Understanding pancreatic development is instrumental to diabetes research and β-cell replacement therapies. Here, we investigate glucocorticoid receptor (GR) signaling during early pancreas development in mice and humans. Previous reports suggest that glucocorticoids do not play a significant role in mouse pancreas development before the second transition. In this study, we demonstrate that, under physiological conditions, the GR is selectively active in mouse pro-acinar and early endocrine cells from embryonic day 11.5, silenced in bipotent progenitors, and reactivated during endocrine commitment. In mouse pancreatic explants, ectopic GR activation globally promotes acinar fate. Surprisingly, GR activation in human in vitro-derived multipotent pancreatic progenitors steers lineage commitment toward a bipotent/endocrine trajectory and upregulates genes for which expression profiles resemble those of SOX9 and HES1 during human embryonic pancreatic bipotential and endocrine progenitor fate choice. Our combined epigenomic and single-cell transcriptomic analyses suggest that these newly identified marker genes may play important roles in human pancreas development. Taken together, our findings position the GR pathway as an endogenous developmental modulator of early-stage pancreatic progenitor cell differentiation and provide insights into the underlying transcriptional mechanisms involved.

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.

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.

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.

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.

Thioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress, which disrupt glucose homeostasis in diabetes. However, the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.

We used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP-/-) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP-/- and TXNIP+/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice.

TXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency, viability, or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP-/- and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes, with enriched apoptosis-related pathways in TXNIP+/+ cells, but minimal transcriptional changes specific to TXNIP deficiency. In HLCs, TXNIP deletion reduced albumin secretion and insulin signalling, as indicated by decreased AKT phosphorylation, while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress, TXNIP-/- HLCs exhibited transiently reduced eIF2α phosphorylation and lower BiP expression, suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP-/- hESCs showed comparable viability, endocrine cell composition, and cytokine responses to TXNIP+/+ islets. Following IFNα or IFNγ treatment, STAT1 phosphorylation was increased in TXNIP-/- SC-islets, indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice, both TXNIP-/- and TXNIP+/+ SC-islets produced human C-peptide and responded to glucose stimulation. However, TXNIP-/- SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls.

Our study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP-/- and TXNIP+/+ H1-hESCs, HLCs, and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.

Pancreatic β cell loss by cellular stress contributes to diabetes pathogenesis. Nevertheless, the fundamental mechanism of cellular stress regulation remains elusive. Here, it is found that elevated zinc transportation causes excessive cellular stress in pancreatic β cells in diabetes. With gene-edited human embryonic stem cell-derived β cells (SC-β cells) and human primary islets, the results reveal that elevated zinc transportation initiates the integrated stress response (ISR), and ultimately leads to β cell death. By contrary, genetic abolishment of zinc transportation shields β cells from exacerbated endoplasmic reticulum stress (ER stress) and concurrent ISR. To target excessive zinc transportation with a chemical inhibitor, an isogenic SC-β cells based drug-screening platform is established. Surprisingly, independent of its traditional role as protein synthesis inhibitor at a high-dose (10 µm), low-dose (25 nm) anisomycin significantly inhibits zinc transportation and effectively prevents β cell loss. Remarkably, in vivo administration of anisomycin in mice demonstrates protective effects on β cells and prevents type 2 diabetes induced by high-fat diet. Overall, elevated zinc transportation is identified as a crucial driver of β cell loss and low-dose anisomycin as a potential therapeutic molecule for diabetes.

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.

Renalase (Rnls) is annotated as an oxidase enzyme. It has been implicated in Type 1 diabetes (T1D) risk via genome-wide association studies (GWAS). We previously discovered through CRISPR screening and validation experiments that Rnls inhibition prevents or delays T1D in multiple mouse models of diabetes in vivo, and protects pancreatic β cells against autoimmune killing, ER and oxidative stress in vitro. The molecular biochemistry and functions of Rnls are largely uncharted. Here we studied the mechanisms of Rnls inhibition that underlie β cell protection during diabetogenic stress.

