Search

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.

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.

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.

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.

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.

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

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.

The identification of new sources of β cells is an important endeavor with therapeutic implications for diabetes. Insulin resistance, in physiological states such as pregnancy or in pathological states such as type 2 diabetes (T2D), is characterized by a compensatory increase in β cell mass. To explore the existence of a dynamic β cell reserve, we superimposed pregnancy on the liver-specific insulin receptor–KO (LIRKO) model of insulin resistance that already exhibits β cell hyperplasia and used lineage tracing to track the source of new β cells. Although both control and LIRKO mice displayed increased β cell mass in response to the relative insulin resistance of pregnancy, the further increase in mass in the latter supported a dynamic source that could be traced to pancreatic ducts. Two observations support the translational significance of these findings. First, NOD/SCID-γ LIRKO mice that became pregnant following cotransplantation of human islets and human ducts under the kidney capsule showed enhanced β cell proliferation and an increase in ductal cells positive for transcription factors expressed during β cell development. Second, we identified duct cells positive for immature β cell markers in pancreas sections from pregnant humans and in individuals with T2D. Taken together, during increased insulin demand, ductal cells contribute to the compensatory β cell pool by differentiation/neogenesis.

When diabetes is diagnosed, the majority of insulin-secreting pancreatic β cells are already dysfunctional or destroyed. This β cell dysfunction/destruction usually takes place over many years, making timely detection and clinical intervention difficult. For this reason, there is immense interest in developing tools to bioimage β cell mass and/or function noninvasively to facilitate early diagnosis of diabetes as well as to assist the development of novel antidiabetic therapies. Recent years have brought significant progress in β cell imaging that is now inching towards clinical applicability. We explore here the need to bioimage human β cells noninvasively in various types of diabetes, and we discuss current and emerging tools for bioimaging β cells. Further developments in this field are expected to facilitate β cell imaging in diabetes.

Summary Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and β cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and β-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and β cell defects in patients., Graphical Abstract, Highlights • HNF4A is downregulated and predominantly mislocalized in the cytoplasm in MODY1 • Foregut markers, pancreatic and hepatic genes, were downregulated in MODY1-HPPs • A reciprocal upregulation of hindgut HOX genes was observed in MODY1-HPPs • Mutant HNF4A resulted in loss of transcriptional activation of target genes, Molecular Mechanism of Gene Regulation; Diabetology; Stem Cells Research; Developmental Biology

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry–based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.

A major goal of diabetes research is to develop strategies that replenish pancreatic insulin-producing beta cells. One emerging strategy is to harness pancreatic plasticity—the ability of pancreatic cells to undergo cellular interconversions—a phenomenon implicated in physiological stress and pancreatic injury. Here, we investigate the effects of inflammatory cytokine stress on the differentiation potential of ductal cells in a human cell line, in mouse ductal cells by pancreatic intraductal injection, and during the progression of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. We find that inflammatory cytokine insults stimulate epithelial-to-mesenchymal transition (EMT) as well as the endocrine program in human pancreatic ductal cells via STAT3-dependent NGN3 activation. Furthermore, we show that inflammatory cytokines activate ductal-to-endocrine cell reprogramming in vivo independent of hyperglycemic stress. Together, our findings provide evidence that inflammatory cytokines direct ductal-to-endocrine cell differentiation, with implications for beta cell regeneration. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Summary Patients with an HNF1BS148L/+ mutation (MODY5) typically exhibit pancreatic hypoplasia. However, the molecular mechanisms are unknown due to inaccessibility of patient material and because mouse models do not fully recapitulate MODY5. Here, we differentiated MODY5 human-induced pluripotent stem cells (hiPSCs) into pancreatic progenitors, and show that the HNF1BS148L/+ mutation causes a compensatory increase in several pancreatic transcription factors, and surprisingly, a decrease in PAX6 pancreatic gene expression. The lack of suppression of PDX1, PTF1A, GATA4, and GATA6 indicates that MODY5-mediated pancreatic hypoplasia is mechanistically independent. Overexpression studies demonstrate that a compensatory increase in PDX1 gene expression is due to mutant HNF1BS148L/+ but not wild-type HNF1B or HNF1A. Furthermore, HNF1B does not appear to directly regulate PAX6 gene expression necessary for glucose tolerance. Our results demonstrate compensatory mechanisms in the pancreatic transcription factor network due to mutant HNF1BS148L/+ protein. Thus, patients typically develop MODY5 but not neonatal diabetes despite exhibiting pancreatic hypoplasia., Graphical Abstract, Highlights • HNF1BS148L/+ mutation elicits a compensatory increase in DE and pancreatic genes • MODY5-mediated pancreatic hypoplasia is independent of PDX1, PTF1A, GATA4, and GATA6 • HNF1BS148L mutation directly causes a compensatory increase in PDX1 gene expression • HNF1BS148L/+ mutation limits PAX6 expression and consequently leads to MODY5, Kulkarni, Teo, and colleagues differentiated MODY5-hiPSCs into pancreatic progenitors and demonstrated that HNF1BS148L/+ mutation elicits a compensatory mechanism in the pancreatic transcription factor network. They report that MODY5-mediated pancreatic hypoplasia is independent of PDX1, PTF1A, GATA4, and GATA6. In addition, a decrease in PAX6 gene expression may lead to MODY5 development.

