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Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencap-sulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions. Keywords: diabetes; porcine islets; beta-cell replacement; immune barrier

© 2017, The Author(s). Aims/hypothesis: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). Methods: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. Results: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate =0.05, fold change =1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and RE

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

© 2017, National Academy of Sciences. All rights reserved. Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencap-sulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.

Copyright © 2017 Endocrine Society. Islet-cell hormone release is modulated by signals from endothelial and endocrine cells within the islet. However, models of intraislet vascularization and paracrine cell signaling are mostly based on the rodent pancreas. We assessed the architecture and endocrine cell interaction of the vascular network in unperturbed human islets in situ and their potential to re-establish their endogenous vascular network after transplantation in vivo.We prepared slices of fresh pancreas tissue obtained from nondiabetic patients undergoing partial pancreatectomy. In addition, we transplanted human donor islets into the anterior chamber of the mouse eye. Next, we performed three-dimensional in situ and in vivo imaging of islet cell and vessel architecture at cellular resolution and compared our findings with mouse and porcine islets. Our data reveal a significantly different vascular architecture with decreased vessel diameter, reduced vessel branching, and shortened total vessel network in human compared with mouse islets. Together with the distinct cellular arrangement in human islets, this limits ß to endothelial cell interactions, facilitates connection of a and ß cells, and promotes the formation of independent ß-cell clusters within islets. Furthermore, our results show that the endogenous vascular network of islets is significantly altered after transplantation in a donor agerelated mechanism. Thus, our study provides insight into the vascular architecture and cellular arrangement of human islets with apparent consequences for intercellular islet signaling. Moreover, our findings suggest that human islet engraftment after transplantation can be improved by using alternative, less mature islet-cell sources.

© 2017 The Author(s). Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

© 2017 John Wiley & Sons Ltd Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper ß-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the ß-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

© 2017 The Author(s) Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.