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Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.

The advent of the technology to isolate or generate human pluripotent stem cells provided the potential to develop a wide range of human models that could enhance understanding of mechanisms underlying human development and disease. These systems are now beginning to mature and provide the basis for the development of in vitro assays suitable to understand the biological processes involved in the multi-organ systems of the human body, and will improve strategies for diagnosis, prevention, therapies and precision medicine. Induced pluripotent stem cell lines are prone to phenotypic and genotypic changes and donor/clone dependent variability, which means that it is important to identify the most appropriate characterization markers and quality control measures when sourcing new cell lines and assessing differentiated cell and tissue culture preparations for experimental work. This paper considers those core quality control measures for human pluripotent stem cell lines and evaluates the state of play in the development of key functional markers for their differentiated cell derivatives to promote assurance of reproducibility of scientific data derived from pluripotent stem cell-based systems.

Summary The discovery of insulin a century ago changed the lives of millions of individuals suffering from diabetes, paving the way for long-term survival. While the availability of recombinant insulin for hormone replacement therapy has served extremely well to help control blood glucose in diabetes, there remains significant room for further improvements for an ultimate "cure" for diabetes patients. In this review, we celebrate the 100th anniversary of the discovery of insulin and consolidate the key milestones and advances in the development of recombinant human insulin. We then summarize recent and current technological developments in terms of insulin gene- and cell-replacement therapies that are promising in greater therapeutic potential. We envision that the next era of insulin replacement therapies will effectively treat diabetes and serve our patients even better for the next century to come.

We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Background and aims: Single nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. Methods and Results: We generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63- 89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of ‘A’ risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. Conclusions: Our study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version The National Research Foundation, Singapore (Project Number 370062002) funded the Singapore Coronary Artery Disease Genetics Study (SCADGENS) and genotyping of the participants. The team from Nanyang Technological University Singapore was funded by an Academic Research Fund Tier 1 grant (2018-T1-001-030) from the Ministry of Education, Singapore, Human Frontier Science Program Research Grant (RGY0069/2019), and the Nanyang Assistant Professorship. K.Y. T. is supported by NTU Research Scholarship. H.H.L. is supported by the Institute of Molecular and Cell Biology (IMCB) Scientific Staff Development Award (SSDA) for her part-time Ph.D. A.K.K.T. is supported by IMCB, A*STAR, Precision Medicine and Personalised Therapeutics Joint Research Grant 2019, the 2nd A*STAR-AMED Joint Grant Call 192B9002 and NUHSRO/2021/035/NUSMed/04/NUS-IMCB Joint Lab/LOA.

Diabetes is a major healthcare burden globally, affecting over 463 million people today, according to the International Diabetes Federation. The most common types of diabetes are Type I diabetes (T1D) and Type II diabetes (T2D), characterized by hyperglycemia due to autoimmune destruction of β cells (T1D) and β cell dysfunction, usually on a background of insulin resistance (T2D). There is currently no cure for diabetes, and patients with T1D require lifelong insulin therapy. Additionally, while most cases of T2D can be managed by lifestyle and diet modifications, with or without antidiabetic drugs, severe cases of T2D may also require insulin therapy. The only means to restore stable euglycemia in these patients is now via whole pancreas or islet transplantation. However, this is limited by the scarcity of donors. In recent years, advances in human pluripotent stem cell (hPSC) technologies and pancreatic β cell differentiation protocols have opened up new potential avenues for cell replacement therapies for diabetes. These advances have also created opportunities to use hPSC-derived β-like cells for studies of disease mechanisms and drug discovery, which in turn have the potential to lead to better therapies for diabetes patients. Here, we describe the protocol used in our laboratory to generate β-like cells from hPSCs to study the mechanisms underlying various types of diabetes.

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived β-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients., Heterozygous HNF1A mutations can give rise to maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. Here the authors show that MODY3-related HNF1A mutation in patient hiPSCderived pancreatic cells decreases glucose transporter GLUT2 expression due to compromised DNA binding.

Chromatin immunoprecipitation (ChIP) is a technique that has been widely used to interrogate DNA-protein interactions in cells. In recent years, human pluripotent stem cell (hPSC)-derived 3D organoids have emerged as a powerful model to understand human development and diseases. Performing ChIP in hPSC-derived 3D organoids is a useful approach to dissect the roles of transcription factors or co-factors and to understand the epigenetic landscape in human development and diseases. However, performing ChIP in 3D organoids is more challenging than monolayer cultures, and an optimized protocol is needed for interpretable data. Hence, in this chapter, we describe in detail a protocol for performing ChIP in hPSC-derived islet-like cells as an example, from organoid harvest to ChIP-qPCR data analysis. This chapter also highlights potential pitfalls and provides recommendations for troubleshooting.