Chemotherapy still remains the major treatment option for cancer patients. However, it is often imperative to use chemotherapy in conjunction with other pharmacological drugs, in order to minimize adverse side effects and to circumvent drug resistance. Natural products are increasingly being applied as an adjuvant therapy in combination with existing chemotherapy regimens to increase treatment efficacy. A promising example of one such natural agent, which may have an enormous potential to be exploited as a chemosensitizer, is a triterpene compound called celastrol. It was first identified from thunder god vine and can negatively regulate various oncogenic molecules such as NF-κB, topoisomerase II, Akt/mTOR, HSP90, STAT3, and Notch-1. These can lead to an antiinflammatory response, inhibition of tumor growth and survival, and abrogation of angiogenesis. This chapter briefly summarizes the potential role of celastrol as a chemosensitizer and the underlying molecular mechanism(s) regulating its reported chemosensitizing effects in diverse cancers.

Type 1 and type 2 diabetes are caused by a destruction and decrease in the number of functional insulin-producing β cells, respectively; therefore, the generation of functional β cells from human embryonic stem cells and human induced pluripotent stem cells, collectively known as human pluripotent stem cells (hPSCs), for potential cell replacement therapy and disease modelling is an intensely investigated area. Recent scientific breakthroughs enabled derivation of large quantities of human pancreatic β-like cells in vitro, although with varied glucose-stimulated insulin secretion kinetics. In the present review, we comprehensively summarize, compare and critically analyze the intricacies of these developing technologies, including differentiation platforms, robustness of protocols, and methodologies used to characterize hPSC-derived β-like cells. We also discuss experimental issues that need to be resolved before these β-like cells can be used clinically.

Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells, leading to insulin deficiency. Although infiltrating immune cells are traditionally considered to negatively impact β-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of β-cells that positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4+ and CD8+ T cells, but not B cells, selectively promoted β-cell proliferation in vivo independent from the effects of blood glucose or circulating insulin or by modulating apoptosis. Complementary to our in vivo approach, coculture of diabetogenic CD4+ and CD8+ T cells with NOD.RAG1−/− islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokines/chemokines (interleukin-2 [IL-2], IL-6, IL-10, MIP-1α, and RANTES) that together enhanced β-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance β-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes.

Because noncommunicable diseases such as type 2 diabetes mellitus have their roots in prenatal development and conditions such as maternal gestational diabetes mellitus (GDM), we aimed to test this hypothesis in primary cells derived from the offspring of mothers with GDM compared with control subjects. We have assessed primary umbilical cord–derived cells such as human umbilical vein endothelial cells (HUVECs) and Wharton’s jelly–derived mesenchymal stem cells from the offspring of mothers with and without GDM. We have compared the primary isolates in cell-based assays measuring proliferation, mitochondrial oxygen consumption, and the ability to support blood vessel growth. We conducted gene expression microarray studies with subsequent pathway analysis and candidate gene validation. We observed striking differences between the two groups, such as lower metabolic rates and impairment of endothelial tube formation in cells with GDM background. HUVECs from subjects with maternal GDM have lower expression of the antiapoptotic protein BCL-xL, suggesting compromised angiogenic capabilities. Comparative gene expression analysis revealed blood vessel formation as a major pathway enriched in the GDM-derived HUVECs with the surface marker CD44 as a gene underexpressed in the GDM group. Functional validation of CD44 revealed that it regulates tube formation in HUVECs, thereby providing insights into a pathway imprinted in primary umbilical cord–derived cells from GDM offspring. Our data demonstrate that primary cells isolated from the umbilical cord of offspring born to mothers with GDM maintain metabolic and molecular imprints of maternal hyperglycemia, reflecting an increased risk for cardiovascular disease later in life.

In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1+ pancreatic progenitors, much less is known about the transition toward Ngn3+ pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells in vitro, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments. Published version

Summary There is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs., Graphical Abstract, Highlights • High dose of WNT3A cooperates with Activin to induce DE without serum • GSK-3 inhibitors, CHIR99021 and BIO, work with Activin to induce DE without serum • Wnt and BMP signaling can induce DE with comparable efficiencies without serum • WNT3A is not as effective as BMP4, CHIR99021, or BIO in suppressing pluripotency, It is yet unclear how Wnt compares with BMP signaling in promoting Activin-induced definitive endoderm formation from human pluripotent stem cells in serum-free conditions. Kulkarni and colleagues demonstrate that Wnt or BMP signaling generates Activin-induced definitive endoderm comparably and that WNT3A is ineffective at suppressing E-CADHERIN and pluripotency.

The landmark discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka has transformed regenerative biology. Previously, insights into the pathogenesis of chronic human diseases have been hindered by the inaccessibility of patient samples. However, scientists are now able to convert patient fibroblasts into iPSCs and differentiate them into disease-relevant cell types. This ability opens new avenues for investigating disease pathogenesis and designing novel treatments. In this review, we highlight the uses of human iPSCs to uncover the underlying causes and pathological consequences of diabetes and metabolic syndromes, multi-factorial diseases whose etiologies have been difficult to unravel using traditional methodologies.