Challenges in Beta Cell Replacement for Type 1 Diabetes

Type 1 diabetes (T1D) is a significant global health challenge even with advancements in treatment and management strategies. Despite sharing common genetic risk factors and autoantibody biomarkers, the considerable variability in clinical presentation and management of the disease complicates achieving glycemic targets, thereby increasing the risk of complications and comorbidities [1]. Choices of ideal therapeutic goals in T1D include prevention of immune destruction, preservation of β-cell mass, replacement or regeneration of β-cells, and automated insulin delivery.

The search for effective β-cell replacement therapies has been marked by significant achievements, from the first successful pancreas transplant to sophisticated genetic manipulations of stem cells, aiming to produce immunologically compatible β-cells [2]. However, these advances are tempered by substantial challenges, reflecting the complex nature of biological and technological interventions in diabetes care. Years of experience with cadaver donor islet transplants have significantly deepened our understanding of how β-cells can successfully engraft and function posttransplant [3]. This knowledge is now being applied to the field of regenerative medicine. It has become feasible to generate functional β-cells from pluripotent stem cells [4].

However, the path from promising preclinical results to effective clinical applications is fraught with technical, biological, and ethical challenges. This journey is often prolonged by the rigorous validations necessary to translate benchtop successes into bedside solutions, a process that can extend over decades and is susceptible to both scientific and public misinterpretations.

The scientific community continues to explore various avenues for beta cell replacement, including pancreas transplantation, islet cell transplantation, and innovative biotechnological approaches like stem cell therapies and bioengineering of pancreatic tissues. Each method presents unique challenges and opportunities, from immunological rejection to issues of long-term viability and integration of transplanted cells. Furthermore, the excitement surrounding early successes often overshadows the intricate, stepwise process required to achieve clinically relevant outcomes, underscoring the need for a balanced perspective that manages patient expectations while fostering continued support for research [5].

This review aimed to discuss the challenges and scientific intricacies of β-cell replacement therapy, exploring both the achievements and obstacles in the field. The goal is to provide a comprehensive overview that not only highlights the potential of β-cell replacement therapy in transforming diabetes care but also addresses the critical need for solutions to enhance the viability and success of these therapeutic interventions.

Pancreas transplantation offers replacement of the dysfunctional pancreas with a healthy donor pancreas. The journey of pancreas transplantation began in 1966 at the University of Minnesota, marking a significant milestone with the first successful procedure with a simultaneous pancreas and kidney transplantation (SPK) [6]. This early success set the foundation for the evolution of the procedure.

Although it can be performed alone (pancreas-alone transplant), SPK is the prevalent and preferred strategy for patients with T1D who also suffer from diabetic nephropathy. This dual-organ transplantation is primarily indicated to reinstate the physiological glucose homeostasis by reestablishing insulin production, alongside addressing renal failure.

Surgical techniques have evolved to improve the success rates and manage complications effectively [7]. The success of pancreas transplants in achieving insulin independence and improving life quality is well documented, with 1-year graft survival rates being notably high (90–100%), especially in SPK procedures [8, 9].

Despite its benefits, like any surgical procedure, pancreas transplantation is associated with certain risks and complications. These include surgical complications such as infection, bleeding, and graft thrombosis, as well as immunological complications such as rejection and graft failure. The long-term effects of immunosuppressants require careful consideration, particularly in younger patients, as prolonged immunosuppression can increase the risk of infections, malignancies, and organ toxicity.

Transplantation of isolated islets provides a safer and more favorable alternative with less surgical invasion. It has proved its efficacy as a viable treatment option for selected patients with T1D, mitigating recurrent severe hypoglycemia and improving quality of life [10], particularly in those with impaired hypoglycemia awareness [11]. Phase III trials have documented the safety and efficacy of transplanting allogeneic islets to restore near-normoglycemia and protect against severe hypoglycemia in both islet-alone and islet-after-kidney recipients [12]. By implanting isolated pancreatic islet cells from cadaveric donors into the hepatic portal vein, these cells functionally replace the patient’s own beta-cells, offering the potential to achieve physiological glycemic control.