The unlimited proliferative capacity of human pluripotent stem cells (hPSCs) fortifies it as one of the most attractive sources for cell therapy application in diabetes. In the past two decades, vast research efforts have been invested in developing strategies to differentiate hPSCs into clinically suitable insulin-producing endocrine cells or functional beta cells (β cells). With the end goal being clinical translation, it is critical for hPSCs and insulin-producing β cells to be derived, handled, stored, maintained and expanded with clinical compliance. This review focuses on the key processes and guidelines for clinical translation of human induced pluripotent stem cell (hiPSC)-derived β cells for diabetes cell therapy. Here, we discuss the (1) key considerations of manufacturing clinical-grade hiPSCs, (2) scale-up and differentiation of clinical-grade hiPSCs into β cells in clinically compliant conditions and (3) mandatory quality control and product release criteria necessitated by various regulatory bodies to approve the use of the cell-based products.

Although IKK-β has previously been shown as a negative regulator of IL-1β secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1β expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1β secretion was elevated in the patient’s stimulated leukocytes, in her induced pluripotent stem cell–derived macrophages, and in murine bone marrow–derived macrophages containing the L34P mutation. The patient’s hypersecretion of IL-1β correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1β secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition. Refereed/Peer-reviewed

Pancreatic β-cells are responsible for maintaining glucose homeostasis. Therefore, their dysregulation leads to diabetes. Pancreas or islet transplants can be used to treat diabetes but these human tissues remain in short supply. Significant progress has now been made in differentiating human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into pancreatic β-like cells for potential cell replacement therapy. Additionally, these hPSC-derived β-like cells represent a new invaluable model for studying diabetes disease mechanisms. Here, we review the use of hPSC-derived β-like cells as a platform to model various types of defects in human β-cells in diabetes, comparing them against existing animal models, ex vivo human islets and human β-cell line. We also discuss how hPSC-derived β-like cells are being used as a platform for screening novel therapeutic compounds. Last but not least, we evaluate the strengths and limitations of this human cell-based platform as an avenue to study and reveal new insights into human β-cell biology.

Beta cells assume a fundamental role in maintaining blood glucose homeostasis through the secretion of insulin, which is contingent on both beta cell mass and function, in response to elevated blood glucose levels or secretagogues. For this reason, evaluating beta cell mass and function, as well as scrutinizing how they change over time in a diabetic state, are essential prerequisites in elucidating diabetes pathophysiology. Current clinical methods to measure human beta cell mass and/or function are largely lacking, indirect and sub-optimal, highlighting the continued need for noninvasive in vivo beta cell imaging technologies such as optical imaging techniques. While numerous probes have been developed and evaluated for their specificity to beta cells, most of them are more suited to visualize beta cell mass rather than function. In this review, we highlight the distinction between beta cell mass and function, and the importance of developing more probes to measure beta cell function. Additionally, we also explore various existing probes that can be employed to measure beta cell mass and function in vivo, as well as the caveats in probe development for in vivo beta cell imaging.

SummarySelection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

The effects of imeglimin, a novel anti-diabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation, after treatment with thapsigargin, and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell (hPSC)-derived β-like cells. Taken together, imeglimin modulates ER homeostasis pathway, which results in the prevention of β-cell apoptosis both in vitro and in vivo.

Type 1 diabetes (T1D) is characterized by pancreatic islet infiltration by autoreactive immune cells and a nearly complete loss of β cells1. Restoration of insulin-producing β cells coupled with immunomodulation to suppress the autoimmune attack has emerged as a potential approach to counter T1D2–4. Here we report that enhancing β-cell mass early in life, in two models of female non-obese diabetic (NOD) mice, results in immunomodulation of T cells, reduced islet infiltration and lower β-cell apoptosis, which together protect them from developing T1D. The animals displayed altered β-cell antigens; islet transplantation studies showed prolonged graft survival in the NOD-liver-specific insulin receptor knockout (LIRKO) model. Adoptive transfer of splenocytes from NOD-LIRKO mice prevented development of diabetes in prediabetic NOD mice. A substantial increase in the splenic CD4+CD25+Foxp3+ regulatory T cell (Treg) population was observed to underlie the protected phenotype since Treg-cell depletion rendered NOD-LIRKO mice diabetic. An increase in Treg cells coupled with activation of transforming growth factor-β/SMAD family member 3 signalling pathway in pathogenic T cells favoured reduced ability to kill β cells. These data support a previously unidentified observation that initiating β-cell proliferation, alone, before islet infiltration by immune cells alters the identity of β cells, decreases pathological self-reactivity of effector T cells and increases Treg cells to prevent the progression of T1D. Type 1 diabetes (T1D) involves immune-mediated destruction of pancreatic β cells. Here, the authors show that inducing β-cell replication before immune cell infiltration of the pancreas alters β-cell antigen expression and prevents T1D disease progression in female NOD mice in a regulatory-T-cell-dependent manner.

