Diabetes mellitus is currently a priority health issue worldwide, but existing therapies suffer from insufficient donors, inability to provide glucose-dependent endogenous insulin secretion, transplantation risks, and immune rejection. Especially, reported engineered cells are mostly promoter-induced glucose-independent insulin producing cells. Here we constructed a closed-loop of insulin secretion with glucose-dependent IRES to achieve glucose-sensitive endogenous insulin secretion. Those cells successfully reversed hyperglycemia in diabetic mice for at least 60 days after transplantation without any significant immune rejection, demonstrating that our constructed engineered cellular grafts have good biocompatibility. Our findings hold great promise in the field of diabetes treatment and provide a new, glucose-dependent genetic engineering approach to insulin production, which is expected to solve many of the current problems faced in the clinical treatment of diabetes mellitus.

Developments in basic stem cell biology have paved the way for technology translation in human medicine. An exciting prospective use of stem cells is the ex vivo generation of hepatic and pancreatic endocrine cells for biomedical applications. This includes creating novel models 'in a dish' and developing therapeutic strategies for complex diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetes. In this review, we explore recent advances in the generation of stem cell-derived hepatocyte-like cells and insulin-producing β-like cells. We cover the different differentiation strategies, new discoveries, and the caveats that still exist regarding their routine use. Finally, we discuss the challenges and limitations of stem cell-derived therapies as a clinical strategy to manage metabolic diseases in humans.

Type 1 diabetes (T1D) remains a significant global health challenge and patients with T1D need lifelong insulin therapy. Islet transplantation holds transformative potential by replacing autoimmune-mediated destruction of insulin-producing beta cells. This review examines the trajectory of islet transplantation for T1D, focusing on the process and benefits of obtaining biologics license application (BLA) approval for cell-based therapies. Following US Food and Drug Administration (FDA) approval, the authors identify key steps urgently needed to foster islet transplantation as a viable treatment for a broader population of patients with T1D. Furthermore, the authors highlight recent advances in encapsulation technologies, stem cell-derived islets, xenogeneic islets, and gene editing as strategies to overcome challenges such as immune rejection and limited islet sources. These innovations are pivotal in enhancing the safety and efficacy of islet transplantation. Ultimately, this review emphasizes that while BLA approval represents a critical milestone, realizing the full potential of cell therapy for T1D requires addressing the scientific, clinical, and logistical challenges of its real-world implementation. By fostering innovation, collaboration, and strategic partnerships, the field can transform T1D care, offering patients a durable, life-changing alternative to traditional insulin therapy.

Advanced protocols show potential for human stem cells (SC)-derived islets generation under planar (2D) alone or three-dimensional (3D) cultures, but show challenges in scalability, cell loss, and batch-to-batch consistency. This study explores Vertical Wheel (VW)® bioreactor suspension technology to differentiate islets from human induced pluripotent stem cells, achieving uniform, transcriptionally mature, and functional SC-islets. A 5x increase in scale from 0.1 L to 0.5 L reactors resulted in a 12-fold (15,005-183,002) increase in islet equivalent count (IEQ) without compromising islet structure. SC-islets show enriched β-cell composition (~63% CPPT+NKX6.1+ISL1+), glucose responsive insulin release (3.9-6.1-fold increase), and reversed diabetes in STZ-treated mice. Single cell RNA sequencing and flowcytometry analysis confirmed transcriptional maturity and functional identity, similar to adult islets. Lastly, harvested SC-islet grafts demonstrate improved islet functionality and mature transcriptomic signatures. Overall, scale-up in VW® bioreactor technology enhances IEQ yield with minimal variability and reduced cell loss, offering a pathway for clinical-grade SC-islet production.

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes, but its efficacy is hindered by low oxygen supply post-transplantation, particularly in subcutaneous spaces and encapsulation devices, leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here, we show that β cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1, FOS, and JUN), which downregulates key β cell transcription factors. We further identified genes important for maintaining β cell fitness in hypoxia, with EDN3 as a potent player. Elevated EDN3 expression preserves β cell identity and function in hypoxia by modulating genes involved in β cell maturation, glucose sensing and regulation. These insights improve the understanding of hypoxia's impact on stem cell-derived islets, offering a potential intervention for clinical applications.

This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.

