Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors.

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

Mitochondrial Dysfunction, Oxidative Stress, and Inter-Organ Miscommunications in T2D Progression.

Type 2 diabetes (T2D) is a heterogenous disease, and conventionally, peripheral insulin resistance (IR) was thought to precede islet ß-cell dysfunction, promoting progression from prediabetes to T2D. New evidence suggests that T2D-lean individuals experience early ß-cell dysfunction without significant IR. Regardless of the primary event (i.e., IR vs. ß-cell dysfunction) that contributes to dysglycemia, significant early-onset oxidative damage and mitochondrial dysfunction in multiple metabolic tissues may be a driver of T2D onset and progression. Oxidative stress, defined as the generation of reactive oxygen species (ROS), is mediated by hyperglycemia alone or in combination with lipids. Physiological oxidative stress promotes inter-tissue communication, while pathological oxidative stress promotes inter-tissue mis-communication, and new evidence suggests that this is mediated via extracellular vesicles (EVs), including mitochondria containing EVs. Under metabolic-related stress conditions, EV-mediated cross-talk between ß-cells and skeletal muscle likely trigger mitochondrial anomalies leading to prediabetes and T2D. This article reviews the underlying molecular mechanisms in ROS-related pathogenesis of prediabetes, including mitophagy and mitochondrial dynamics due to oxidative stress. Further, this review will describe the potential of various therapeutic avenues for attenuating oxidative damage, reversing prediabetes and preventing progression to T2D.

Deficiency of WTAP in islet beta cells results in beta cell failure and diabetes in mice.

AIMS/HYPOTHESIS: N6-methyladenosine (m6A) mRNA methylation and m6A-related proteins (methyltransferase-like 3 [METTL3], methyltransferase-like 14 [METTL14] and YTH domain containing 1 [YTHDC1]) have been shown to regulate islet beta cell function and the pathogenesis of diabetes. However, whether Wilms' tumour 1-associating protein (WTAP), a key regulator of the m6A RNA methyltransferase complex, regulates islet beta cell failure during pathogenesis of diabetes is largely unknown. The present study aimed to investigate the role of WTAP in the regulation of islet beta cell failure and diabetes. METHODS: Islet beta cell-specific Wtap-knockout and beta cell-specific Mettl3-overexpressing mice were generated for this study. Blood glucose, glucose tolerance, serum insulin, glucose-stimulated insulin secretion (both in vivo and in vitro), insulin levels, glucagon levels and beta cell apoptosis were examined. RNA-seq and MeRIP-seq were performed, and the data were well analysed. RESULTS: WTAP was downregulated in islet beta cells in type 2 diabetes, due to lipotoxicity and chronic inflammation, and islet beta cell-specific deletion of Wtap (Wtap-betaKO) induced beta cell failure and diabetes. Wtap-betaKO mice showed severe hyperglycaemia (above 20 mmol/l [360 mg/dl]) from 8 weeks of age onwards. Mechanistically, WTAP deficiency decreased m6A mRNA modification and reduced the expression of islet beta cell-specific transcription factors and insulin secretion-related genes by reducing METTL3 protein levels. Islet beta cell-specific overexpression of Mettl3 partially reversed the abnormalities observed in Wtap-betaKO mice. CONCLUSIONS/INTERPRETATION: WTAP plays a key role in maintaining beta cell function by regulating m6A mRNA modification depending on METTL3, and the downregulation of WTAP leads to beta cell failure and diabetes. DATA AVAILABILITY: The RNA-seq and MeRIP-seq datasets generated during the current study are available in the Gene Expression Omnibus database repository ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE215156 ; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE215360 ).

Revisiting the Pathogenesis of Type 1 Diabetes: Importance of Neural Input to Pancreatic Islets and the Therapeutic Capability of Stem Cell Educator TM Therapy to Restore Their Integrity.