Akita mice were treated with oral Pargyline (PG) in vivo to bind and inhibit Rnls, and pancreas or islets were harvested for β cell mass and β cell function analyses. Genetic and pharmacological tools were used to inhibit Rnls in β cell lines. RNA sequencing, metabolomics and metabolic function experiments were conducted in vitro in NIT-1 mouse β cell lines and human stem cell-derived β cells.

In vivo, PG improved glycemia and mildly preserved β cell mass and function in females. Genetic strategies to mutate (Rnlsmut) or knockout (Rnls KO) Rnls induced a robust metabolic shift towards glycolysis in both mouse and human β cell lines, in vitro. Stress protection was abolished when glycolysis was blocked with 2-deoxyglucose (2-DG). Pharmacological Rnls inhibition with PG did not strongly mimic these newly identified metabolic mechanisms.

Our work illustrates a role for Rnls in regulating cell metabolism. We show that inhibiting Rnls protects against chronic stress in vivo, and shields against acute stress in β cell lines in vitro by rewiring cell metabolism towards glycolysis.

The large-scale production of mammalian cells is pivotal for various applications; however, current bioreactor technologies encounter significant technical and economic challenges. Scaling up cell cultures remains problematic due to excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates. Nevertheless, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. To address these limitations, this study reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Utilizing a novel micro-extruder, we efficiently produced cell-loaded ColAlgTubes. Collagen formed a dense nanofiber network interwoven with the alginate mesh, enhancing the hydrogel's mechanical properties while providing cell adhesion sites. ColAlgTubes protected cells from hydrodynamic stress and maintained cell mass within a 400 μm diameter, ensuring efficient nutrient exchange and waste removal. This optimized microenvironment resulted in high cell viability, rapid proliferation, and exceptional yields of 5×10 8 cells/mL - 200 times higher than conventional culture methods. With their scalability, cost-effectiveness, and efficiency, ColAlgTubes offers a transformative solution for large-scale cell production with broad applications in biotechnology, regenerative medicine, and therapeutic manufacturing.

The role of regulatory factor X 3 (RFX3) in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from induced pluripotent stem cells (iPSCs), hypothesising that RFX3 regulates human islet cell differentiation.

We generated RFX3 knockout (RFX3 KO) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation and glucose-stimulated insulin secretion. Single-cell RNA-seq datasets from human embryonic stem cell-derived pancreatic islet differentiation were re-analysed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA-seq was conducted to further assess transcriptomic changes. RFX3 overexpression was implemented to reverse dysregulated gene expression.

RFX3 was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs) and mature islet stages derived from iPSCs. Single-cell RNA-seq further confirmed RFX3 expression across different endocrine cell clusters during differentiation. The loss of RFX3 disrupted pancreatic endocrine gene regulation, reduced the number of hormone-secreting islet cells and impaired beta cell function and insulin secretion. Despite a significant reduction in the expression levels of pancreatic islet hormones, the pan-endocrine marker chromogranin A remained unchanged at both EP and islet stages, likely due to an increase in the abundance of enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression levels in RFX3 KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated thioredoxin-interacting protein levels and increased apoptosis in EPs and islets. Furthermore, RFX3 overexpression rescued the expression of dysregulated genes in RFX3 KO at the PP and EP stages.

These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing EC specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility.

The RNA-seq datasets have been submitted to the Zenodo repository and can be accessed via the following links: DOI https://doi.org/10.5281/zenodo.13647651 (PPs); and DOI https://doi.org/10.5281/zenodo.13762055 (SC-islets).