The paired box 6 (PAX6) transcription factor is crucial for normal pancreatic islet development and function. Heterozygous mutations of PAX6 are associated with impaired insulin secretion and early-onset diabetes mellitus in humans. However, the molecular mechanism of PAX6 in controlling insulin secretion in human beta cells and its pathophysiological role in type 2 diabetes (T2D) remain ambiguous. We investigated the molecular pathway of PAX6 in the regulation of insulin secretion and the potential therapeutic value of PAX6 in T2D by using human pancreatic beta cell line EndoC-βH1, the db/db mouse model, and primary human pancreatic islets. Through loss- and gain-of-function approaches, we uncovered a mechanism by which PAX6 modulates glucose-stimulated insulin secretion (GSIS) through a cAMP response element-binding protein (CREB)/Munc18-1/2 pathway. Moreover, under diabetic conditions, beta cells and pancreatic islets displayed dampened PAX6/CREB/Munc18-1/2 pathway activity and impaired GSIS, which were reversed by PAX6 replenishment. Adeno-associated virus-mediated PAX6 overexpression in db/db mouse pancreatic beta cells led to a sustained amelioration of glycemic perturbation in vivo but did not affect insulin resistance. Our study highlights the pathophysiological role of PAX6 in T2D-associated beta cell dysfunction in humans and suggests the potential of PAX6 gene transfer in preserving and restoring beta cell function.

Metformin is becoming a popular treatment before and during pregnancy but current literature on in utero exposure to metformin lacks long-term clinical trials and mechanistic studies. Current literature on the effects of metformin on mature pancreatic β cells highlighted its dual, opposing, protective or inhibitory, effects depending on metabolic environments. However, the impact of metformin on developing human pancreatic β cells remains unknown. Here, we investigated the potential effects of metformin exposure on human pancreatic β cell development and function in vitro. In the absence of metabolic challenges such as high levels of glucose and fatty acids, metformin exposure impaired the development and function of pancreatic β cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic β cells. These findings call for further in-depth evaluation of the exposure of human embryonic and fetal tissue during pregnancy to metformin, and its implications on long-term offspring health.

There is now a sizeable number of single cell transcriptomics studies performed on human and rodent pancreatic islets that have shed light on the unique gene signatures and level of heterogeneity within each individual islet cell type. Following closely from these studies, there is also rapidly-growing activity on characterizing islet-like cells derived from in vitro differentiation of human pluripotent stem cells (hPSCs) at the single cell level. The overall consensus across the studies so far suggests that the first few stages of differentiation are largely uniform, whereas during pancreatic endocrine commitment, cell trajectories start to diverge, resulting in multiple end-stage pancreatic cells that include progenitor-like, endocrine and non-endocrine cells. Comprehensive transcriptional profiling is important for understanding how and why islet cells, especially the insulin-secreting beta cells, exist in subpopulations that differ in maturity, proliferation rate, sensitivity to stress, and insulin secretion function. For hPSC-derived beta cells to be used confidently for cell therapy, optimal differentiation and thorough characterization is required. The key questions to address are-What is the trajectory of differentiation? Is heterogeneity a natural occurrence or is it a consequence of imperfect differentiation protocols? Can lessons be drawn from the extensive single cell transcriptomic data to help guide maturation of beta cells in vitro? This book chapter seeks to address some of these questions, and facilitate ongoing efforts in improving the beta cell differentiation pipeline or enriching for desired beta cell populations following differentiation, to make way for better mechanistic studies and future clinical translation.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing beta cells in the pancreas. Consequently, T1D patients produce little to no insulin, resulting in absolute insulin deficiency and an inability to regulate blood glucose levels effectively. Insulin replacement is the mainstream treatment for T1D, but it comes with a major drawback—the risk of hypoglycemia unawareness. As such, replenishment of glucose-sensing and insulin-producing beta cells via cell replacement therapy is believed to have great therapeutic value for T1D patients. Human pluripotent stem cells (hPSCs) are considered an attractive source for the derivation of insulin-producing beta-like cells for cell replacement therapy. However, several challenges need to be addressed before hPSC-derived beta-like cells can be used for T1D treatment. In this chapter, we provide a snapshot of the current progress of hPSC therapy with an emphasis on T1D, discuss some of the clinical considerations and technical strategies in the generation of current good manufacturing practice (cGMP)-compliant hPSC-derived beta-like cells and the future outlook for T1D hPSC therapy.

