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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

Interferon (IFN)-α and IFN-β are the central regulators of antiviral immunity but little is known about their roles in viral glomerulonephritis (eg, HIV nephropathy). We hypothesized that IFN-α and IFN-β would trigger local inflammation and podocyte loss. We found that both IFNs consistently activated human and mouse podocytes and parietal epithelial cells to express numerous IFN-stimulated genes. However, only IFN-β significantly induced podocyte death and increased the permeability of podocyte monolayers. In contrast, only IFN-α caused cell-cycle arrest and inhibited the migration of parietal epithelial cells. Both IFNs suppressed renal progenitor differentiation into mature podocytes. In Adriamycin nephropathy, injections with either IFN-α or IFN-β aggravated proteinuria, macrophage influx, and glomerulosclerosis. A detailed analysis showed that only IFN-β induced podocyte mitosis. This did not, however, lead to proliferation, but was associated with podocyte loss via podocyte detachment and/or mitotic podocyte death (mitotic catastrophe). We did not detect TUNEL-positive podocytes. Thus, IFN-α and IFN-β have both common and differential effects on podocytes and parietal epithelial cells, which together promote glomerulosclerosis by enhancing podocyte loss while suppressing podocyte regeneration from local progenitors.

Podocyte apoptosis as a pathway of podocyte loss is often suspected but rarely detected. To study podocyte apoptosis versus inflammatory forms of podocyte death in vivo, we targeted murine double minute (MDM)-2 for three reasons. First, MDM2 inhibits p53-dependent apoptosis; second, MDM2 facilitates NF-κB signalling; and third, podocytes show strong MDM2 expression. We hypothesized that blocking MDM2 during glomerular injury may trigger p53-mediated podocyte apoptosis, proteinuria, and glomerulosclerosis. Unexpectedly, MDM2 blockade in early adriamycin nephropathy of Balb/c mice had the opposite effect and reduced intra-renal cytokine and chemokine expression, glomerular macrophage and T-cell counts, and plasma creatinine and blood urea nitrogen levels. In cultured podocytes exposed to adriamycin, MDM2 blockade did not trigger podocyte death but induced G2/M arrest to prevent aberrant nuclear divisions and detachment of dying aneuploid podocytes, a feature of mitotic catastrophe in vitro and in vivo. Consistent with these observations, 12 of 164 consecutive human renal biopsies revealed features of podocyte mitotic catastrophe but only in glomerular disorders with proteinuria. Furthermore, delayed MDM2 blockade reduced plasma creatinine levels, blood urea nitrogen, tubular atrophy, interstitial leukocyte numbers, and cytokine expression as well as interstitial fibrosis. Together, MDM2-mediated mitotic catastrophe is a previously unrecognized variant of podocyte loss where MDM2 forces podocytes to complete the cell cycle, which in the absence of cytokinesis leads to podocyte aneuploidy, mitotic catastrophe, and loss by detachment. MDM2 blockade with nutlin-3a could be a novel therapeutic strategy to prevent renal inflammation, podocyte loss, glomerulosclerosis, proteinuria, and progressive kidney disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Kidney remodeling is a response to intrinsic or extrinsic triggers of kidney injury. Injury initiates a set of universal response programs that were positively selected through evolution to control potentially life-threatening dangers and to regain homeostasis, including tissue repair. These danger control programs are (i) clotting, to control the risk of bleeding; (ii) inflammation, to control the risk of infection; (iii) epithelial repair; (iv) mesenchymal repair; and (v) scar resolution or minimization. In this review we focus on the role of mesangial cells in glomerular disorders and how their behaviors follow these danger control programs. We review the role of mesangial cells in glomerular coagulation and fibrinolysis, as well as their role in triggering glomerular inflammation and mesangioproliferative disorders. Furthermore, we discuss how the mesangium self-repairs, how podocyte injury triggers a "mesenchymal healing"-kind of response that leads to glomerular fibrosis and sclerosis. Thus, we can better appreciate the contribution of mesangial cells to glomerular pathology when we understand their behavior as an attempt to support the evolutionally conserved universal danger control programs. However, these mechanisms often result in maladaptive processes that destroy the complex glomerular ultrastructure rather than help to regain tissue homeostasis.

Nephrocalcinosis, acute calcium oxalate (CaOx) nephropathy, and renal stone disease can lead to inflammation and subsequent renal failure, but the underlying pathological mechanisms remain elusive. Other crystallopathies, such as gout, atherosclerosis, and asbestosis, trigger inflammation and tissue remodeling by inducing IL-1β secretion, leading us to hypothesize that CaOx crystals may induce inflammation in a similar manner. In mice, intrarenal CaOx deposition induced tubular damage, cytokine expression, neutrophil recruitment, and renal failure. We found that CaOx crystals activated murine renal DCs to secrete IL-1β through a pathway that included NLRP3, ASC, and caspase-1. Despite a similar amount of crystal deposits, intrarenal inflammation, tubular damage, and renal dysfunction were abrogated in mice deficient in MyD88; NLRP3, ASC, and caspase-1; IL-1R; or IL-18. Nephropathy was attenuated by DC depletion, ATP depletion, or therapeutic IL-1 antagonism. These data demonstrated that CaOx crystals trigger IL-1β–dependent innate immunity via the NLRP3/ASC/caspase-1 axis in intrarenal mononuclear phagocytes and directly damage tubular cells, leading to the release of the NLRP3 agonist ATP. Furthermore, these results suggest that IL-1β blockade may prevent renal damage in nephrocalcinosis.