Type 1 diabetes (T1D) is a chronic hyperglycemia disorder emerging from beta-cell (insulin secreting cells of the pancreas) targeted autoimmunity. As the blood glucose levels significantly increase and the insulin secretion is gradually lost, the entire body suffers from the complications. Although various advances in the insulin analogs, blood glucose monitoring and insulin application practices have been achieved in the last few decades, a cure for the disease is not obtained. Alternatively, pancreas/islet transplantation is an attractive therapeutic approach based on the patient prognosis, yet this treatment is also limited mainly by donor shortage, life-long use of immunosuppressive drugs and risk of disease transmission. In research and clinics, such drawbacks are addressed by the endocrine tissue engineering of the pancreas. One arm of this engineering is scaffold-free models which often utilize highly developed cell-cell junctions, soluble factors and 3D arrangement of islets with the cellular heterogeneity to prepare the transplant formulations. In this review, taking T1D as a model autoimmune disease, techniques to produce so-called pseudoislets and their applications are studied in detail with the aim of understanding the role of mimicry and pointing out the promising efforts which can be translated from benchside to bedside to achieve exogenous insulin-free patient treatment. Likewise, these developments in the pseudoislet formation are tools for the research to elucidate underlying mechanisms in pancreas (patho)biology, as platforms to screen drugs and to introduce immunoisolation barrier-based hybrid strategies.

Artificial insulin-producing cells (IPCs) used to treat diabetes mellitus type 1 (DMT1) are naturally hampered by their carcinogenicity. This in vitro study aimed to examine the carcinogenic potential of IPCs produced by the differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs).

hADSCs were transformed into IPCs by administering insulin-transferrin, selenium (ITS), and nicotinamide in a 14-day differentiation protocol. The cells were transfected with 20 μg of pure Pdx1-pIRES recombinant vector on the tenth day of differentiation. The successful transfection was confirmed by Pdx1 overexpression and GFP fluorescence activity. The differentiated cells' capacity to release insulin and glucose-dependent C-peptide was used to evaluate their functionality. Gene expression was assessed using real-time PCR. Meanwhile, protein expression was investigated using western blotting. The transfected cells exhibited fluorescence activity and Pdx1 overexpression. The differentiated IPCs were able to secrete C-peptide and insulin. The artificial IPCs showed significantly reduced Oct4 and Nanog expression. However, the differentiation process induced a noticeable elevation in tPA expression. The artificial IPCs expressed much lower c-MYC expression compared to undifferentiated hADSCs. The differentiated cells exhibited a significant elevation in Glut2, MMP-2, CD24, P16, and P21 expression.

The differentiation technique used in this work produced functional beta-like cells devoid of typical markers of stem cells. The synthetic IPCs displayed characteristics of newly generated β-like cells. The artificial IPCs showed no signs of expressing tumor-associated markers. The findings imply that the artificial IPC cells lack tumor characteristics in vitro.

Cystic fibrosis related diabetes (CFRD) is the most common comorbidity of cystic fibrosis (CF) still, its pathogenesis is poorly understood. Recent studies have suggested that although pancreatic insufficiency is an important explanation for CFRD development, inherent pancreatic islet cell dysfunction may play a role. This study aimed to systematically compile current data regarding the impact of pancreatic islet cell dysfunction on the development of CFRD.

A systematic search was conducted in PubMed and Embase. The resulting articles were screened for relevant experimental design and outcomes. Articles underwent data extraction and quality assessment before compilation and analysis of the results.

A total of 268 articles were initially screened and 19 studies conducted between 2006-2022 were finally included in this review. Half of the studies in human tissue and most of the studies in animal tissue could detect CFTR in the islets. Similarly, half of the publications in human islets and most studies in animal islets detect decreased insulin secretion with inhibition/mutation of CFTR.

The literature on the role of islet CFTR is contradictory. However, a pattern emerges where CFTR loss-of-function mutations have the potential to negatively affect islet cell function in a way that, together with previously described exocrine damage occurring in CF, could play a part in the development of CFRD.

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.