Islet cell transplantation is regulated differently across countries, reflecting diverse approaches to classification and oversight. In many countries, clinical islet allotransplantation is governed as tissue or organ transplantation, acknowledged as a safe and effective therapy for select patients with type 1 diabetes mellitus. These countries, including Canada, Australia, and several European nations, integrate islet transplants within their established organ transplantation frameworks, ensuring accessibility and insurance coverage. Recently, France and Japan have also excluded islets from drug regulations based on current scientific evidence, reinforcing this global consensus. For example, over 600 islet infusions in 255 patients have been successfully conducted at the University of Alberta in Canada over the past 20 years. Conversely, in the USA, pancreatic islets are classified as a biologic drug by the Food and Drug Administration (FDA), requiring a Biological License Application for clinical use. This requirement has hindered the progression of islet transplantation from experimental to routine clinical practice over the past 2 decades. The Biological License Application process is expensive and lengthy, and even when islets meet all predefined release criteria, their sterility and potency cannot be verified. This has led to advocacy for the USA to harmonize its regulatory framework with those of other countries by including islets within existing organ transplantation regulations, facilitating broader clinical adoption and ensuring patient safety and effectiveness [13].

Patient survival is reported to be greater than 80% at 20 years post islet transplant according to experience at the center in Miami [14] and 74% according to the center in Edmonton, AB [15], while median time of graft survival is estimated to be 5.9 (interquartile: 3.0–9.5) years [15]. Despite its potential, the widespread clinical adoption of islet transplantation faces significant challenges. The scarcity of cadaveric human organ donors severely limits the availability of pancreatic islets, creating a substantial gap between supply and demand. This issue underscores the need for alternative sources of islets. Moreover, recipients of islet transplantation currently require lifelong immunosuppressive therapy to prevent immune-mediated rejection of the transplanted cells, posing substantial risks, especially for vulnerable populations.

The long-term success of transplanted islets is often undermined by a gradual decline in graft mass and function, which can be attributed partly to the suboptimal microenvironment at the liver implantation site. Furthermore, other factors contributing to reduced function over time include the high metabolic demand, leading to exhaustion of beta-cells, potentiated by the peripheral insulin resistance induced by prolonged immunosuppressive drug treatment. Additionally, significant variability in the morphology, function, and gene expression of the islets has been observed, initially identified through differences in nuclear and organelle sizes [16]. This heterogeneity has been linked to factors such as islet location, developmental origins, maturation state, and stress responses. For instance, proximity to other cells, blood vessels, and nerve endings can affect β-cell morphology and glucose sensitivity. Moreover, β-cells exhibit dynamic responses under stress conditions, such as varying levels of insulin unfolding protein response, which helps them adapt to oxidative and endoplasmic reticulum stress [17]. These characteristics can change during metabolic stress or diabetes progression and are influenced by conditions like aging, obesity, and consequent insulin resistance [18].

This highlights the ongoing need for a deeper understanding of beta-cell behavior and for exploring the factors contributing to the loss of function. To enhance the success of islet transplantation, it is essential to identify these mechanisms and develop new strategies to mitigate these challenges and improve transplantation outcomes. Table 1 shows the milestones in pancreas and islet transplantation.

The alloimmune response, which causes allograft rejection during transplantation, is driven by the adaptive immune response involving T and B lymphocytes. Generally, the adaptive immune response necessitates three types of signals: the first through antigen-specific receptors (T-cell receptor [TCR] and B-cell receptor or [BCR]), the second via costimulation through different pathways (such as CD28 and CD40), and the third from cytokines (e.g., TNFα, IL-1) that encourage both autocrine and paracrine signaling [19].

The primary challenge in islet transplantation lies in managing the alloimmune reactions, which if left unchecked ultimately leads to graft rejection. This requires the administration of immunosuppressive therapies that, while necessary to prevent rejection, introduce risks such as increased susceptibility to infections and, with long-term use, potential of malignancies, among other side effects.

Additionally, the recurrence of autoimmunity in T1D presents a significant challenge in islet transplantation. Considering the autoimmune nature of T1D, and despite advances in immunosuppressive treatments designed to prevent graft rejection, autoimmunity can recur, targeting the newly transplanted islets for destruction and undermining the longevity and functionality of the graft (Fig. 1) [20]. This autoimmune response mirrors the original destruction of pancreatic β-cells seen in native T1D. It is exemplified by cases where, despite initial transplant success, there was a marked decline in graft function. Liver biopsies from these cases, looking for the transplanted cells, revealed that the transplant had a predominance of glucagon-expressing cells and a lack of insulin-producing cells, suggesting possible recurrence of autoimmunity [21].

Such recurrences are characterized by the reactivation of islet cell autoantibodies and the infiltration of T cells, which lead to β-cell destruction. Allosensitization has been observed in patients posttransplant following the discontinuation of immunosuppression, persisting for 7–15 years after graft failure [22]. This ongoing risk necessitates careful consideration of autoimmunity in the posttransplant care of T1D patients and underscores the need for immunomodulation strategies that more effectively target specific autoimmune mechanisms, in addition to targeting alloimmunity.