Pancreatic endocrine cells are categorized in to 5 types (alpha, beta, delta, pancreatic polypeptide cells and epsilon), which expresses glucagon, insulin, somatostatin, pancreatic polypeptide, and ghrelin, respectively. Several studies including lineage tracing in Ins2Akita diabetic mice have been done to investigate the identities of pancreatic endocrine cells which concludes, alpha cells have enormous plasticity, which enables them to be reprogrammed by specific transcription factors into insulin secreting beta like cells. Gene therapy has provided the beneficial outcome. Pdx1, MaFA and PAX4 (the transcription factors) in alpha cells can be over expressed which results in reprogramming the targeted alpha cells into beta cells. This trans-differentiation may be induced by infusing an adeno-associated virus (AAV) loaded with distinct transcription factors in the duct of pancreas. Several researches have demonstrated the successful restoration of enhanced insulin secretion in diabetes induced mice. Additionally ductal neurogenin3 (Ngn3), Sglt2 inhibitors, Igfbp1, GLP1 and several clinical and non-clinical agents has been postulated as a basis of beta cell neogenesis. Alpha cell owing to its high plasticity, on prolonged exposure to GABA reprogrammed into beta-like cell due to downregulation of Arx expression by GABA. The various approaches for beta cell neogenesis open a new window towards the establishment of novel gene therapy accession to treat diabetes. However, broad studies are still needed to improve and optimize this treatment methodology. The potentiality of endogenous pancreatic alpha cell to beta cell conversion methods and its outcomes are invigorating. This accomplishment is presently being under trial in non-human primates.

Here, we present a protocol to generate key pancreatic cell types in vitro using human pluripotent stem cell (hPSC)-based Matrigel-overlay organoid differentiation. These include multipotent and bipotent progenitors, endocrine progenitors, and hormone-producing endocrine cells. We describe steps for culturing hPSCs as a 2D monolayer, applying a Matrigel overlay to create a 3D epithelial niche, and guiding stepwise differentiation. This system supports live-cell imaging and real-time tracking of morphogenesis and fate decisions, providing a platform for studying organ development and disease. For complete details on the use and execution of this protocol, please refer to Ulf et al.1.

Stem cell-derived islets (SC-islets) consist of multiple hormone-producing cell types and offer a promising therapeutic avenue for treating type 1 diabetes (T1D). Currently, the composition of cell types generated within these SC-islets cannot be controlled via soluble factors during this differentiation process and consist of off-target cell types. In this study, we devised a magnetic-activated cell sorting protocol to enrich SC-islets for CD49a, a marker associated with functional insulin-producing β cells. SC-islets were generated from human pluripotent stem cells using an adherent differentiation protocol and then sorted and aggregated into islet-like clusters to produce CD49a-enriched, CD49a-depleted, and unsorted SC-islets. Single-cell RNA sequencing (scRNA-seq) and immunostaining revealed that CD49a-enriched SC-islets had higher proportions of β cells and improved transcriptional identity compared to other cell types. Functional assays demonstrated that CD49a-enriched SC-islets exhibited enhanced glucose-stimulated insulin secretion both In vitro and In Vivo following transplantation into diabetic mice. These findings suggest that CD49a-based sorting significantly improves β cell identity and the overall function of SC-islets, improving their effectiveness for T1D cell replacement therapies.

Inducing the neogenesis of pancreatic insulin-producing β cells holds great promise for diabetes research. However, non-toxic compounds with such activities remain to be discovered. Herein, we report the identification of RSPO1, a key agonist of the Wnt/β-catenin pathway, as an inducer of β cell replication. Specifically, we provide evidence that RSPO1 promotes a significant increase in β cell neogenesis in vitro, ex vivo, and in vivo. Importantly, RSPO1 administration is sufficient to activate Wnt/β-catenin signaling in β cells and counter chemically induced or autoimmune-mediated diabetes. Similarly, an optimized analog of RSPO1, allowing for weekly administration, also prevents diabetes in vivo. Lastly, the treatment of transplanted human islets with RSPO1 induces a significant 2.78-fold increase in human β cell numbers in only 60 days, these cells being functional. Such activities of RSPO1 to promote β cell neogenesis could therefore represent an unprecedented hope in the continued search for diabetes alternative therapies.

Blood vessels play a critical role in pancreatic islet function, yet current methods for deriving islet organoids from human pluripotent stem cells (SC-islets) lack vasculature. We engineered three-dimensional (3D) vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs), and fibroblasts in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-β cells, a hallmark of β cell function that is blunted in non-vascularized SC-islets. Moreover, vascularization accelerated diabetes reversal post engraftment of a subtherapeutic SC-islet dose into mice. We show that vasculature leads to the formation of an islet-like basement membrane that contributes to the functional improvement of SC-β cells. Furthermore, single-cell RNA sequencing (scRNA-seq) data predicted BMP2/4-BMPR2 signaling from ECs to SC-β cells, and correspondingly, BMP4 enhanced the SC-β cell Ca2+ response and insulin secretion. Vascularized SC-islets will enable further studies of crosstalk between β cells and ECs and will serve as an in vitro platform for disease modeling and therapeutic testing.