Type 1 diabetes (T1D) is an autoimmune disease with a shortage of islet ß cells. To date, the etiology of T1D remains elusive. Increasing clinical evidence and animal studies demonstrate that autoimmune cells are directed against the nervous system of pancreatic islets, contributing to the development of T1D. Therefore, it highlights the necessity to explore novel clinical approaches to fundamentally correct the T1D autoimmunity not only focusing on islet ß cells but also on protecting the islet nervous system. This allows the restoration of the integrity of islet innervation and the normal islet ß-cell function. To address these issues, we developed a novel technology designated the Stem Cell Educator TM therapy, based on immune education by human cord-blood-derived multipotent stem cells (CB-SC). International amulticenter clinical trials demonstrated its clinical safety and efficacy to treat T1D and other autoimmune diseases. Stem Cell Educator TM therapy may have the potential to revolutionize the treatment of T1D, without the safety and ethical concerns associated with conventional immune and/or stem cell-based therapies.

ATF5 is a regulator of ER stress and ß-cell apoptosis in different mouse models of genetic- and diet-induced obesity and diabetes mellitus.

Endoplasmic reticulum (ER) stress is closely associated with type 2 diabetes (T2D). Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element binding protein (CREB) family whose levels are increased upon stress in pancreatic islets from mice. Intriguingly, ATF5 deficiency has been shown to contribute to increased ER stress and apoptosis in mouse islet micro-organs. We hypothesized that either deficiency or overexpression of ATF5 is equally deleterious for pancreatic islets in terms of ER stress and apoptosis. To test this, we used a number of in vitro and in vivo models whereby ATF5 levels were overexpressed. We also determined the regulation of ATF5 in the context of metabolic derangements by using various mouse models of obesity and T2D. Our in vitro results show that ATF5 overexpression promoted palmitic acid (PA)-induced lipotoxic apoptosis. In vivo, global ATF5 overexpression in mice was lethal and pancreas-specific ATF5 overexpressing mice exhibit increased ß-cell apoptosis. Interestingly, ATF5 is downregulated in all mouse models of severe obesity and T2D used in the current study. In conclusion, a tight control on ATF5 levels might be considered when developing novel agents targeting ATF5 for prevention and treatment of metabolic diseases.

Therapeutic Strategies Targeting Pancreatic Islet ß-Cell Proliferation, Regeneration, and Replacement.

Diabetes results from insufficient insulin production by pancreatic islet ß-cells or a loss of ß-cells themselves. Restoration of regulated insulin production is a predominant goal of translational diabetes research. Here, we provide a brief overview of recent advances in the fields of ß-cell proliferation, regeneration, and replacement. The discovery of therapeutic targets and associated small molecules has been enabled by improved understanding of ß-cell development and cell cycle regulation, as well as advanced high-throughput screening methodologies. Important findings in ß-cell transdifferentiation, neogenesis, and stem cell differentiation have nucleated multiple promising therapeutic strategies. In particular, clinical trials are underway using in vitro-generated ß-like cells from human pluripotent stem cells. Significant challenges remain for each of these strategies, but continued support for efforts in these research areas will be critical for the generation of distinct diabetes therapies.

Berberine protects against palmitate induced beta cell injury via promoting mitophagy.

BACKGROUND: Destruction of pancreatic beta cells is the most typical characteristic of diabetes. OBJECTIVE: We aimed to evaluate the effect of berberine (BBR), a bioactive isoquinoline derivative alkaloid, on beta cell injury. METHODS: Rodent pancreatic beta cell line INS-1 was treated with 0.5 mM palmitate (PA) for 24 h to establish an in vitro beta cell injury model. RESULTS: BBR at 5 µM promoted cell viability, inhibited cell apoptosis and enhanced insulin secretion in PA-induced INS-1 cells. BBR treatment also suppressed PA-induced oxidative stress in INS-1 cells, as evidenced by the decreased ROS production and increased activities of antioxidant enzymes. In addition, suppressed ATP production and reduced mitochondrial membrane potential were restored by BBR in PA-treated INS-1 cells. It was further determined that BBR affected the expressions of mitophagy-associated proteins, suggesting that BBR promoted mitophagy in PA-exposed INS-1 cells. Meanwhile, we found that BBR facilitated nuclear expression and DNA-binding activity of Nrf2, an antioxidative protein that can regulate mitophagy. Finally, a rescue experiment was performed and the results demonstrated that the effect of BBR on cell viability, apoptosis and mitochondrial function in PA-induced INS-1 cells were cancelled by PINK1 knockdown. CONCLUSIONS: BBR protects islet ß cells from PA-induced injury, and this protective effect may be achieved by regulating mitophagy. The present study may provide a novel therapeutic strategy for ß cell injury in diabetes mellitus.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Stem Cell Educator therapy in type 1 diabetes: From the bench to clinical trials.