Beyond these immunological concerns, several non-immunological factors also play crucial roles in the success of islet transplants. Donor characteristics and the isolation process itself can be traumatic for islets.

Previous studies have identified that donor characteristics, such as BMI and cold ischemia time, along with enzymatic and mechanical stresses during extraction, significantly influence the success rates of attaining adequate islet numbers for transplantation. These factors can compromise the viability and function of the islets, impacting their overall effectiveness in transplant procedures [23, 24].

After transplantation, adequate vascularization of the islets is a crucial aspect, impacting their survival, function, and the overall success of the transplantation. Upon transplantation, islet grafts are typically avascular and rely on the immediate surrounding microenvironment for oxygen and nutrient supply. The process of neovascularization begins almost immediately but can take several days to weeks to become fully established. During this period, the islets are particularly vulnerable to hypoxia and nutrient deprivation, which can lead to cellular stress, dysfunction, and apoptosis [25].

The revascularization process involves the migration and proliferation of endothelial cells [26], which form new blood vessels that integrate with the host’s vascular system. This neovascularization is critical for restoring the islet’s ability to respond adequately to blood glucose levels as it facilitates direct access to blood-borne glucose and the rapid release of insulin into the circulation.

One of the major challenges in posttransplant islet survival is the initial period of hypoxia before adequate vascularization is established. The lack of immediate blood supply can lead to significant losses in islet mass and function. Additionally, the inflammatory response triggered by the transplantation can further exacerbate islet injury and impede the vascularization process.

The pursuit of effective beta cell replacement therapies for diabetes management is fraught with challenges, including donor islet availability, the use of stem cells as alternative sources, and the consistency of cell quality and functionality in laboratory-generated islets. The scarcity of donor islets and the variability in their quality remain significant challenges. Islet cell transplantation depends heavily on the availability of high-quality human pancreatic islets, which are typically sourced from cadaveric donors [27]. However, the number of suitable donors is limited, and the viability and functional capacity of isolated islets can vary significantly due to donor-related factors such as age, health status at the time of death, and conditions during islet processing and preservation [28]. These variabilities affect the outcomes of transplantation, with a direct impact on the efficacy and longevity of the treatment.

To address the combined challenges of T1D management effectively, an integrated research strategy is essential, encompassing advanced immunomodulation techniques and improvements in islet isolation, preservation, and transplantation procedures. This includes enhancing vascularization posttransplant, refining the handling and treatment of islets during isolation, and exploring the impact of various factors on transplant outcomes to maximize the therapeutic potential of islet transplantation [26, 29].

The significant progress in these areas has been driven by the transformative experiences of recipients of successful islet transplants, which have fueled a deeper commitment to research and development. Optimizations in islet banking, along with advancements in islet implantation and cellular co-transplantation approaches, are enhancing islet cell engraftment [30, 31]. Furthermore, the development of an unlimited supply of islet cells for transplant, now being evaluated in clinical trials, and strategies to circumvent the risks associated with chronic immunosuppression, such as novel islet micro- and macroencapsulation technologies, induction of immune tolerance, and the creation of hypoimmunogenic islets through gene editing, are pivotal advancements in the field.

To tackle these challenges, a comprehensive, multipronged research strategy is necessary. Future developments in cellular therapy for T1D should focus on the following areas.

To prevent the immune rejection of the grafts, considerable efforts are focused on immunosuppressive and immunomodulatory therapies. These drugs are needed for attenuating the immune response of the recipient, enabling the transplanted cells to survive and function effectively.

The human body naturally mounts a defense against transplanted tissue, recognizing it as foreign due to the expression of donor-specific major histocompatibility complex (MHC) molecules on graft cells. This recognition triggers T-cell activation, leading both directly and indirectly to graft rejection. Historically, the primary strategy to manage this immune response has been the administration of a regimen that broadly suppresses T-cell activity [32]. This includes the use of T-cell depletion (e.g., with antithymocyte globulin), calcineurin inhibitors to inhibit TCR signaling, antiproliferatives like mycophenolate to halt T-cell proliferation, and steroids to reduce inflammation [12, 15, 33, 34].

Moreover, patients receive induction therapies immediately posttransplant, aiming to minimize early immunogenic responses and facilitate native immune regulation mechanisms. Induction immunosuppression is designed to robustly deplete T-cells and block inflammatory cytokines such as TNFα and IL-1, enhancing the long-term function of the islet grafts. Techniques such as the use of biologic agents like anti-thymocyte globulin and costimulatory blockers have shown promise in reducing acute rejection episodes and sparing the use of toxic long-term immunosuppressants.