Despite recent advancements in technology for the treatment of type 1 diabetes (T1D), exogenous insulin delivery through automated devices remains the gold standard for treatment. This review will explore progress made in pancreatic islet bioengineering within the field of beta-cell replacement for T1D treatment.

First, we will focus on the use of decellularized extracellular matrices (dECM) as a platform for pancreatic organoid development. These matrices preserve microarchitecture and essential biochemical signals for cell differentiation, offering a promising alternative to synthetic matrices. Second, advancements in 3D bioprinting for creating complex organ structures like pancreatic islets will be discussed. This technology allows for increased precision and customization of cellular models, crucial for replicating native pancreatic islet functionality. Finally, this review will explore the use of stem cell-derived organoids to generate insulin-producing islet-like cells. While these organoids face challenges such as functional immaturity and poor vascularization, they represent a significant advancement for disease modeling, drug screening, and autologous islet transplantation.

These innovative approaches promise to revolutionize T1D treatment by overcoming the limitations of traditional therapies based on human pancreatic islets.

β-Cell replacement for type 1 diabetes (T1D) can restore normal glucose homeostasis, thereby eliminating the need for exogenous insulin and halting the progression of diabetes complications. Success in achieving insulin independence following transplantation of cadaveric islets fueled academic and industry efforts to develop techniques to mass produce β cells from human pluripotent stem cells, and these have now been clinically validated as an alternative source of regulated insulin production. Various encapsulation strategies are being pursued to contain implanted cells in a retrievable format, and different implant sites are being explored with some strategies reaching clinical studies. Stem cell lines, whether derived from embryonic sources or reprogrammed somatic cells, are being genetically modified for designer features, including immune evasiveness to enable implant without the use of chronic immunosuppression. Although hurdles remain in optimizing large-scale manufacturing, demonstrating efficacy, durability, and safety, products containing stem cell-derived β cells promise to provide a potent treatment for insulin-dependent diabetes.

Diabetes mellitus is a significant and fast-growing health problem worldwide. Cost, donor shortages, and immune rejection limit current treatment strategies. While considerable progress has been made in creating β-cells in vitro with remarkable morphological and functional resemblance to those in primary pancreatic islets, exploring alternative sources for β-cell replacement is crucial. With adult pancreatic stem cells still not conclusively identified, researchers focus their attention on heterogeneity within pancreatic ductal epithelial cells, exploring these cells as a potential source of progenitor cells for pancreatic regeneration and β-cell formation. Recent studies using techniques such as fluorescence-activated cell sorting, immunostaining and single cell RNA-sequencing have identified ductal cell heterogeneity with several subpopulations of ductal cells with progenitor-like properties and their capacity for differentiation into insulin producing cells. Here, we have reviewed the most recent studies on pancreatic ductal cell subpopulations that offer insights into potential stem-cell populations to form β-cells in diabetes treatment.

Immunotherapy is a crucial treatment for type 1 diabetes (T1D), yet analyses focusing on research priorities and trends in this field are limited. Therefore, this study employs bibliometric methods to systematically explore the current research status of immunotherapy for T1D.

Based on the Web of Science Core Collection Database, 1573 articles and review articles related to immunotherapy for T1D published from 2004 to 2023 were screened for bibliometric analysis. VOSviewer, CiteSpace, and R software were applied to comprehensively analyze the number of publications, journals, countries, authors, institutions, keywords, and references.

In the past two decades, the global annual publication rate has seen a significant increase of 238.24%. Almost 40% of all publications have appeared in the last 5 years, accounting for over 50% of total citations. Journals such as Diabetes, Journal of Autoimmunity and Frontiers in Immunology have exerted substantial influence. Collaboration across nations has been notably strong, with the United States leading the way. The University of Florida is the most productive institution. Terms like "nivolumab," "ipilimumab," "pembrolizumab," and "immune checkpoint inhibitor(s)" gain considerable traction. The majority of research has clustered around themes such as immunomodulation, autoimmune diseases, immune checkpoint inhibitors, mesenchymal stem cells, and cell therapy. Precision medicine, immune checkpoint inhibitors, and nanotechnology are trending focal points in contemporary research.

The outcomes of the study are instrumental in enabling scholars to comprehend the evolving trajectory of immunotherapeutic approaches for T1D and facilitate the swift recognition of emerging research pathways.

