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Multiple pancreatic islet single-cell RNA sequencing (scRNA-seq) datasets have been generated to study development, homeostasis, and diabetes. However, there is no consensus on cell states and pathways across conditions as well as the value of preclinical mouse models. Since these challenges can only be resolved by jointly analyzing multiple datasets, we present a scRNA-seq cross-condition mouse islet atlas (MIA). We integrated over 300,000 cells from nine datasets with 56 samples, varying in age, sex, and diabetes models, including an autoimmune type 1 diabetes (T1D) model (NOD), a gluco-/lipotoxicity T2D model (db/db), and a chemical streptozotocin (STZ) β-cell ablation model. MIA is a curated resource for interactive exploration and computational querying, providing new insights inaccessible from individual datasets. The β-cell landscape of MIA revealed new disease progression cell states and cross-publication differences between previously suggested marker genes. We show that in the STZ model β-cells transcriptionally correlate to human T2D and mouse db/db, but are less similar to human T1D and mouse NOD. We observe different pathways shared between immature, aged, and diabetes model β-cells. In conclusion, our work presents the first comprehensive analysis of β-cell responses to different stressors, providing a roadmap for the understanding of β-cell plasticity, compensation, and demise.

Tremendous progress has been made over the last two decades in the field of pancreatic beta cell replacement therapy as a curative measure for diabetes. Transplantation studies have demonstrated therapeutic efficacy, and cGMP-grade cell products are currently being deployed for the first time in human clinical trials. In this perspective, we discuss current challenges surrounding the generation, delivery, and engraftment of stem cell-derived islet-like cells, along with strategies to induce durable tolerance to grafted cells, with an eye toward a functional cellular-based therapy enabling insulin independence for patients with diabetes.

Pancreatic beta cells differ in terms of glucose responsiveness, insulin secretion and proliferative capacity; however, the molecular pathways that regulate this cellular heterogeneity are unknown. We have identified the Wnt-planar cell polarity (PCP) effector Flattop (FLTP) as a biomarker that identifies mature beta cells in the islets of Langerhans. Interestingly, three-dimensional architecture and Wnt-PCP ligands are sufficient to trigger mouse and human beta cell maturation. These results highlight the fact that novel biomarkers shed light on the long-standing mystery of beta cell heterogeneity and identify the Wnt-PCP pathway as triggering beta cell maturation. Understanding heterogeneity in the islets of Langerhans might allow targeting of beta cell subpopulations for regenerative therapy and provide building principles for stem cell-derived islets. This review summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Harry Heimberg and colleagues, DOI: 10.1007/s00125-016-3879-6 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ).

In this review, we focus on the processes guiding human pancreas development and provide an update on methods to efficiently generate pancreatic progenitors (PPs) and β-like cells in vitro from human pluripotent stem cells (hPSCs). Furthermore, we assess the strengths and weaknesses of using PPs and β-like cell for cell replacement therapy for the treatment of Type 1 diabetes with respect to cell manufacturing, engrafting, functionality, and safety. Finally, we discuss the identification and use of specific cell surface markers to generate safer populations of PPs for clinical translation and to study the development of PPs in vivo and in vitro.

In pancreatic β-cells, mitochondrial bioenergetics control glucose-stimulated insulin secretion. Mitochondrial dynamics are generally associated with quality control, maintaining the functionality of bioenergetics. By acute pharmacological inhibition of mitochondrial fission protein

In pancreatic β-cells, mitochondrial bioenergetics control glucose-stimulated insulin secretion. Mitochondrial dynamics are generally associated with quality control, maintaining the functionality of bioenergetics. By acute pharmacological inhibition of mitochondrial fission protein Drp1, we demonstrate in this study that mitochondrial fission is necessary for glucose-stimulated insulin secretion in mouse and human islets. We confirm that genetic silencing of Drp1 increases mitochondrial proton leak in MIN6 cells. However, our comprehensive analysis of pancreatic islet bioenergetics reveals that Drp1 does not control insulin secretion via its effect on proton leak but instead via modulation of glucose-fueled respiration. Notably, pyruvate fully rescues the impaired insulin secretion of fission-deficient β-cells, demonstrating that defective mitochondrial dynamics solely affect substrate supply upstream of oxidative phosphorylation. The present findings provide novel insights into how mitochondrial dysfunction may cause pancreatic β-cell failure. In addition, the results will stimulate new thinking in the intersecting fields of mitochondrial dynamics and bioenergetics, as treatment of defective dynamics in mitochondrial diseases appears to be possible by improving metabolism upstream of mitochondria.