Immunosuppression is, therefore, a critical aspect to prevent graft rejection. By sparing long-term immunosuppression, patients are not subjected to the risk of malignancies and only face a transient risk of infections.

One alternative would be the use of localized immunosuppression. The use of localized delivery systems for anti-inflammatory and immunomodulatory drugs can significantly mitigate the adverse effects associated with systemic immunosuppression, targeting the transplant site specifically, reducing the systemic exposure and minimizing side effects [35].

However, the ideal scenario would involve reprogramming the immune system to recognize the transplanted tissue as its own, thus obviating the need for lifelong immunosuppression. Achieving this level of immune tolerance has been demonstrated in preclinical studies [36, 37], and research is ongoing to translate these successes into human treatments. T-cell tolerance is achieved through two distinct mechanisms. The first mechanism involves the elimination of self-reactive T cells during their development in the thymus. This process, known as T-cell clonal deletion, involves the apoptosis of T cells whose TCRs recognize host antigens, serving as the primary mechanism of tolerance during T-cell development in the thymus [38, 39]. The second mechanism occurs in the periphery and involves the suppression or removal of self-reactive mature T cells. This can be mediated by immunologically active suppressive cells like regulatory T cells (Tregs) or by the inactivation of autoreactive T-cell clones through inhibitory molecules [40].

Current immunosuppressive drugs, though effective in suppressing effector immune responses, can also diminish the regulatory pathways crucial for establishing tolerance, such as those mediated by Tregs. Certain drugs, such as calcineurin inhibitors, reduce the prevalence of Tregs, whereas others like rapamycin can increase their numbers. Immunomodulatory drugs such as the co-stimulation blocker CTLA-4Ig (belatacept) can inhibit effector T-cell activation but may also disrupt Treg function at high doses.

To induce a more selective and sustainable tolerance, some strategies have been explored over the past 2 decades. The use of adoptive Treg therapy aims to enhance the recipient’s immune regulation but requires careful balancing to prevent overpowering by donor-reactive effector T cells. Emerging evidence suggests that combining Treg therapy with other methods might provide a more reliable and durable immune tolerance, potentially transforming transplantation medicine by significantly reducing the need for global immunosuppression [41].

As the demand for organ transplants continues to exceed supply in the USA, xenotransplantation emerges as a promising solution. This technique involves transplanting cells or organs across species, potentially offering an unlimited source of lifesaving organs. Pigs are considered the most suitable donor species due to advancements in genetic modifications that minimize immune rejection issues. Notably, pig kidneys genetically altered to lack the alpha-gal epitope – responsible for rapid transplant rejection – have shown promising results in nonhuman primates, with survival extending beyond a year. This approach is further enhanced by the development of “thymokidneys,” pig thymus tissue combined with kidney transplants, which helps in promoting immune tolerance in recipients. Initial tests in human recipients showed potential [42].

Xenotransplantation represents a potential frontier in beta cell replacement, employing pancreatic tissues or cells from genetically engineered nonhuman sources. This approach is underscored by advancements in immunomodulation and genetic engineering, aiming to curtail immunogenic rejection and zoonotic transmissions, thus broadening the therapeutic potential of this modality.

Recent findings have demonstrated prolonged diabetes reversal in nonhuman primates following intraportal xenotransplantation of wild-type porcine islets. In two studies, diabetes was reversed in cynomolgus macaques after the transplantation of cultured porcine islets, with reasonably long graft survival (Fig. 2). The immunosuppressive regimens used in these studies effectively minimized indirect T-cell activation and reduced pig-specific antibody responses, allowing the islets to maintain their function. However, despite preventing graft rejection, chronic systemic inflammation persisted in many recipients, indicated by elevated neutrophil-to-lymphocyte ratios and increased levels of IL-6, MCP-1, CD40, and CRP, which were associated with severe adverse events [43, 44].

As these modalities continue to evolve, their integration into clinical practice requires understanding of their respective biological intricacies, immunological challenges, and potential for patient-specific approaches. Ongoing research and clinical trials are fundamental in refining these interventions, ensuring their safety and efficacy for broader application in patients with diabetes. Table 2 summarizes the beta cell replacement approaches.

To address the limitations arising from the shortage of donor organs for transplantation, significant research has focused on using stem cells, specifically induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), as alternative sources of beta cells. These pluripotent cells have the remarkable capacity to self-renew indefinitely and differentiate into any cell type, offering a promising avenue for regenerative medicine aimed at creating insulin-producing beta cells from an unlimited cell source [45].