Beta cell replacement therapy via pancreatic islet transplantation offers a promising treatment for type 1 diabetes as an alternative to insulin injections. However, posttransplantation oxygenation remains a critical challenge; isolated islets from donors lose vascularity and rely on slow oxygen diffusion for survival until revascularization occurs in the host tissue. This often results in significant hypoxia-induced acute graft loss. Overcoming the oxygenation barrier is crucial for advancing islet transplantation. This review is structured in three sections: the first examines oxygen dynamics in islet transplantation, focusing on factors affecting oxygen supply, including vascularity. It highlights oxygen dynamics specific to both transplant sites and islet grafts, with particular attention to extrahepatic sites such as subcutaneous tissue. The second section explores current oxygen delivery strategies, categorized into two main approaches: augmenting oxygen supply and enhancing effective oxygen solubility. The final section addresses key challenges, such as the lack of a clearly defined oxygen threshold for islet survival and the limited precision in measuring oxygen levels within small islet constructs. Recent advancements addressing these challenges are introduced. By deepening the understanding of oxygen dynamics and identifying current obstacles, this review aims to guide the development of innovative strategies for future research and clinical applications. These advancements are anticipated to enhance transplantation outcomes and bring us closer to a cure for type 1 diabetes.

Although originating from different germ layers, pancreatic islet cells and neurons share extensive similarities, both physiological (e.g., voltage-dependent release of a bioactive molecule) and molecular (e.g., highly similar composition of transcription factors and structural genes). Here we propose that two seemingly unrelated phenomena recognized in these cell types-neurotransmitter switching in neurons and the expression of two or more hormones in individual islet cells-share a deep resemblance, potentially reflecting an ancient molecular circuit of cell plasticity. Comparing and contrasting dynamic hormone expression in islet cells and transmitter switching in neurons may provide insights into the functions and underlying mechanisms of these phenomena.

Endothelial cells (ECs) play pivotal roles in the development and maintenance of tissue homeostasis. During development, vasculature actively involves in organ morphogenesis and functional maturation, through the secretion of angiocrine factors and extracellular matrix components. Islets of Langerhans, essential functional units of glucose homeostasis, are embedded in a dense endothelial capillary network. Islet vasculature not only supplies nutrients and oxygen to endocrine cells but also facilitate the rapid delivery of pancreatic hormones to target tissues, thereby ensuring precise glucose regulation. Diabetes mellitus is a major disease burden and is caused by islet dysfunction or depletion, often accompanied by vessel loss and dysregulation. Therefore, elucidating the regulatory mechanisms of ECs within islets hold profound implications for diabetes therapy. This review provides an overview of recent research advancements on the functional roles of ECs in islet biology, transplantation, and in vitro islet organoid culture.

Human induced pluripotent stem cell (hiPSC)-derived insulin-producing β cell therapy shows promise in treating type 1 diabetes and potentially type 2 diabetes. Understanding the genetic factors controlling hiPSC differentiation could optimize this therapy. In this study, we investigated the role of glucose-regulated protein 94 (GRP94) in human β cell development by generating HSP90B1/GRP94 knockout (KO) hiPSCs, re-expressing GRP94 in the mutants and inducing their β cell differentiation. Our results revealed that GRP94 depletion hindered β cell generation by promoting cell death induced by endoplasmic reticulum (ER) stress and other stressors during definitive endoderm (DE) differentiation. Moreover, GRP94 deletion resulted in decreased activation of WNT/β-catenin signaling, which is critical for DE specification. Re-expression of GRP94 in GRP94 KO iPSCs partially reversed DE differentiation deficiency and alleviated cell death. These findings highlight the previously unrecognized indispensable role of GRP94 in human DE formation and consequent β cell development from hiPSCs. GRP94 mitigates ER stress-induced cell death and regulates the WNT/β-catenin signaling pathway, which is both crucial for successful β cell differentiation. These results provide new insights into the molecular mechanisms underlying β cell differentiation from hiPSCs and suggest that targeting GRP94 pathways could enhance the efficiency of hiPSC-derived insulin-producing cell therapies for diabetes treatment.

Type 2 diabetes (T2D) is a chronic disease characterized by long-term insulin resistance (IR) and pancreatic β-cell dysfunction. Conventional T2D medication ignores pancreatic β-cell damage. In this study, we designed an oral glucose-responsive nanoparticle for pancreatic β-cell regeneration and treatment of T2D. It was formed by carboxymethyl chitosan (CMC) grafted with 3-aminophenylboronic acid (APBA) as the shell and small-molecule citrus pectin (MCP) spheres encapsulating artemisinin (Art) connected by borate ester bonds. The prepared CMC-APBA wrapped Art-loaded MCP nanoparticles (CAM@Art) had therapeutic effects for the treatment of IR, antioxidant and promotion of pancreatic α-cell differentiation in vitro experiments. In addition, in vivo experiments showed that CAM@Art could reduce blood glucose, oxidative stress and inflammation levels and reverse IR in diabetic rats. Importantly, pancreatic β-cell regeneration was found in islets in vivo. Mechanistically, CAM@Art promotes pancreatic α-cell differentiation by promoting overexpression of the transcription factor Pax4 and ectopic expression of Arx. The results suggest that the present study provides a promising therapeutic strategy for the treatment of diabetic pancreatic β-cell dysfunction.

