitro, so it has been widely concerned and applied in the field of tissue engineering and regeneration. MSCs can improve the efficacy of IT in animal models, especially in regulating immune responses and protecting islet transplants (81–83). MSCs can improve insulin resistance in peripheral tissues through potential immunomodulatory and anti-inflammatory effects and promote pancreatic β-cell regeneration and protection (84, 85). Multiple studies have shown that, when co-cultured or co-transplanted with islet cells, MSCs can protect islet cells from apoptosis due to hypoxia and inflammatory cytokines through their secretory function, thus improving the survival of islet grafts in vivo and promoting the early recovery of the islet function (86, 87). In 2021, Kenyon et al. reported that islet cells and MSCs could be co-transplanted in non-human primate IT experiments. The results showed that the rejection-free survival and overall survival of treated islet grafts were significantly extended (88). Wang et al. used engineered MSCs as helper cells for islet co-transplantation and obtained similar results in diabetic mice. MSCs can induce local immune regulation and are potentially suitable for IT (89). Another study in patients with chronic pancreatitis showed that co-transplantation of autologous MSCs and islets is a safe and potential strategy for improving the islet function after transplantation (90). Generally speaking, co-transplantation with islet cells, it was found that mesenchymal stem cells had the functions of nutrition, support and protection to islet β-cells, as well as anti-inflammatory and immune regulation.

Regulatory T cells constitute a subset of T cells characterized by the presence of typical biological markers such as CD4+CD25+FoxP3+. These cells wield potent immunomodulatory functions and are pivotal in regulating immune homeostasis, upholding self-tolerance, and preventing excessive activation of the immune system (91). Tregs are considered a promising alternative to pharmacological agents that promote the engraftment and survival of transplanted organs/tissues (92–94). Tregs mainly produce self-tolerance, tolerance to alloantigens, and transplantation tolerance by inhibiting the activation and function of reactive effector T-cells (94). Currently, Treg therapy can be applied in two situations in IT: to promote the survival of islets during the initial transplantation and to induce peripheral tolerance to eliminate immunosuppression. The addition of Tregs at the time of islet infusion has been explored as a method to reduce the initial islet graft loss and improve islet engraftment (95–97). It has been reported that, in clinical models, Treg expansion in vitro and subsequent reinjection into patients can induce long-term remission of T1DM (98, 99). Although there are few relevant reports, a large amount of preclinical evidence shows that Treg-based treatment has benefits (100–102). Zielinski et al. recently reported a two-year study using a combined infusion of Tregs and rituximab to treat pediatric patients with T1DM. The study results show that combination therapy can delay disease progression compared with Treg or rituximab alone, and patients who received combination therapy were able to maintain higher insulin sensitivity and fasting C-peptide levels than patients in the single-treatment and control groups. Furthermore, patients who received Tregs alone had higher C-peptide levels than those in the untreated control group. Another ongoing clinical trial (NCT03182426) is observing the benefits of T cell depletion and dual anti-inflammatory treatment. If successful, it will provide new benefits to islet transplant patients.

With the development of IT, most traditional immunosuppressive drugs require continuous medication and cannot completely solve the problem of immune rejection in islet transplants. Cell therapy co-transplanted with MSCs/Tregs and islets has shown great advantages, although it is still in the experimental stage, and its application scenarios are broad.

Islet encapsulation represents a promising approach to tackle host immune rejection, employing biomaterials to envelop islets in a protective barrier. This allows oxygen and nutrients to permeate islet cells while enabling secreted insulin to disseminate into the bloodstream. Concurrently, it shields islet cells from assault by the host immune system (103–105). This technology has developed rapidly over the past century and can be categorized into micro and macro-encapsulation based on different processes.

Micro-encapsulation technology encapsulates islets in a thin layer of biomaterials, facilitating exchange of nutrients, oxygen, and metabolites. Transplantation of these micro-encapsulated islets is also simplified. Alginate stands out as a particularly promising biomaterial due to its superior biocompatibility and ease of manufacture. Studies confirm alginate reduces post-transplantation immune rejection and enhances survival of encapsulated islet cells (105). For instance, incorporating chemokine CXCL12 into alginate micro-encapsulation protects islets and boosts islet cell function even without immunosuppressants (106). This alginate-based micro-encapsulation method has also been applied to encapsulate SC-β-cells, exhibiting no excessive fibrosis post-transplantation sans immunosuppressive therapy (107). It has emerged as a key biomaterial for β-cell encapsulation studies. Recently, research teams have modified extracellular matrix (ECM) components into alginate, simulating the pancreatic microenvironment to safeguard coated islet cells from immune cell and inflammatory factor impacts while promoting insulin secretion by islet β-cells (108, 109). Nevertheless, several challenges persist in leveraging micro-encapsulation, especially post-implantation, presenting potential issues.

Another macro-encapsulation technique can prevent direct graft-host immune cell contact and spread, and enable easy removal of any post-transplantation safety issues, and evaluating graft efficacy at any time, unavailable with micro-encapsulation (110). Macro-encapsulation has combated host immune rejection but is limited by inadequate oxygen and nutrient exchange before blood vessel formation around the device (30). Adding vascular endothelial growth factor (VEGF) and pre-vascularization improved this (111, 112). Recently, Wang et al. developed a new device with immunoprotective hydrogel and thermoplastic silica gel-polycarbonate-polyurethane maintaining islet function for up to 200 days (113) in allogeneic rodent islet transplant models. Another macro-encapsulation type encapsulated SC-β cells with amphoteric modified alginate gel, reversing hyperglycemia for 238 days (114) post-implantation in severe combined immunodeficiency (SCID) mice. Many research teams are studying islet packaging, and we summarize recent progress in Table 4 .