The process of generating functional beta cells from hESCs uses a differentiation process that mimics natural pancreatic development, starting with the induction of definitive endoderm and progressing through stages where cells gradually express key pancreatic progenitor markers like PDX1 and NGN3. The final stage involves the maturation of these progenitors into insulin-producing beta cells, marked by the expression of crucial beta cell markers such as MAFA and NKX6 [46] (Fig. 3). Achieving functional maturity requires a highly controlled environment, often enriched with specific growth factors and a glucose-rich milieu [47].

One can reprogram an individual’s cells by taking cells (e.g., liver cells or fibroblasts) and reprogramming them to a pluripotent state and then reprogramming them to a differentiated state. This autologous approach involves using factors that induce a return to an embryonic-like state, enabling the cells to differentiate into any cell type, including pancreatic beta cells. This method, known as transdifferentiation, sidesteps many of the immune rejection issues commonly associated with allogeneic transplants as the cells are patient-specific [48].

In contrast, generic stem cells derived from hESCs or a bank of iPSCs offer advantages in scalability and commercial viability as these can be uniformly produced and applied across multiple patients. Despite the potential of stem cells, translating these into clinically viable treatments presents its own set of challenges. The differentiation of iPSCs and hESCs into fully functional, mature beta cells that accurately mimic physiological insulin production and glucose regulation is highly complex. Achieving consistency in the quality and functionality of lab-generated islets involves overcoming numerous hurdles. These include mastering the intricate cues for cell differentiation, ensuring the reproducibility of the differentiation process, and eliminating the risk of tumorigenicity. Furthermore, lab-generated islets must demonstrate robust and sustained functionality in glucose sensing and insulin secretion under physiological conditions, a milestone that has proven challenging [49].

The clinical application of stem cell-derived islets involves overcoming four main steps:

• (1)

  Generating highly functional stem cell islets that can effectively mimic the regulated insulin secretion of natural pancreatic islets: The functional integrity of the differentiated islets is crucial. For successful transplantation, the derived islets must exhibit sufficient and appropriate insulin secretion, which includes both the first and second phases of glucose-stimulated insulin secretion. Achieving this requires not only the correct differentiation of beta cells but also maintaining intact islet paracrine relationships, particularly between beta cells and alpha cells. This is essential because in therapeutic scenarios such as treating patients with recurrent severe hypoglycemia, the glucagon response must be adequately restored to prevent such episodes. Efforts to create pure beta cell preparations have been insufficient due to the lack of these crucial intercellular relationships. Furthermore, gene expression and chromatin accessibility within these islets must be precisely regulated to prevent off-target effects, such as teratoma formation or other undesirable outcomes. This adds another layer of complexity to developing stem cell-derived therapies that are both safe and effective.

• Previous study explored the generation of functional human pancreatic β-cells from stem cells in vitro, presenting a potential revolutionary cell source for both diabetes treatment and drug discovery. Despite prior attempts, insulin-producing cells derived from hPSCs often lacked key functional characteristics. Researchers introduced a scalable differentiation protocol that successfully produces hundreds of millions of glucose-responsive β-cells from hPSCs. These cells not only express mature β-cell markers but also respond to glucose by increasing calcium influx and secreting insulin comparable to adult β-cells. Significantly, when transplanted into diabetic mice, these cells promptly secreted insulin in response to glucose and effectively mitigate hyperglycemia, demonstrating their potential in diabetes therapy [50].

• (2)

  Immune protection to prevent rejection of transplanted cells, which might require encapsulation techniques or genetic engineering to enhance immune compatibility: Immune protection of the stem cell-derived islets is another critical challenge. Ideally, treatments would avoid or minimize the use of immunosuppressive drugs, potentially through the use of innovative strategies such as encapsulation. Encapsulation technology, however, introduces its own set of challenges, including ensuring adequate oxygenation and vascularization of the cells, avoiding fibrosis around the encapsulation device, and allowing proper nutrient and insulin exchange. Recent advancements are exploring the use of immunomodulating biomaterials and gene-edited hypoimmune stem cell islets, which may offer new ways to enhance the biocompatibility and effectiveness of these treatments.

• (3)

  Effective transplantation techniques that ensure the islets connect sufficiently with blood vessels and nerves to function properly long-term: The optimal dose and placement of stem cell-derived islets need careful consideration to maximize therapeutic efficacy. Historically, islets have been transplanted into the liver via the portal vein due to its accessibility and the initial success of islet engraftment, but several immunologic, anatomical, and physiological drawbacks contribute significantly to early graft loss., Alternative sites have been tested in experimental animal models and in some clinical settings, including the pancreas, gastric submucosa, genitourinary tract, muscle, omentum, bone marrow, kidney capsule, peritoneum, anterior eye chamber, testis, and thymus [51]. Each site is analyzed for its potential to minimize early inflammatory reactions, support revascularization, and provide an immune-privileged environment conducive to long-term islet function. Despite the exploration of these sites, few have moved into clinical practice, indicating a need for more research to establish the ideal transplantation site that overcomes both early and late challenges of islet transplantation. The omentum, e.g., offers an attractive site due to its vascular nature and direct connection to the portal circulation, potentially improving the insulin dynamics posttransplantation.