The extracellular matrix-F-actin-Yes-associated protein 1 (YAP1)-Notch mechanosignaling axis is a gatekeeper in the fate decisions of bipotent pancreatic progenitors (bi-PPs). However, the link between F-actin dynamics and YAP1 activity remains poorly understood. Here, we identify salt-inducible kinases (SIKs) as mediators of F-actin-triggered changes in YAP1 activity. Interestingly, sodium chloride treatment promotes the differentiation of bi-PPs into NEUROG3+ endocrine progenitors (EPs) through enhanced SIK expression. Consistently, the pan-SIK inhibitor HG-9-09-01 (HG) inhibits latrunculin B (LatB)-induced EP differentiation via nuclear YAP1 accumulation. Unexpectedly, withdrawal of HG after a 12-h treatment increased SIK expression by a negative feedback mechanism, leading to significantly enhanced endocrinogenesis. Therefore, the combined treatment of bi-PPs with LatB and HG for 12 h boosted endocrinogenesis, ultimately leading to an increased number of beta cells. In summary, we identify SIKs as new transducers of mechanotransduction-triggered induction of pancreatic endocrine cell fates.

Gene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes, NDM) can provide unique insights into the development and function of human pancreatic beta-cells. We describe the identification of homozygous PAX4 loss-of-function variants in 2 unrelated individuals with NDM: a p.(Arg126*) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126*) variant causes nonsense mediated decay in CRISPR-edited human induced pluripotent stem cell (iPSC)-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN and RNA-sequencing in PAX4-depleted islet cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion. Both probands had transient NDM which remitted in early infancy but relapsed between the ages of 2 and 7 years, demonstrating that in contrast to mouse models, PAX4 is not essential for the development of human pancreatic beta-cells.

Diabetes is a complex disease affecting over 500 million people worldwide. Traditional approaches, such as insulin delivery, are mainstay treatments, but do not cure the disease. Recent advances in biofabrication and synthetic biology offer new hope for the development of tissue constructs recapitulating salient organ functions. Here, we discuss recent progress in bioprinting a functional endocrine pancreas, ranging from cell sources to main advances in biomaterials. We review innovative areas for the development of this field, with a particular focus on the convergence of synthetic biology and cell engineering with bioprinting, which opens new avenues for developing advanced in vitro models and regenerative, transplantable grafts, with the potential to provide independence from exogenous insulin administration.

Human pluripotent stem cell-derived islet (SC-islet) transplantation is a promising β cell replacement therapy for patients with type 1 diabetes, offering a potential unlimited cell supply. Yet, the heterogeneity of the final cell product containing non-target cell types has relevant implications for SC-islet function, transplant volume, and cell product safety. Here, we present a clinically compliant, full three-dimensional differentiation protocol that includes a purification step of endocrine cell-rich clusters, relying on the principle of isopycnic centrifugation (density gradient separation). Enriched SC-islets displayed signs of functionality in vitro and in vivo. In contrast with antibody-based single-cell sorting approaches, this method does not destroy the islet cytoarchitecture associated with alterations of islet function and cell loss. Furthermore, it is fast, is easily scalable to large cell volumes, and can be applied during cell manufacturing. This method may also contribute to the generation of improved cell-based therapies for regenerative medicine purposes beyond the SC-islet field.

Metformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti-diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)-derived pancreatic islet-like spheroid (SC-islet) models.

Human iPSCs are differentiated into SC-islets by three-dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC-islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC-islets. SC-islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP.

Metformin perturbed SC-islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF-β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin.

Imeglimin does not perturb differentiation of SC-islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.

