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Features of type 1 diabetes (T1D) include loss of first-phase insulin secretion in response to glucose, declining c-peptide, and glucose intolerance; indicating β cell dysfunction. Here, we investigate the mechanisms of this dysfunction with the hypothesis that immune dysregulation during the early stages of T1D development impacts β cell dysfunction in pathways affecting glucose metabolism. The live pancreas tissue slice (LPTS) model is ideal for the study of T1D pathogenesis due to its preservation of the pancreatic environment. LPTS were generated from organ donors without diabetes or autoantibodies (ND, n=9) , donors positive for one or more autoantibodies without a diagnosis of T1D (AAb+, n=6) , and donors with T1D (T1D, n=4) . Imaging studies were conducted to assess the impact of T cell infiltration on β cell function through Ca2+ imaging and slice perifusion. Islets from ND and AAb+ donors had no insulitis and exhibited Ca2+ responses to both high glucose (HG) and potassium chloride (KCl) . A majority of β cell+ islets in tissue from donors with T1D had infiltrating T cells with several having insulitis. Islet HG response from donors with T1D was heterogenous (∼50% failed to respond) while all T1D islets exhibited responses to KCl. Insulin secretion was significantly higher in LPTS from ND donors than in donors with T1D (p=0.0079) . To determine the basis for this loss of function, we assessed transcriptomics of islets within these cohorts. When comparing gene expression levels in islets from ND versus T1D, significant differences were observed in genes involved in glycolysis (GAPDH, p=3.27x10-21) , the citric acid cycle components (OGDH, p=1.88x10-18) , and the electric transport chain (F1FoATP synthase, p=2.69x10-13) . The decreased expression of glucose metabolism genes during T1D development along with the loss of β cell function in LPTS demonstrates β cell dysfunction before their demise and may be a contributory mechanism towards the pathogenesis of disease. Disclosure M.Huber: None. M.Slak rupnik: None. D.M.Drotar: None. H.Hiller: None. M.Beery: None. I.Kusmartseva: None. M.A.Atkinson: None. E.Phelps: Research Support; Immunocore, Ltd. C.E.Mathews: None. Funding National Institutes of Health (5T32DK108736-03) National Institutes of Health (PO1 AI42288)

Within the human pancreas, exocrine and endocrine cells control secretion of digestive enzymes and production of hormones to maintain metabolic homeostasis, respectively. While the vast majority of type 1 diabetes research efforts have focused on endocrine function and autoimmunity, recent studies identified a series of unique features (e.g., reduced weight and volume, increased density of leukocytes) within the exocrine pancreas in this disease, but the mechanisms underlying these aberrancies are unknown. Therefore, we histologically assessed amylase, insulin, glucagon, lipase, and/or trypsinogen in 78 organ donor pancreata from birth through adulthood in control subjects and those at various stages of type 1 diabetes. While amylase-positive (AMY(+)) acinar cells were detectable in pancreata from all study groups, tissues from individuals >2 years of age contained clusters of acinar cells devoid of amylase (AMY(−)). A majority of these AMY(−) cell clusters localized proximal to islets (i.e., peri-islet). Additionally, most AMY(−) clusters were positive for the exocrine enzymes lipase and trypsinogen. Interestingly, type 1 diabetes pancreata displayed significant reductions in the frequency of these AMY(−) cell clusters. These results support a contribution of the islet-acinar axis in pancreatic development and underscore a potential role for the exocrine pancreas in the pathogenesis of type 1 diabetes.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas., The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic beta cells leads to insulin deficiency and loss of glycemic control. However, knowledge about human pancreas pathophysiology in T1D remains incomplete. To address this limitation, we established a pancreas tissue slice platform of donor organs with and without diabetes, facilitating the first live cell studies of human pancreas in T1D pathogenesis to our knowledge. We show that pancreas tissue slices from organ donors allow thorough assessment of processes critical for disease development, including insulin secretion, beta cell physiology, endocrine cell morphology, and immune infiltration within the same donor organ. Using this approach, we compared detailed pathophysiological profiles for 4 pancreata from donors with T1D with 19 nondiabetic control donors. We demonstrate that cell loss, beta cell dysfunction, alterations of beta cell physiology, and islet infiltration contributed differently to individual cases of T1D, allowing insight into pathophysiology and heterogeneity of T1D pathogenesis. Thus, our study demonstrates that organ donor pancreas tissue slices represent a promising and potentially novel approach in the search for successful prevention and reversal strategies of T1D.