Islet organoids derived from pluripotent stem cells offer a promising solution for the shortage of cadaveric donors in diabetes treatment. However, challenges remain in improving their differentiation, viability, functional maturity, and engraftment. Here, we generated improved islet organoids with high viability and functionality by employing extracellular matrix (ECM) hydrogel of decellularized amniotic membrane (dAM). The dAM sheet facilitates islet organoid engraftment and rapidly restores normoglycemia in diabetic mice, accompanied by increased body weight and augmented insulin release in response to glucose. Interestingly, collagen VI (Col VI) was identified as a key component of islet niche, enhancing islet cell viability and biological function. Col-VI-based biomimetic ECM recapitulates the native environment and exhibits superior physiological properties. Importantly, the cellular composition and endocrine function of optimized induced pluripotent stem cell (iPSC)-derived islet organoids are comparable with those of human islets. Our findings offer a valuable platform for future endeavors in organoid-transplantation-based therapy of diabetes.

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.

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.

Transplantation of insulin-secreting cells provides a promising method for re-establishing the autonomous blood glucose control ability of type 1 diabetes (T1D) patients, but the low survival of the transplanted cells hinder the therapeutic efficacy. In this study, we 3D-printed an encapsulation system containing β-like cells and microvascular fragments (MVF), to create a retrivable microdevice with vascularized islets in vivo for T1D therapy. The functional β-like cells were differentiated from the urine epithelial cell-derived induced pluripotent stem cells (UiPSCs). Single-cell RNA sequencing provided an integrative study and macroscopic developmental analyses of the entire process of differentiation, which revealed the developmental trajectory of differentiation in vitro follows the developmental pattern of embryonic pancreas in vivo. The MVF were isolated from the epididymal fat pad. The microdevice with a groove structure were rapidly fabricated by the digital light processing (DLP)-3D printing technology. The β-like cells and MVF were uniformly distributed in the device. After subcutaneous transplantation into C57BL/6 mice, the microdevice have less collagen accumulation and low immune cell infiltration. Moreover, the microdevice encapsulated vascularized islets reduced hyperglycemia in 33 % of the treated mice for up to 100 days without immunosuppressants, and the humanized C-peptide was also detected in the serum of the mice. In summary, we described the microdevice-protected vascularized islets for long-term treatment of T1D, with high safety and potential clinical transformative value, and may therefore provide a translatable solution to advance the research progress of β cell replacement therapy for T1D.

Given their capability to differentiate into each cell type of the human body, human pluripotent stem cells (hPSCs) provide a unique platform for developmental studies. In the current study, we employed this cell system to understand the role of pancreatic progenitor differentiation and proliferation factor (PPDPF), a protein that has been little explored so far. While the zebrafish orthologue exdpf is essential for exocrine pancreas specification, its importance for mammalian and human development has not been studied yet. We implemented a four times CRISPR/Cas9 nicking approach to knockout PPDPF in human embryonic stem cells (hESCs) and differentiated PPDPFKO/KO and PPDPFWT/WT cells towards the pancreatic lineage. In contrast to data obtained from zebrafish, a very modest effect of the knockout was observed in the development of pancreatic progenitors in vitro, not affecting lineage specification upon orthotopic transplantation in vivo. The modest effect is in line with the finding that genetic variants near PPDPF are associated with random glucose levels in humans, but not with type 2 diabetes risk, supporting that dysregulation of this gene may only result in minor alterations of glycaemic balance in humans. In addition, PPDPF is less organ- and cell type specifically expressed in higher vertebrates and its so far reported functions appear highly context-dependent.

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.

Bulk RNA sequencing is one type of RNA sequencing technique, as well as targeted RNA sequencing and whole transcriptome sequencing. It provides valuable insights into gene expression in specific cell populations or regions. However, these methods often miss the diversity of cells within complex tissues. This restriction is overcome by single-cell RNA sequencing, which records gene expression at the single-cell level. It offers a detailed picture of the diversity of cells. It is essential to study glucose homeostasis. It offers thorough explanations of cellular variation. Networks and Governance Dynamics The use of scRNA-seq in islet cells is reviewed in this study, along with sample preparation, sequencing, and computational analysis. It highlights advances in understanding cell types. Gene activity and cell interactions. Along with the challenges and limitations of scRNA-seq, this review highlights the importance of scRNA-seq in understanding complex biological processes and diseases. It is an essential resource for future research and method development in this field, which will help to build personalized treatment.