Beta cell replacement therapy for type 1 diabetes (T1D) is undergoing a transformative shift, driven by advances in stem cell biology, gene editing, and tissue engineering. While islet transplantation has demonstrated proof-of-concept success in restoring endogenous insulin production, its clinical impact remains limited by donor scarcity, immune rejection, and procedural complexities. The emergence of stem cell-derived beta-like cells represents a paradigm shift, with initial clinical trials showing promising insulin secretion in vivo. However, translating these breakthroughs into scalable, widely accessible treatments poses significant challenges. Drawing parallels to space exploration, this paper argues that while scientific feasibility has been demonstrated, true accessibility remains elusive. Without a strategic shift, beta cell therapy risks becoming an elite intervention, restricted by cost and infrastructure. Lessons from gene and cell therapies for rare diseases highlight the dangers of unsustainable pricing and limited market viability. To bridge the "last mile" a Quality by Design approach is proposed, emphasizing scalability, ease of use, and economic feasibility from the outset. By emphasizing practical implementation over academic achievements, corporate interests, market economics, or patent constraints, beta cell therapy can progress from proof-of-concept to a viable, widely accessible treatment.

Diabetes mellitus is characterized by the loss of pancreatic insulin-secreting β-cells in the Islets of Langerhans. Understanding the regenerative potential of human islet cells is relevant in the context of putative restoration of islet function after damage and novel islet cell replacement therapies. Adult human pancreatic tissue can be cultured as three-dimensional organoids with the capacity for long-term expansion and the promise of endocrine cell formation. Here, we characterize the endocrine differentiation potential of human adult pancreatic organoids. Because exocrine-to-endocrine differentiation is dependent on the expression of Neurogenin 3 (NEUROG3), we first generated NEUROG3-inducible organoid lines. We show that doxycycline-induced NEUROG3 expression in the organoids leads to the formation of chromogranin A positive (CHGA+) endocrine progenitor cells. The efficiency of this differentiation was improved with the addition of thyroid hormone T3 and the AXL inhibitor R428. Further, compound screening demonstrated that modifying the pivotal embryonic endocrine pancreas signalling pathways driven by Notch, YAP, and EGFR led to increased NEUROG3 expression in organoids. In a similar fashion to embryonic development, adult ductal cells delaminated from the organoids after NEUROG3 induction. Thus, mechanisms in islet (re)generation including the initiation of endocrine differentiation and delamination can be achieved by NEUROG3 induction.

Innovative solutions have entered the routine management of patients with type 1 diabetes or are making the headlines and this is shaking the world of beta cell replacement therapies. Above all, allogeneic islet transplantation is enthusiastically doomed to extinction by the aficionados of "closed loop" artificial insulin delivery systems or those convinced of the imminent large scale availability of stem-cell derived insulin-producing tissues. This opinion paper will propose that neither will be a universal solution in the very near future and will argue that xenogeneic islet transplantation may be a serious outsider in the race for new therapies. In the meantime, the odds are in favor of allogeneic islet (and pancreas) transplantation remaining first line options in the treatment of complicated type 1 diabetes. There is no question that "closed loop" systems have already greatly improved the management of type 1 diabetes, but, while "unlimited" sources of insulin-producing cells are jockeying for approval as standard-of-care, these improvements are more likely to drive a shift of indications -from islet transplant alone to simultaneous islet-kidney transplantation- than to herald the demise of islet transplantation.

Type 3 iodothyronine deiodinase (Dio3) converts triiodothyronine (T3) to diiodothyronine, thereby reducing intracellular T3 levels. In this study, we investigated the potential roles of Dio3 in the differentiation of human pancreatic β cells, using β cells derived from human induced pluripotent stem cells (hiPSCs).

hiPSCs were differentiated to β cells in a stepwise manner over 29 days. The differentiation medium was supplemented with B27, which contains T3 but not T4, instead of serum. The T3 levels in the differentiated cells were determined based on the amount of T3 supplied to the medium and the activity of Dio3 within the cells. Iopanoic acid (IOP) was used as the Dio3 inhibitor.

Dio3 expression is substantially altered during differentiation. IOP treatment reduced Dio3 activity on day 4 and increased T3 levels in the medium on day 29. To investigate the involvement of Dio3 during differentiation, we used IOP, in which cells differentiated in the presence of IOP (+IOP) were compared to those differentiated without IOP (-IOP). On day 29, the proportion of β cells expressing C-peptide, NKX6 homeobox 1, and both markers was considerably higher in the presence than in the absence of IOP. Furthermore, on day 29, the insulin content of differentiated + IOP cells was considerably higher than that of differentiated -IOP cells.

An increase in intracellular T3 content promoted via the inhibition of Dio3 activity by IOP from day 0-29 enhances the differentiation of hiPSCs to β cells.

In primary cell types, intracellular deoxynucleotide triphosphate (dNTP) levels are tightly regulated in a cell cycle-dependent manner. We report that prime editing efficiency is increased by mutations that improve the enzymatic properties of Moloney murine leukemia virus reverse transcriptase and treatments that increase intracellular dNTP levels. In combination, these modifications produce substantial increases in precise editing rates.