There is now a sizeable number of single cell transcriptomics studies performed on human and rodent pancreatic islets that have shed light on the unique gene signatures and level of heterogeneity within each individual islet cell type. Following closely from these studies, there is also rapidly-growing activity on characterizing islet-like cells derived from in vitro differentiation of human pluripotent stem cells (hPSCs) at the single cell level. The overall consensus across the studies so far suggests that the first few stages of differentiation are largely uniform, whereas during pancreatic endocrine commitment, cell trajectories start to diverge, resulting in multiple end-stage pancreatic cells that include progenitor-like, endocrine and non-endocrine cells. Comprehensive transcriptional profiling is important for understanding how and why islet cells, especially the insulin-secreting beta cells, exist in subpopulations that differ in maturity, proliferation rate, sensitivity to stress, and insulin secretion function. For hPSC-derived beta cells to be used confidently for cell therapy, optimal differentiation and thorough characterization is required. The key questions to address are—What is the trajectory of differentiation? Is heterogeneity a natural occurrence or is it a consequence of imperfect differentiation protocols? Can lessons be drawn from the extensive single cell transcriptomic data to help guide maturation of beta cells in vitro? This book chapter seeks to address some of these questions, and facilitate ongoing efforts in improving the beta cell differentiation pipeline or enriching for desired beta cell populations following differentiation, to make way for better mechanistic studies and future clinical translation.

We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells.We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers.The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INSThe results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Objective Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. Methods We used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. Results βStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of “rod-like” dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3’ position. Lipidomic analysis of βStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in βStard10KO islets. Conclusion Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing., Highlights • βStard10KO β-cells show altered granule morphology. • Deletion of Stard10 increased basal secretion of newly synthesised proinsulin. • βStard10KO islets had an altered lipidomics profile, including phosphatidylinositols. • STARD10 bound to phosphoinositides in a lipid overlay assay. • STARD10 structure reveals that its cavity readily accommodates phosphatidylinositols.

Current complexities in human pluripotent stem cell (hPSC)-based studies. hPSC studies begin with the recruitment of patients harboring disease-associated gene variant(s) that may increase susceptibility to disease development. Somatic reprogramming is then performed to derive patient-specific human induced pluripotent stem cells (hiPSCs), followed by step-wise directed differentiation to a relevant cell type before qualitative/quantitative assays are performed to assess for phenotypic or gene expression differences between the healthy and diseased hiPSCs.

Pancreatic β cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic β cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic β cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic β cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

For more than 40 years, metformin has been used before and during pregnancy. However, it is important to note that metformin can cross the placenta and circulate in the developing foetus. Recent studies reported that the concentration of metformin in foetal cord blood ranges from half to nearly the same concentration as in the maternal plasma. Since metformin has anti-cell growth and pro-apoptotic effects, there are persistent concerns over the use of metformin in early pregnancy. Current human studies are limited by sample size, lack of controls or, short follow-up durations. In this review, we examine the settings in which metformin can be passed on from mother to child during pregnancy and address the current controversies relating to the cellular and molecular mechanisms of metformin. Our efforts highlight the need for more data on the effects of metformin on general offspring health as well as further scrutiny into foetal development upon exposure to metformin. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

The differentiation of human pluripotent stem cells into pancreatic cells involves cellular proliferation and apoptosis during cell fate transitions. However, their implications for establishing cellular identity are unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification and observed an upregulation of BCL-xL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. Experimental inhibition of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in anti-apoptotic protein BCL-2. RNA-Seq analyses then revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL. Follow-up bioenergetics assays revealed broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL was inhibited. Early perturbation of BCL-xL during pancreatic specification also had subsequent detrimental effects on the formation of INS+ pancreatic beta-like cells. In conclusion, the more differentiated pancreatic progenitors are dependent on anti-apoptotic BCL-xL for survival, whereas the less differentiated pancreatic progenitors that survived after WEHI-539 treatment would exhibit a more immature phenotype. Therefore, modulation of the expression level of BCL-xL can potentially increase the survival and robustness of pancreatic progenitors that ultimately define human pancreatic beta cell mass and function.

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.