Glomerular crescents are most common in rapidly progressive glomerulonephritis but also occur in non-inflammatory chronic glomerulopathies; thus, factors other than inflammation should trigger crescent formation, eg vascular damage and plasma leakage. Here we report that Alport nephropathy in Col4A3-deficient Sv129 mice is complicated by diffuse and global crescent formation in which proliferating parietal epithelial cells are the predominant cell type. Laminin staining and transmission and acellular scanning electron microscopy of acellular glomeruli documented disruptions and progressive disintegration of the glomerular basement membrane in Col4A3-deficient mice. FITC-dextran perfusion further revealed vascular leakage from glomerular capillaries into Bowman's space, further documented by fibrin deposits in the segmental crescents. Its pathogenic role was validated by showing that the fibrinolytic activity of recombinant urokinase partially prevented crescent formation. In addition, in vitro studies confirmed an additional mitogenic potential of serum on murine and human parietal epithelial cells. Furthermore, loss of parietal cell polarity and unpolarized secretion of extracellular matrix components were evident within fibrocellular crescents. Among 665 human Alport nephropathy biopsies, crescent formation was noted in 0.4%. We conclude that glomerular vascular injury and GBM breaks cause plasma leakage which triggers a wound healing programme involving the proliferation of parietal cells and their loss of polarity. This process can trigger cellular and fibrocellular crescent formation even in the absence of cellular inflammation and rupture of the Bowman's capsule.

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.

Foreign nucleic acids are recognized by germ-line-encoded receptors expressed in immune and nonimmune cells. Activation of the nucleic acid-specific pattern recognition receptors by foreign nucleic acid promotes production of inflammatory cytokines (mostly type I IFNs) and at the later stage leads to cell death. Here, we describe reliable and simple methods to quantify cell death caused by nucleic acid recognition. Additionally, we report two different methods to discriminate between two cell death modalities: apoptosis and necrosis.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells resulting in failure of metabolic control. Even though type 1 and 2 diabetes differ in their pathogenesis, restoring β-cell function is the overarching goal for improved therapy of both diseases. This could be achieved either by cell-replacement therapy or by triggering intrinsic regenerative mechanisms of the pancreas. For type 1 diabetes, a combination of β-cell replacement and immunosuppressive therapy could be a curative treatment, whereas for type 2 diabetes enhancing endogenous mechanisms of β-cell regeneration might optimize blood glucose control. This review will briefly summarize recent efforts to allow β-cell regeneration where the most promising approaches are currently (1) increasing β-cell self-replication or neogenesis from ductal progenitors and (2) conversion of α-cells into β-cells.

Lupus nephritis is a complication of systemic lupus erythematosus, a heterogeneous autoimmune syndrome involving multiple pathways. Accumulating data from the fields of genetics, clinical science, transcriptomics and basic immunology indicate that antiviral immunity has relevance in the pathogenesis of lupus nephritis. This idea is based on the existence of genetic variants that promote the persistence of nuclear particles in the extracellular space or inside lysosomes. Such nuclear particles mimic viral particles and their RNA or DNA components activate viral nucleic acid recognition receptors in antigen-presenting cells. These autoadjuvant effects of endogenous nucleic acids promote an inappropriate immune interpretation of the nuclear particles during antigen presentation. This process fosters the expansion of autoreactive T cells and B cells, which promotes autoantibody production and immune complex glomerulonephritis. The release of interferon α sets off an antiviral immune response with a coordinated induction of hundreds of antiviral genes both inside and outside the kidney. In this article we summarize the available data indicating that innate immunity triggers antiviral immunity in systemic lupus erythematosus. We also discuss the related implications for innovative therapeutic strategies.

The long pentraxin PTX3 has multiple roles in innate immunity. For example, PTX3 regulates C1q binding to pathogens and dead cells and regulates their uptake by phagocytes. It also inhibits P-selectin-mediated recruitment of leukocytes. Both of these mechanisms are known to be involved in autoimmunity and autoimmune tissue injury, e.g. in systemic lupus erythematosus, but a contribution of PTX3 is hypothetical. To evaluate a potential immunoregulatory role of PTX3 in autoimmunity we crossed Ptx3-deficient mice with Fas-deficient (lpr) C57BL/6 (B6) mice with mild lupus-like autoimmunity. PTX3 was found to be increasingly expressed in kidneys and lungs of B6lpr along disease progression. Lack of PTX3 impaired the phagocytic uptake of apoptotic T cells into peritoneal macrophages and selectively expanded CD4/CD8 double negative T cells while other immune cell subsets and lupus autoantibody production remained unaffected. Lack of PTX3 also aggravated autoimmune lung disease, i.e. peribronchial and perivascular CD3+ T cell and macrophage infiltrates of B6lpr mice. In contrast, histomorphological and functional parameters of lupus nephritis remained unaffected by the Ptx3 genotype. Together, PTX3 specifically suppresses autoimmune lung disease that is associated with systemic lupus erythematosus. Vice versa, loss-of-function mutations in the Ptx3 gene might represent a genetic risk factor for pulmonary (but not renal) manifestations of systemic lupus or other autoimmune diseases.