• Furthermore, islets derived from these stem cells have shown superior vascular and neural engraftment when compared to traditional human islet transplants [52]. This enhanced integration is critical for the long-term viability and functionality of transplanted cells, suggesting that iPSC-derived islets could provide a more effective and sustainable solution for managing diabetes. By improving integration within the host’s body, these stem cell-derived islets could potentially revolutionize the efficacy of beta cell replacement.

• (4)

  Scalable manufacturing to produce a consistent and reliable supply of functional islets, which involves sophisticated bioreactor technologies and process engineering to maintain cell quality and viability on an industrial scale.

• Manufacturing process for these therapies must be scalable to meet clinical demand. This involves producing a sufficient number of cells per batch, fully characterizing these cells to ensure quality and safety, and enabling cryopreservation for wider distribution. Attention must also be given to genetic stability, avoiding mutations that may pose safety risks and compromise the therapeutic integrity of the cells.

Additionally, patient-specific stem cell strategies represent a cutting-edge approach in regenerative medicine. Tailored to address the genetic and molecular causes of an individual’s condition, such approaches are particularly valuable in diabetes, where iPSCs are used to study why beta cells fail and to determine the most effective therapeutic strategies for that individual. Employing patient-specific iPSCs allows researchers to create model systems to enhance understanding of disease mechanisms, screen for potential drugs, and develop more effective, personalized treatments.

The integration of advanced technologies such as 3D bioprinting, microfluidics, and genetic engineering has the potential to enhance the maturation and functionality of stem cell-derived beta cells. However, rigorous clinical trials are essential to ensure that these lab-generated islets are safe and effective for long-term clinical use. As such, the field continues to evolve with a focus on optimizing these technologies to meet clinical needs.

The development of stem cell derived from human islets has taken a step forward with the creation of a current Good Manufacturing Practice (cGMP) compliant iPSC variant. These cells are fully characterized for clinical use and are under evaluation for therapeutic application. One of the critical advancements in this regard is the ability to produce large-scale, reproducible, and consistent batches of islets with 95–100% purity. This high level of purity is crucial for clinical applications where uniformity in cell product is necessary to ensure predictable therapeutic outcomes. Notably, these manufacturing processes do not require cell sorting or selection, which simplifies production and reduces costs.

The iPSC-derived islet clusters generated through this technology contain a high percentage of insulin-secreting beta cells, typically ranging from 60 to 90%. This high proportion of functional beta cells is essential for effective diabetes management as these cells play a critical role in regulating blood glucose levels.

A significant advantage of this new stem cell variant is that its production process involves no use of viruses or integrating plasmids, which are common in some other types of cell therapy manufacturing. This omission greatly reduces the risk of insertional mutagenesis, a concern where foreign DNA may disrupt normal genetic processes, potentially leading to complications. The development of this iPSC variant for the generation of pancreatic islets marks a promising direction in regenerative medicine, offering a potentially limitless supply of essential beta cells for diabetes treatment.

A method has been developed to map the molecular steps involved in transforming stem cells into islet-like cells capable of producing insulin. By utilizing single-cell RNA sequencing, researchers analyzed over 100,000 cells at various stages of differentiation, from stem cells to pancreatic progenitor cells and finally to hormone-producing cells. This detailed profiling enabled the construction of a comprehensive roadmap of the differentiation process, identifying the specific conditions that favor the development of insulin-producing β-cells [53].

Beyond insulin-producing cells, the grafts contain a variety of other cell types. Recognizing the importance of the microenvironment that surrounds the islets, there is an understanding that creating a cellular composition, which mimics natural islets more closely, is essential, including endogenous islet structure, cell-cell contacts, and communication with the islet environment. This supports the idea that effective diabetes therapy requires a reconstruction of the entire islet architecture, not solely focusing on beta-cells, contributing with prevention of hypoglycemic events and paracrine regulation needed for the glycemic homeostasis [54, 55].

While iPSC-derived differentiated cells have the potential to provide an unlimited cell supply, the induction process carries the risk of contamination by undifferentiated or off-target cells. Recent research has highlighted the emergence of proliferative off-target cells, termed proliferative mesenchymal stem cells (PMSCs), after the implantation of iPSC-derived pancreatic islet cells. These PMSCs can proliferate unexpectedly and pose significant risks, including tumorigenesis. The study developed an in vitro detection system and effective removal methods for PMSCs, enhancing the safety of clinical applications of PSC-derived islet-like cells [56] (Table 3).