Although β-cell heterogeneity was discovered more than 50 years ago, the underlying principles have been explored only during the past decade. Islet-cell heterogeneity arises during pancreatic development and might reflect the existence of distinct populations of progenitor cells and the developmental pathways of endocrine cells. Heterogeneity can also be acquired in the postnatal period owing to β-cell plasticity or changes in islet architecture. Furthermore, β-cell neogenesis, replication and dedifferentiation represent alternative sources of β-cell heterogeneity. In addition to a physiological role, β-cell heterogeneity influences the development of diabetes mellitus and its response to treatment. Identifying phenotypic and functional markers to discriminate distinct β-cell subpopulations and the mechanisms underpinning their regulation is warranted to advance current knowledge of β-cell function and to design novel regenerative strategies that target subpopulations of β cells. In this context, the Wnt/planar cell polarity (PCP) effector molecule Flattop can distinguish two unique β-cell subpopulations with specific transcriptional signatures, functional properties and differential responses to environmental stimuli. In vivo targeting of these β-cell subpopulations might, therefore, represent an alternative strategy for the future treatment of diabetes mellitus.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.

Tcf7l2, also known as Tcf4, is a member of the HMG-box containing T-cell factor (Tcf)/Lymphoid enhancer factor (Lef) transcription factor family of DNA binding proteins downstream of the canonical Wnt pathway [1]. In addition to its well known role during development recent evidence suggests that the Wnt pathway is implicated in stem cell homeostasis, cancer development and metabolic disorders [2]. Thus, it is not surprising, that genome wide association studies (GWAS) have identified TCF7L2 as a locus conveying an increased risk for developing type 2 diabetes (T2D) for the homozygous carrier of the minor allele [3]. Since this finding, several studies have focused on understanding the mechanism underlying the metabolic function of Tcf7l2 in organs and in pancreatic β-cells. In the small intestine, Tcf7l2-dependent Wnt signaling is essential for maintenance of proliferating cells located in the “intervillus pockets” as well as for the differentiation towards the hormone-producing enteroendocrine lineage [2]. In these cells, Tcf7l2 regulates pro-glucagon expression, precursor of glucagon-derived hormones. In the adult pancreas, cumulative evidence suggests that Tcf7l2/β-catenin signaling is critical for Glucagon-like peptide 1 (Glp-1) induced β-cell proliferation and Stromal-derived factor 1 (Sdf-1)-mediated β-cell survival [4]. Moreover, recent data indicate that alternative splicing isoforms of TCF7L2 differently regulate β-cell proliferation and glucose-dependent insulin secretion in adult human islets [5]. Despite all these efforts, many questions remain, e.g. which metabolic organ requires Wnt/Tcf7l2 signaling for its function, when is Tcf7l2 function required for organ development and homeostasis, and is Tcf7l2 function conserved between the pre-clinical mouse model and human patients? Answers to these questions will unravel the contribution of TCF7L2 to the development of diabetes. In the April 2015 issue of Molecular Metabolism, Shao et al. provide new insight on the function of TCF7L2 in pancreatic β-cells [6]. The authors used an adenoviral vector system expressing a dominant-negative (DN) variant of TCF7L2 (TCF7L2DN) to attenuate canonical WNT/β-catenin signaling in insulinoma cells (Ins-1) and during pancreas development as well as in the adult islet. Specifically, forced expression of TCF7L2DN in Ins-1 cells represses β-cell proliferation, glucose-dependent insulin secretion and down-regulates key β-cell genes, such as MafA, Isl1, Pdx1 and the canonical Wnt target gene Axin2. Despite the lack of mechanistic data, this is an interesting starting point to investigate if MafA, Isl1 and Pdx1 represent novel pancreas specific target genes of TCF7L2/β-catenin signaling pathway. Additionally, Shao et al. observed the down regulation of Glp-1 receptor and Glucose-dependent insulin tropic polypeptide (Gip) receptor following