Human induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However, their differentiation into specific cell lineages, such as definitive endoderm (DE) and pancreatic progenitor (PP), often suffers from poor reproducibility, due partially to their pluripotency. In this work, we investigated the impact of iPSC confluency during cell self-renewal and seeding density on cell metabolic activity, glycolysis to oxidative phosphorylation shift, and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual-positive cells in PPs. Interestingly, we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification, offering new insights into the robustness of iPSC self-renewal and differentiation for creating human tissues.

The search for an effective cell replacement therapy for diabetes has driven the development of "perfect" pancreatic islets from human pluripotent stem cells (hPSCs). These hPSC-derived pancreatic islet-like β cells can overcome the limitations for disease modelling, drug development and transplantation therapies in diabetes. Nevertheless, challenges remain in generating fully functional and mature β cells from hPSCs. This review underscores the significant efforts made by researchers to optimize various differentiation protocols aimed at enhancing the efficiency and quality of hPSC-derived pancreatic islets and proposes methods for their improvement. By emulating the natural developmental processes of pancreatic embryogenesis, specific growth factors, signaling molecules and culture conditions are employed to guide hPSCs towards the formation of mature β cells capable of secreting insulin in response to glucose. However, the efficiency of these protocols varies greatly among different human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) lines. This variability poses a particular challenge for generating patient-specific β cells. Despite recent advancements, the ultimate goal remains to develop a highly efficient directed differentiation protocol that is applicable across all genetic backgrounds of hPSCs. Although progress has been made, further research is required to optimize the protocols and characterization methods that could ensure the safety and efficacy of hPSC-derived pancreatic islets before they can be utilized in clinical settings.

Background: Type 1 diabetes (T1D) is a chronic autoimmune disorder characterized by the destruction of pancreatic β-cells, leading to absolute insulin deficiency. Despite advancements in insulin therapy and glucose monitoring, achieving optimal glycemic control remains a challenge. Emerging technologies and novel therapeutic strategies are transforming the landscape of T1D management, offering new opportunities for improved outcomes. Methods: This review synthesizes recent advancements in T1D treatment, focusing on innovations in continuous glucose monitoring (CGM), automated insulin delivery systems, smart insulin formulations, telemedicine, and artificial intelligence (AI). Additionally, we explore biomedical approaches such as stem cell therapy, gene editing, immunotherapy, gut microbiota modulation, nanomedicine-based interventions, and trace element-based therapies. Results: Advances in digital health, including CGM integration with hybrid closed-loop insulin pumps and AI-driven predictive analytics, have significantly improved real-time glucose management. AI and telemedicine have enhanced personalized diabetes care and patient engagement. Furthermore, regenerative medicine strategies, including β-cell replacement, CRISPR-based gene editing, and immunomodulatory therapies, hold potential for disease modification. Probiotics and microbiome-targeted therapies have demonstrated promising effects in maintaining metabolic homeostasis, while nanomedicine-based trace elements provide additional strategies to regulate insulin sensitivity and oxidative stress. Conclusions: The future of T1D management is shifting toward precision medicine and integrated technological solutions. While these advancements present promising therapeutic avenues, challenges such as long-term efficacy, safety, accessibility, and clinical validation must be addressed. A multidisciplinary approach, combining biomedical research, artificial intelligence, and nanotechnology, will be essential to translate these innovations into clinical practice, ultimately improving the quality of life for individuals with T1D.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells within pancreatic islets, the specialized endocrine cell clusters of the pancreas. Islet transplantation has emerged as a β cell replacement therapy, involving the infusion of cadaveric islets into a patient's liver through the portal vein. This procedure offers individuals with T1D the potential to restore glucose control, reducing or even eliminating the need for exogenous insulin therapy. However, it does not address the underlying autoimmune condition responsible for T1D. The need for systemic immunosuppression remains the primary barrier to making islet transplantation a more widespread therapy for patients with T1D. Here, we review recent progress in addressing the key limitations of islet transplantation as a viable treatment for T1D. Concerns over systemic immunosuppression arise from its potential to cause severe side effects, including opportunistic infections, malignancies, and toxicity to transplanted islets. Recognizing the risks, the Edmonton protocol (2000) marked a shift away from glucocorticoids to prevent β cell damage specifically. This transition led to the development of combination immunosuppressive therapies and the emergence of less toxic immunosuppressive and anti-inflammatory drugs. More recent advances in islet transplantation derive from islet encapsulation devices, biomaterial platforms releasing immunomodulatory compounds or surface-modified with immune regulating ligands, islet engineering and co-transplantation with accessory cells. While most of the highlighted studies in this review remain at the preclinical stage using mouse and non-human primate models, they hold significant potential for clinical translation if a transdisciplinary research approach is prioritized.