IER3IP1 mutations are linked to the development of microcephaly, epilepsy, and early-onset diabetes syndrome 1. However, the underlying molecular mechanisms of cell dysfunction are unknown. Using targeted genome editing, we generated specific IER3IP1 mutations in human embryonic stem cell lines that were differentiated into pancreatic islet lineages. Loss of IER3IP1 resulted in a threefold reduction in endoplasmic reticulum-to-Golgi trafficking of proinsulin in stem cell-derived β-cells, leading to β-cell dysfunction both in vitro and in vivo. Loss of IER3IP1 also triggered increased markers of endoplasmic reticulum stress, indicating the pivotal role of the endoplasmic reticulum-to-Golgi trafficking pathway for β-cell homeostasis and function.

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.

Cell reprogramming represents a powerful approach to achieve the conversion cells of one type into cells of another type of interest, which has substantially changed the landscape in the field of developmental biology, regenerative medicine, disease modeling, drug discovery and cancer immunotherapy. Cell reprogramming is a complex and ordered process that involves the coordination of transcriptional, epigenetic, translational and metabolic changes. Over the past two decades, a range of questions regarding the facilitators/barriers, the trajectories, and the mechanisms of cell reprogramming have been extensively investigated. This review summarizes the recent advances in cell reprogramming mediated by transcription factors or chemical molecules, followed by elaborating on the important roles of biophysical cues in cell reprogramming. Additionally, this review will detail our current understanding of the mechanisms that govern cell reprogramming, including the involvement of the recently discovered biomolecular condensates. Finally, the review discusses the broad applications and future directions of cell reprogramming in developmental biology, disease modeling, drug development, regenerative/rejuvenation therapy, and cancer immunotherapy.

G protein-coupled receptors (GPCRs) are cell-surface proteins that are targeted therapeutically for a range of disorders, including diabetes. Adhesion GPCRs (aGPCRs) are the second largest class of the GPCR superfamily and some members of this family have been implicated in appropriate organ development. However, the role of aGPCRs in endocrine pancreas specification is not yet known.

Here, we systematically characterised expression of mRNAs encoding aGPCRs and their ligands in developing mouse and human pancreas using our own and publicly available single-cell RNA sequencing and spatial transcriptomics data, and we conducted qPCR analysis of aGPCR expression in human pancreas at different gestational stages. We then investigated the role of GPR56 (ADGRG1), the most abundant aGPCR in pancreatic endocrine progenitors, in islet development using Gpr56 null mice and their wildtype littermates.

We demonstrated that aGPCRs are dynamically expressed during mouse and human pancreas development, with specific aGPCR mRNAs expressed in distinct endocrine, endothelial, mesenchymal, acinar, ductal, and immune cell clusters. aGPCR ligand mRNAs were mostly expressed by non-endocrine cells, and the most highly expressed receptor-ligand interacting mRNA pairs were those encoding GPR56 and COL3A1. Deletion of Gpr56 in neonatal mice was associated with an altered α-/β-/δ-cell ratio and reduced β-cell proliferation.

Our data show that aGPCRs are expressed at key stages of human and mouse pancreas endocrine lineage decisions, and analysis of pancreases from Gpr56 knockout mice implicate this aGPCR in the development of a full complement of β-cells.