Currently, most clinical IT methods involve injecting islet cells through the hepatic portal vein under ultrasound guidance. This is a conventional, mature method (11, 121). However, portal vein IT can cause postoperative bleeding, vascular emboli formation, portal hypertension, and periportal fatty degeneration. In particular, the blood-mediated acute inflammatory response (IBMIR) caused by portal vein transplantation can result in massive graft loss in the very early stages of transplantation (122), suggesting that the liver is not the most suitable site for IT. Researchers are exploring different organs and sites ( Figure 2B ) to determine the best location for islet cell transplantation ( Table 5 ).

The omentum represents a potentially valuable transplant site, offering avoidance of IBMIR compared to traditional portal vein inflow. This richly vascularized tissue secretes various growth factors (e.g. CXCR4, VEGF, and SDF-1) that promote islet vascularization and survival (131, 132). In addition, omentum possesses immunomodulatory capabilities and can monitor the graft for prompt removal if adverse reactions occur. Omental transplantation using biological scaffolds has been used for clinical applications. A US trial (NCT02213003) transplanted pancreatic islets into the omentum of T1DM patients (133). Insulin independence was achieved by day 17 post-transplant but declined approximately one year later. Another ongoing trial (NCT02821026) has shown limited success. However, in 2023, Deng et al. reported a method of omental allogeneic IT in nonhuman primates using locally applied recombinant thrombin (Recothrom) and the recipient’s autologous plasma to design a degradable matrix for islet fixation. Normal blood sugar and insulin independence were achieved at one week post-transplant, with stable expression thereafter. This study provides strategies for the clinical translation of omental transplantation.

The subcutaneous space is another ideal transplant site. It is a relatively avascular region that is easily accessible to biomaterials or macroscopically encapsulated islets. In 2020, Yu et al. reported successful subcutaneous IT in various immune-competent and immune-naïve animal models using a device-free islet survival matrix to achieve long-term normoglycemia. This method has been used for mice, pigs, and humans. Islet cell transplant models have the advantages of simplicity, safety, and reproducibility (134). With the clinical application of ESCs/iPSC-derived islet-like cells and islet encapsulation technology, the subcutaneous cavity can be easily monitored and removed, making it a promising transplant method. However, the skin lacks relative blood vessels and cannot obtain early-stage nutrients and oxygen, which limits its clinical application. To address this, Darling et al. tested a biodegradable temporary matrix based on a polyurethane scaffold that forms good blood vessels within the skin. In a porcine islet transplant model, grafts maintained normal function and survived for over three months (128). In addition, the immune response hinders subcutaneous transplantation. Therefore, the development of advanced biomaterials with angiogenesis and immune modulation capabilities may be the next step for the long-term islet survival and function in the skin.

In addition to the two aforementioned research hotspots of transplant sites, studies on transplanting islets into the intrapleural (135), skeletal muscle (136), anterior chamber of the eye (ACE) (137), and other sites have been reported (138–140). However, research on these aspects is still in its infancy, and there is a large gap in clinical applications. Due to the application of bioengineering materials and macro-encapsulated islet grafts, the greater omentum and subcutaneous space seem to be ideal sites for IT in the future.

Although several of the above options are effective in mitigating the immune rejection caused by islet transplantation and are the way forward, immunosuppressive therapy is still required at this time to ensure islet survival and function. The goal of immunosuppression is to provide effective and sustained immune protection in the smallest effective amount without suffering from the side effects associated with immunosuppression. Since inflammation leads to significant islet loss, anti-inflammatory drugs reduce damage from pro-inflammatory factors and may improve islet cell function in the early post-transplant period (141). Therefore, in order to attenuate the IBMIR response that occurs after islet transplantation and thereby reduce islet loss, several anti-inflammatory therapies have been used in the perioperative period of islet transplantation, including TNF-α inhibitors (etanercept), IL-1 receptor antagonists (anabolic acid), and α1-antitrypsin. Enalcipro, which targets TNF-α, is a potent antitumor agent that is widely used in T1D patients with allogeneic transplantation (17), and its use in mouse animal models results in a reduction of inflammatory markers and has been shown to have a sustained effect on autoimmunity (142). And in another study in an immunodeficient mouse islet transplant model, it was found that the percentage of mice achieving normal blood glucose levels after transplantation with the combination of etanercept and anabolic acid was 87.5%, compared to 45.45% with etanercept alone, and 53.9% with anabolic acid alone, suggesting that the combined use of etanercept and anabolic acid significantly improves the function of islet grafts (143). However, a recent study showed that although the use of etanercept demonstrated better islet function in the pre-transplant period, this advantage was not found to be sustained at the subsequent 1- or 2-year follow-up, and therefore, different doses or prolonged use of etanercept need to be explored to benefit patients (144). Another promising anti-inflammatory is α1-antitrypsin, which is a serine protease inhibitor, has been shown in several preclinical studies in animal islet transplantation models to attenuate the IBMIR response and prevent islet cell apoptosis while inhibiting cytokine-induced islet inflammatory responses (145, 146).