Type 1 diabetes (T1D) is an autoimmune disease that causes a deficit of pancreatic islet ß cells. Millions of individuals worldwide have T1D, and its incidence increases annually. Recent clinical trials have highlighted the limits of conventional immunotherapy in T1D and underscore the need for novel treatments that not only overcome multiple immune dysfunctions, but also help restore islet ß-cell function. To address these two key issues, we have developed a unique and novel procedure designated the Stem Cell Educator therapy, based on the immune education by cord-blood-derived multipotent stem cells (CB-SC). Over the last 10 years, this technology has been evaluated through international multi-center clinical studies, which have demonstrated its clinical safety and efficacy in T1D and other autoimmune diseases. Mechanistic studies revealed that Educator therapy could fundamentally correct the autoimmunity and induce immune tolerance through multiple molecular and cellular mechanisms such as the expression of a master transcription factor autoimmune regulator (AIRE) in CB-SC for T-cell modulation, an expression of Galectin-9 on CB-SC to suppress activated B cells, and secretion of CB-SC-derived exosomes to polarize human blood monocytes/macrophages into type 2 macrophages. Educator therapy is the leading immunotherapy to date to safely and efficiently correct autoimmunity and restore ß cell function in T1D patients.

On the causal relationships between hyperinsulinaemia, insulin resistance, obesity and dysglycaemia in type 2 diabetes.

Hundreds of millions of people are affected by hyperinsulinaemia, insulin resistance, obesity and the dysglycaemia that mark a common progression from metabolic health to type 2 diabetes. Although the relative contribution of these features and the order in which they appear may differ between individuals, the common clustering and seemingly progressive nature of type 2 diabetes aetiology has guided research and clinical practice in this area for decades. At the same time, lively debate around the causal relationships between these features has continued, as new data from human trials and highly controlled animal studies are presented. This 'For debate' article was prompted by the review in Diabetologia by Esser, Utzschneider and Kahn ( https://doi.org/10.1007/s00125-020-05245-x ), with the purpose of reviewing established and emerging data that provide insight into the relative contributions of hyperinsulinaemia and impaired glucose-stimulated insulin secretion in progressive stages between health, obesity and diabetes. It is concluded that these beta cell defects are not mutually exclusive and that they are both important, but at different stages.

Smad3 deficiency promotes beta cell proliferation and function in db/db mice via restoring Pax6 expression.

Rationale: Transforming Growth Factor-beta (TGF-ß) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown. Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function. Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro. Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Pancreatic Duct Cells Isolated From Canines Differentiate Into Beta-Like Pancreatic Islet Cells.

In this study, we isolated and cultured pancreatic ductal cells from canines and revealed the possibility for using them to differentiate into functional pancreatic beta cells in vitro. Passaged pancreatic ductal cells were induced to differentiate into beta-like pancreatic islet cells using a mixture of induced factors. Differentiated pancreatic ductal cells were analyzed based on intracellular insulin granules using transmission electron microscopy, the expression of insulin and glucagon using immunofluorescence, and glucose-stimulated insulin secretion using ELISA. Our data revealed that differentiated pancreatic ductal cells not only expressed insulin and glucagon but also synthesized insulin granules and secreted insulin at different glucose concentrations. Our study might assist in the development of effective cell therapies for the treatment of type 1 diabetes mellitus in dogs.

Inhibition of heparanase protects against pancreatic beta cell death in streptozotocin-induced diabetic mice via reducing intra-islet inflammatory cell infiltration.