Fluorescence anisotropy immunoassays (FAIAs) are widely used to quantify the concentration of target proteins based on competitive binding to a monoclonal antibody with a tracer. We recently designed an FAIA to measure mouse C-peptide secretion from living islets in a continuous-flow microfluidic device (InsC-chip). To develop a similar assay for human C-peptide, we selected two monoclonal antibodies (Ab1 and Ab2) that initially showed a low dynamic range and slow kinetics. One option to measure this assay on-chip was to extend the length of the mixing channels. However, this strategy would increase dispersion and ultimately lower the temporal resolution of secreted C-peptide. To shorten the time-to-reach equilibrium for Ab1, we reengineered the tracer based on a comparison between the human and mouse C-peptide sequences, resulting in >30-fold shorter time-to-reach equilibrium. To increase the relatively small dynamic range for Ab2, we used partial epitope mapping and targeted point mutations to increase the dynamic range by 45%. Finally, we validated both FAIAs by measuring depolarization-induced secretion from individual human stem cell-derived islets in our InsC-chip. These data demonstrate a strategy to optimize FAIA kinetics to be measured in continuous-flow microfluidic devices.

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.

Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. Alternative β cell sources and transplantation sites are needed. We demonstrate the feasibility to repurpose a decellularized lung as an endocrine pancreas for β cell replacement. We bioengineer an induced pluripotent stem cell (iPSC)-based version, fabricating a human iPSC-based vascularized endocrine pancreas (iVEP) using iPSC-derived β cells (iPSC-derived islets [SC-islets]) and endothelial cells (iECs). SC-islets and iECs are aggregated into vascularized iβ spheroids (ViβeSs), and over 7 days of culture, spheroids integrate into the bioengineered vasculature, generating a functional, perfusable human endocrine organ. In vitro, the vascularized extracellular matrix (ECM) sustained SC-islet engraftment and survival with a significantly preserved β cell mass and a physiologic insulin release. In vivo, iVEP restores normoglycemia in diabetic NSG mice. We report a human iVEP providing a controlled in vitro insulin-secreting phenotype and in vivo function.

In this study, we demonstrate that cytoskeletal state at the onset of directed differentiation is critical for the specification of human pluripotent stem cells (hPSCs) to all three germ layers. In particular, a polymerized actin cytoskeleton facilitates directed ectoderm differentiation, while depolymerizing F-actin promotes mesendoderm lineages. Applying this concept to a stem cell-derived islet (SC-islet) differentiation protocol, we show that depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of definitive endoderm formation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway signaling dynamics. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers associated with other endodermal lineages. Continued differentiation generates islets containing a higher percentage of β cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling at the onset of directed differentiation.

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However, stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature, which leads to the immaturity of SC-derived islets, reducing their ability to detect glucose fluctuations and insulin release. Here, we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature, supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development, maturation, and diabetic disease modeling, with future implications for translational applications.

Type 1 diabetes (T1D) affects over 2 million people in the United States and has no known cure. The discovery and first use of insulin in humans 102 years ago marked a revolutionary course of treatment for the disease, and although the formulations and delivery systems have advanced, insulin administration remains the standard of care today. While improved treatment options represent notable progress in T1D management, finding a functional cure for the disease remains the ultimate goal. Approaches to curing T1D have historically focused on blunting the autoimmune response, although sustained effects of immune modulation have proven elusive. Islet transplant therapies have also proven effective, although a lack of available donor tissue and the need for immunosuppression to prevent both host-graft rejection and the autoimmune response have reserved such treatments for those who already require immunosuppressive regimens for other reasons or undergo severe hypoglycemic events in conjunction with hypoglycemic unawareness. With the advent of human stem cell research, the focus has shifted toward generating an abundance of allogeneic, functional beta-like cells that can be transplanted into the patients. Immunoisolation devices have also shown some promise as a method of preventing immune rejection and the autoimmune destruction of transplanted cells. Finally, advances in new immune therapies, if used in the early stages of T1D progression, have proven to delay the onset of diabetes. Stem cell-based therapies are a promising approach to curing T1D. The ongoing clinical trials show some success, although they currently require immunosuppressant agents. Encapsulation devices provide a method of immunoisolation that does not require immunosuppression; however, the devices tested thus far eventually lead to cell death and fibrotic tissue growth. Substantial research efforts are underway to develop new approaches to protect the stem cell-derived beta cells upon transplantation.