The field of stem cell therapy has seen transformative advancements, driven by innovative technologies aimed at improving the efficacy, safety, and scalability of treatments. These technologies encompass a variety of approaches, including the precise reprogramming of cells, the development of immune-evasive stem cell lines, advanced gene editing, and detailed molecular profiling techniques. As shown in Table 4, the challenges and opportunities with stem cell islets present significant considerations for future research and application.

An approach harnessed stem cell technology to create a renewable source of functional islet cells derived from hESCs. These cells were engineered to replicate the insulin-producing capabilities of natural pancreatic cells, potentially restoring autonomous glycemia regulation in patients with diabetes. The integration of advanced cryopreservation techniques ensures the long-term viability and stability of the islet cells, allowing for extensive quality control testing and reliable transport to treatment centers globally. Additionally, non-immunosuppressive delivery methods, such as cutting-edge encapsulation technologies, localized delivery of immunomodulatory drugs, and genetic engineering, are being explored to shield the transplanted cells from the immune system without the associated risks and side effects of immunosuppression.

HLA compatibility is crucial for stem cell-derived β-cell transplantation as mismatches can result in graft rejection. Advancements in gene editing provide opportunities to enhance the immune evasion capabilities of stem cell-derived β-cells [57, 58]. Technologies like the creation of hypoimmune pluripotent stem cells show promise in overcoming immune rejection without the need for immunosuppressive drugs. These innovations enhance the safety and effectiveness of stem cell therapies, paving the way for universal, off-the-shelf therapeutic products. For example, hypoimmune pluripotent stem cells are engineered by depleting HLA class I and II molecules and overexpressing CD47, allowing them to evade immune rejection even in fully immunocompetent recipients [59].

Genome editing is achieved by various methodologies like transcription activator-like endonucleases (TALEN), zinc finger nucleases (ZFN), and the clustered regularly interspaced short palindromic repeats (CRISPR) Cas (CRISPR-associated) enzymes [60, 61]. By editing specific genes related to immune recognition, these modified cells can potentially evade detection by the host immune system [62, 63]. This approach aims to reduce the likelihood of rejection and improve the longevity and function of the transplanted cells.

As a novel approach, researchers have engineered SC-islet cells to secrete cytokines IL-10, transforming growth factor β (TGF-β), and a modified version of IL-2, which recruit Tregs to the islet grafts, fostering a tolerogenic microenvironment. This adaptation enabled cytokine-secreting human SC-β-cells to resist xeno-rejection and effectively correct diabetes for up to 8 weeks posttransplantation in NOD mice, highlighting the potential of this method to deliver SC-islet cells for diabetes treatment without the need for encapsulation or ongoing immunosuppression [64].

The development and implementation of implantable devices that can house transplanted cells offer a promising avenue to address some of these challenges. Such devices are designed to encapsulate islets, potentially shielding them from the immune response without the need for systemic immunosuppression while allowing proper vascularization and function.

Effective delivery devices are essential for the transplantation of insulin-producing cells. These devices must facilitate the precise placement of cells in targeted anatomical sites, ensuring optimal cell survival and function. Advanced delivery devices are designed to minimize mechanical stress during transplantation, provide a supportive microenvironment for the cells, and enable easy retrieval if necessary.

Subcutaneous delivery involves implanting the beta cells under the skin, an area readily accessible and minimally invasive, making it a possible option for cell transplantation. Open delivery systems are designed to promote vascularization, crucial for the survival and function of the transplanted cells. These systems allow direct contact between the host’s blood vessels and the implanted cells, facilitating the necessary exchange of oxygen, nutrients, and insulin. Conversely, closed delivery systems provide immune protection by encasing the beta cells in a semipermeable barrier, protecting them from the immune response while still permitting the diffusion of insulin and glucose. This design is critical for preventing immune rejection without the need for continuous immunosuppressive therapy. Each component plays a significant role in advancing the field of beta cell replacement.