Islet β-cell transplantation offers a promising treatment for repairing pancreatic damage in diabetes, with the transcription factor pancreatic duodenal homeobox-1 (PDX1) being crucial for β-cell function and insulin secretion. Mammalian threonine protein kinase (MST1) is recognized for its role in regulating PDX1 during cell apoptosis, yet its function in embryonic stem cell (ESC) differentiation into insulin-producing cells (IPCs) remain underexplored. This study investigated the effect of MST1-silencing on the differentiation of ESC into IPCs.

ESCs were transfected utilizing a recombinant MST1-silencing lentiviral vector (shMST1). qRT-PCR, immunofluorescence, flow cytometry, western blot and ELISA assays were performed to examine function of IPCs in vitro. Furthermore, these IPCs were transplanted into type 1 diabetic mellitus (T1DM) rats. Measuring the changes in blood glucose concentration of animals before and after IPCs transplantation. Intraperitoneal glucose tolerance test (IPGT) was used to determine the regulatory effect of IPCs transplantation on blood glucose stimulation and immunohistochemistry was used to detect the expression of pancreatic Insulin protein in T1DM rats.

It was observed that IPCs from the shMST1 group exhibited notably improvement in insulin secretion and glucose responsiveness, suggesting MST1 suppression may enhance IPC maturity. The rats demonstrated significant normalization of blood sugar levels and increased insulin levels, akin to non-diabetic controls. This implies that MST1-silencing not only augments IPC function in vitro but also their therapeutic efficacy in vivo.

The findings indicate that targeting MST1 offers a novel approach for deriving functionally mature IPCs from ESCs, potentially advancing cell replacement therapies for diabetes. This research underscores the importance of developing IPCs with competent insulin secretion for diabetes treatment in vitro.

This opinion paper explores the path forward for islet transplantation as a cell therapy for type 1 diabetes, following the Biologics License Application (BLA) approval. The authors review key challenges and opportunities that lie ahead. After a brief overview of the history of human islet transplantation, the paper examines the FDA's regulatory stance on isolated islet cells and the requirements for obtaining a BLA. The authors discuss the significance of this approval and the critical steps necessary to broaden patient access, such as scaling up production, clinical integration, reimbursement frameworks, post-marketing surveillance, and patient education initiatives. The paper highlights that the approval of LANTIDRA as an allogeneic cell transplant for uncontrolled type 1 diabetes marks the beginning of new chapters in improving islet transplantation. The authors emphasize essential areas for development, including advancements in islet manufacturing, optimization of transplant sites, islet encapsulation, exploration of unlimited cell sources, and gene editing technologies. In conclusion, the future of islet transplantation beyond the BLA approval presents challenges and opportunities. While significant regulatory milestones have been reached, hurdles remain. Innovations in stem cell-derived islets, cell encapsulation, and gene editing show promise in enhancing graft survival, expanding the availability of transplantable cells, and reducing the reliance on immunosuppressive drugs. These advancements could pave the way for more accessible, durable, and personalized diabetes treatments.

The gold standard for assessing the function of human islets or β-like cells derived from stem cells involves their engraftment under the kidney capsule of hyperglycemic, immunodeficient mice. Current models, such as streptozotocin treatment of severely immunodeficient mice or the NRG-Akita strain, are limited due to unstable and variable hyperglycemia and/or high morbidity. To address these limitations, we developed the IsletTester mouse via CRISPR/Cas9-mediated gene editing of glucokinase (Gck), the glucose sensor of the β-cells, directly in NSG zygotes. IsletTester mice are heterozygous for an Arg345→stop mutation in Gck and present with stable random hyperglycemia (∼250 mg/dL [14 mmol/L]), normal lifespan, and fertility. We demonstrate the utility of this model through functional engraftment of both human islets and human embryonic stem cell-derived β-like cells. The IsletTester mouse will enable the study of human islet biology over time and under different physiological conditions and can provide a useful preclinical platform to determine the functionality of stem cell-derived islet products.

Current mouse models for assessing islet function in vivo are limited due to unstable and variable hyperglycemia and/or high morbidity. We derived the IsletTester mouse to address these limitations. Leveraging a previously characterized glucokinase mutation and CRISPR/Cas9 technology, we successfully developed a moderately hyperglycemic and immunodeficient mouse model for the in vivo assessment of islet function. Our IsletTester mouse has stable, moderate hyperglycemia that can be corrected with primary human islets or stem cell-derived insulin-producing cells. The IsletTester mouse provides a reliable, easy-to-use platform for the preclinical assessment of stem cell-derived islet products or islet-targeted drugs.