the expression of TCF7L2DN. This is consistent with a previous study where the correlation between Glp-1 and TCF7L2/β-catenin pathway was already established [7]. Specifically, it was shown that activation of Glp-1 increased canonical Wnt signaling in Ins-1 cells and overexpression of TCF7L2DN was found to repress Glp-1 mediated β-cell proliferation. Existing data from human T2D islets revealed the correlation between decreased levels of TCF7L2 and incretin hormone receptor expression [8]. Collectively, these data support the hypothesis that WNT/β-catenin signaling via TCF7L2 regulates GLP-1 effects in β-cells by transcriptionally controlling its receptor. Surprisingly, the in vitro results from the Ins-1 insulinoma system cannot be confirmed directly in vivo by the postnatal expression of TCF7L2DN under the Insulin 2 promoter (βTCFDN mice) [6]. Specifically, attenuation of Wnt/β-catenin signaling shortly after birth resulted in down regulation of MafA and Pdx1 expression in islets. No alteration of β-cell mass, homeostasis and glucose control during adulthood was noted either when TCF7L2DN was expressed before or after weaning. Boj et al. found similar results when using a rat insulin promoter (RIP)-driven inducible Cre-ERT2 recombinase approach to ablate Tcf7l2 specifically in adult β-cells at weaning and did not observe β-cell dysfunction either on normal chow or high-fat diet [9]. Moreover, this study revealed that full body knock-out of Tcf7l2 had no effect on embryonic development of the endocrine pancreas, the expression of β-cell genes and β-cell proliferation. The authors of this study conclude that Tcf7l2 is not important for β-cell function in mice but controls the hepatic response to perinatal and adult metabolic demand, besides its function for intestinal stem cell homeostasis and endocrine lineage formation in the gut. This is in stark contrast to the study of Xavier et al. who reported that the ablation of Tcf7l2 in Pdx1+ progenitor cells during pancreas development resulted in glucose intolerance and altered β-cell function [10]. The work of Shao et al. is in line with these results and found that embryonic expression of TCFL2DN affects β-cell development and homeostasis [6]. In particular, a reduced number of pancreatic Pdx1+/Nkx6.1+ double positive cells were formed, which caused an altered β-cell mass and abnormal insulin secretion in adult mice. These findings are comparable to previous data from Mitchell et al. where embryonic Tcf7l2 ablation using an Ins1-Cre driver line which is active already at embryonic day (E) 11.5 led to impaired β-cell mass and secretory function [6,11]. These observations emphasize that Tcf7l2 function is required when β-cells are born during pancreas development, but might also be needed to maintain β-cell homeostasis in the adult islet. Specifically, ectopic stabilization of β-catenin in pancreatic epithelium during the first transition leads to loss of Pdx1 expression, whereby inducing the stabilized form of β-catenin during the second transition positively influences pancreatic progenitor proliferation, resulting in increased pancreas organ size [12]. Collectively, these findings suggest that temporal and spatial regulation of the Wnt/β-catenin signaling pathway plays a critical role during pancreatic development and islet homeostasis and that compensatory mechanisms might be in place in the Tcf7l2 knock-out model [4,13]. Additional mechanistic studies are needed to explain the discrepancies among the different mouse models in terms of compensation in the Lef/Tcf transcription factor family, genetic background and efficiency of gene ablation or inhibition. Overall, accumulating evidence has emphasized the importance of Wnt/β-catenin signaling pathway in β-cell development and homeostasis. In human, genetic and metabolic phenotyping clearly reveals that subjects carrying TCF7L2 polymorphisms display insulin secretion defects in the presence of normal incretin plasma levels [14], suggesting an autonomous role of WNT/TCF7L2 signaling in β-cells and making it an attractive target for development of novel therapies for diabetes. In the future, the key to mechanistic understanding will lie in the investigation of a suitable human model system to unravel WNT/TCF7L2 function in β-cell homeostasis.