However, the design and functionality of these devices still bring additional challenges, including ensuring adequate nutrient and oxygen supply to the encapsulated cells and managing the immune response, which can lead to fibrosis and impair device function. A multigenerational family of products was designed for beta cell replacement, focusing on different stages of development and targeting various needs within the diabetic population. These three products – PEC-Direct, PEC-Encap, and PEC-QT – utilize PEC-01 cells with differing delivery mechanisms to optimize therapeutic outcomes. PEC-Direct is clinically targeted at high-risk T1D patients and employs an open device facilitating direct vascularization, necessitating long-term immunosuppression to prevent immune rejection. PEC-Encap advances the technology by incorporating the Encaptra device, which allows cell surface diffusion to manage glucose levels without requiring immunosuppression, potentially broadening its application to all T1D and insulin-requiring T2D patients. PEC-QT, still in the discovery stage, employs immune-evasive PEC-01 cells in an open device setup, promising direct vascularization with an anticipated elimination of long-term immunosuppression needs, aiming to universally treat all T1D and insulin-requiring T2D patients.

Pepper et al. [65] investigated the long-term efficacy of PECs derived from hESCs in maintaining normoglycemia posttransplant. Mice with induced diabetes who received PEC transplants into a subcutaneous device-less site were able to maintain normoglycemia up to the day of graft retrieval. PEC grafts excised 365 days posttransplant possess similar dynamic glucose-regulated insulin secretory capacity compared with adult human islets. Notably, small intragraft cysts, palpable in all mice, resolved but recurred post-aspiration, showing monomorphic neuroendocrine proliferation and ductal epithelium lining. The study confirmed minimal proliferation and the absence of neoplastic changes within the grafts throughout the evaluated period.

Encapsulation techniques using permeable hydrogels have also been studied for their potential to eliminate the need for systemic immunosuppression and restore insulin production in patients with T1D. One method is the emulsion cross-linking technique. This method involves the conformal coating of islets with a hydrogel at physiological pH, enhancing cell viability and insulin secretion performance. This method achieved hydrogel coatings around islets through a process that maintains the biocompatibility and cytocompatibility of the encapsulated cells [66].

Despite significant progress, clinical success remains limited due to challenges such as biocompatibility, inflammatory responses, and fibrosis at the transplant site. Recent advancements have focused on reducing the size of encapsulation capsules to minimize the inflammatory effect while ensuring adequate nutrient and insulin exchange.

Transforming the treatment of T1D through beta cell replacement therapies embodies a promising frontier fraught with scientific challenges and ethical dilemmas, demanding a nuanced balance between the innovation and the reality of clinical application. Yet, the advances in immune protection (e.g., encapsulation), stem cell technology, gene editing, and cellular engineering have brought us closer to creating functional, immune-evasive beta cells that can be transplanted without the need for lifelong immunosuppression.

The detailed molecular profiling of the differentiation process from stem cells to insulin-producing beta cells has provided invaluable insights, enabling the construction of comprehensive roadmaps that guide the optimization of these processes. The successful implementation of beta cell replacement therapies will also depend on overcoming the technical challenges associated with scalable manufacturing, ensuring adequate vascularization and oxygenation of transplanted cells, and refining transplantation techniques to promote long-term cell viability and function.

With the significant progress made, rigorous clinical trials are essential to translate these scientific advancements into viable clinical solutions. The journey from bench to bedside is complex and requires a balanced approach that manages patient expectations while fostering continued research and development.

In summary, while the path to effective beta cell replacement therapy is challenging, the strides made thus far are encouraging. Continued innovation and collaboration within the scientific community are critical to realizing the full potential of these therapies, ultimately aiming to provide durable reversal of diabetes and improvement in the quality of life for millions of patients worldwide.

J.R.N.L. has no conflicts of interest to declare. J.S.S. has been an advisor to 4Immune, AbbVie, Abvance, ActoBiotics, Adocia, Aerami/Dance Biopharma, AiTA, Altheia, Applied Therapeutics, Arecor, AstraZeneca, Avotres, Bayer, Biomea Fusion, Catalyst Bio, COUR Pharmaceuticals, Dexcom, Diasome, Dompe, Eli Lilly, Enthera, Imcyse, Immunomolecular Therapeutics, Kriya Therapeutics, Levicure, Novo Nordisk, Oramed, Orgenesis, Provention Bio, Quell Therapeutics, RegCell, Remedy Plan Inc., Sanofi, Shoreline Biosciences, Signos, Vertex, ViaCyte, vTv Therapeutics, and WiNK. He is a member of the Board of Directors of Applied Therapeutics and SAB Biotherapeutics. J.S.S. is Chair of the Strategic Advisory Board of the EU EDENT1FI consortium. J.S.S. has equity in 4Immune, Abvance, AiTA, Applied Therapeutics, Avotres, Dexcom, Immunomolecular Therapeutics, Oramed, Orgenesis, Signos, vTv Therapeutics, and WiNK.

The authors are supported by the Diabetes Research Institute Foundation, which had no role in this paper.

J.R.N.L. and J.S.S. worked together in reviewing the literature and writing and editing the paper.