BACKGROUND AND PURPOSE: Intra-islet heparan sulfate (HS) plays an important role in the maintenance of pancreatic islet function. The aim of this study was to investigate the effect mechanism of HS loss on the functioning of islets in diabetic mice. EXPERIMENTAL APPROACH: The hypoglycaemic effect of a heparanase inhibitor, OGT2115, was tested in a streptozotocin-induced diabetic mouse model. The islets in pancreatic sections were also stained to reveal their morphology. An insulinoma cell line (MIN6) and primary isolated murine islets were used to investigate the effect of OGT2115 in vitro. KEY RESULTS: Intra-islet HS was clearly lost in streptozotocin-induced diabetic mice due to the increased heparanase expression in damaged islets. OGT2115 prevented intra-islet HS loss and improved the glucose profile and insulin secretion in streptozotocin-treated mice. The apoptosis of pancreatic beta cells and the infiltration of mononuclear macrophages, CD4- and CD8-positive T-cells in islets was reduced by OGT2115 in streptozotocin-treated mice, but OGT2115 did not alter the direct streptozotocin-induced damage in vitro. The expression of heparanase was increased in high glucose-treated isolated islets but not in response to direct streptozotocin stimulation. Further experiments showed that high glucose stimuli could decreased expression of PPARγ in cultured islets, thereby relieving the PPARγ-induced inhibition of heparanase gene expression. CONCLUSION AND IMPLICATIONS: Hyperglycaemia could cause intra-islet HS loss by elevating the expression of heparanase, thereby aggravating inflammatory cell infiltration and islet damage. Inhibition of heparanase might provide benefit for pancreatic beta cell protection in Type 1 diabetes.

Relationship between circular RNA and type 2 diabetes and its clinical application / 中国药科大学学报

@#Circular RNA (circRNA) is a novel type of non-coding RNA with covalently closed loops which plays an important role in the occurrence and development of type 2 diabetes. In this paper, the relationship between circRNA and type 2 diabetes in terms of the regulation of pancreatic β-cell function by circRNA and the metabolic activity of heart, kidney and other organs is reviewed, and the possibility of circRNA as a clinical diagnostic marker for type 2 diabetes and its complications is emphasized, hoping to provide reference and clues for the prevention, diagnosis and treatment of type 2 diabetes.

Mechanisms of the amplifying pathway of insulin secretion in the ß cell.

Pancreatic islet ß cells secrete insulin in response to nutrient secretagogues, like glucose, dependent on calcium influx and nutrient metabolism. One of the most intriguing qualities of ß cells is their ability to use metabolism to amplify the amount of secreted insulin independent of further alterations in intracellular calcium. Many years studying this amplifying process have shaped our current understanding of ß cell stimulus-secretion coupling; yet, the exact mechanisms of amplification have been elusive. Recent studies utilizing metabolomics, computational modeling, and animal models have progressed our understanding of the metabolic amplifying pathway of insulin secretion from the ß cell. New approaches will be discussed which offer in-roads to a more complete model of ß cell function. The development of ß cell therapeutics may be aided by such a model, facilitating the targeting of aspects of the metabolic amplifying pathway which are unique to the ß cell.

Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic ß cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 ß cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence ß-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in ß cells will have implications for diabetes and other emergent fields involving these receptors.

The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds.

The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive 'omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes.

Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells.

OBJECTIVE: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. METHODS: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. RESULTS: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. CONCLUSIONS: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

Adjusting the 17ß-Estradiol-to-Androgen Ratio Ameliorates Diabetic Nephropathy.

Diabetes is manifested predominantly in males in experimental models, and compelling evidence suggests that 17ß-estradiol (E2) supplementation improves hyperglycemia in humans. We previously generated a severely diabetic transgenic (Tg) mouse model by ß-cell­specific overexpression of inducible cAMP early repressor (ICER) and found that male but not female ICER-Tg mice exhibit sustained hyperglycemia and develop major clinical and pathologic features of human diabetic nephropathy (DN). Thus, we hypothesized that differences in circulating hormone levels have a key role in determining susceptibility to diabetes. Here, we examined whether DN in male ICER-Tg mice is rescued by adjusting the androgen-to-E2 ratio to approximate that in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX), E2 pellet implantation, or both. E2 pellet implantation at an early stage of DN with or without ORX caused a rapid drop in blood glucose and a dramatic increase in ß-cell number, and it markedly inhibited DN progression [namely, E2 reduced glomerulosclerosis, collagen 4 deposition and albuminuria, and prevented hyperfiltration]. Furthermore, E2 pellet implantation was more effective than ORX alone and induced a remarkable improvement, even when initiated at advanced-stage DN. In contrast, induction of normoglycemia by islet transplant in ICER-Tg mice eliminated albuminuria but was less effective than E2 + ORX in reducing glomerulosclerosis, collagen 4 deposition, and hyperfiltration. These findings indicate that E2 treatment is effective, even after establishment of DN, whereas glucose normalization alone does not improve sclerotic lesions. We propose that E2 intervention is a potential therapeutic option for DN.