AIMS Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. METHODS We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). RESULTS Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas. The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

Diabetes research studies routinely rely upon the use of tissue samples from human organ donors. It remains unclear whether the length of hospital stay prior to organ donation affects the presence of cells infiltrating the pancreas or the frequency of replicating beta cells. To address this, 39 organ donors without diabetes were matched for age, sex, BMI and ethnicity in groups of three. Within each group, donors varied by length of hospital stay immediately prior to organ donation (

Introduction: The exocrine compartment of the pancreas has been hypothesized to harbor progenitor cells. While their existence remains the subject of debate, the widespread consensus is that any such putative progenitors should express the pancreatic-duodenal homeobox 1 protein (PDX1). Progenitor cell stimulation often depends on concurrent TGFb inhibition and BMP activation. We further hypothesized that PDX1-expressing putative b-cell progenitors may respond to BMP-7. Our data (1) indicate that extrainsular PDX1+/ALK3+ cells do so by proliferating and subsequently, upon BMP-7 withdrawal, differentiating in a multilineage fashion. Here we present additional mechanistic data on b-cell regeneration using a novel human pancreatic slice culture system that allows for the observation of pancreatic tissue for 2 weeks. We further show single cell RNAseq analyses of sorted ALK3+ from the human pancreas, confirming the heterogeneity of the ductal compartment. Methods: HPS: Low melting point agarose-embedded, vibratome-sectioned live pancreatic slices were generated from human donors. Transduction with a CMV-[loxP]-dsRed-[loxP]-EGFP adenovirus + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear perfluorcarbon (PFC)-based membranes. scRNAseq of ALK3+ sorted cells from human non-endocrine tissue (hNEPT, the leftover of islet isolations) was done on 1,000 cells/sample (250K reads/cell). Conclusions: We report the use of live HPS as a novel tool for the study of pancreatic regeneration. Our preliminary results confirm the feasibility of tracing and monitoring discrete transitional events in response to BMP signalling stimulation, as well as the existence of multiple ductal populations at various degrees of differentiation in healthy donor pancreata. References: 1. Qadir MMF, et al. P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics. Cell Reports. 2018 Disclosure M. Qadir: None. S. Alvarez-Cubela: None. J. van Dijk: None. J. Weitz: None. S. Cechin: None. D. Klein: None. A.M. Tamayo: None. J. Almaca: None. A. Caicedo: None. I. Kusmartseva: None. H. Hiller: None. M. Beery: None. K.B. Johnson: None. Y.B. Moreno Hernandez: None. M.B. Navarro Rubio: None. S. Sadiq: None. C. Ricordi: Advisory Panel; Self; Zone Labs. A. Pugliese: None. C. Fraker: None. R. Pastori: Other Relationship; Self; Ophysio Inc. J. Domínguez-Bendala: None. Funding Diabetes Research Institute Foundation; Inserra Family Foundation; Fred and Mabel R. Parks Foundation; Foundation for Diabetes Research; Tonkinson Foundation; Michael J. and Katherine E. Franco Foundation; Frank Strick Foundation; Mildred Graff; National Institutes of Health (R43DK105655-01, R44DK105655-02, U01DK120393-01); Fulbright Scholarship Board/International Institute of Education (to M.M.F.Q.)

Histopathology based studies of the pancreas obtained from organ donors are increasing our awareness of islet phenotypic heterogeneity during development and aging, as well as in settings of type 1 diabetes, type 2 diabetes, monogenic diabetes or other forms of this metabolic disease. Islet amyloidosis represents a histopathological feature classically ascribed to patients with type 2 diabetes. Herein, the occurrence of islet amyloidosis and its severity are reported in a child with type 1 diabetes along with histological comparisons of islet amyloidosis in two young adults with recent-onset type 1 diabetes. Islet amyloidosis was infrequent yet widely distributed throughout the pancreas in the child with type 1 diabetes and both adults with type 1 diabetes, with no such pathology seen in matched control donors. Analysis of these cases add to the increasing appreciation of islet heterogeneity in children and young adults with type 1 diabetes. Such knowledge also supports a notion that multiple pathophysiological mechanisms underlie the loss of functional β-cell mass in the spectrum of clinical phenotypes in patients with type 1 diabetes.

Summary Human tissue phenotyping generates complex spatial information from numerous imaging modalities, yet images typically become static figures for publication, and original data and metadata are rarely available. While comprehensive image maps exist for some organs, most resources have limited support for multiplexed imaging or have non-intuitive user interfaces. Therefore, we built a Pancreatlas resource that integrates several technologies into a unique interface, allowing users to access richly annotated web pages, drill down to individual images, and deeply explore data online. The current version of Pancreatlas contains over 800 unique images acquired by whole-slide scanning, confocal microscopy, and imaging mass cytometry, and is available at https://www.pancreatlas.org. To create this human pancreas-specific biological imaging resource, we developed a React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND), which can be adapted beyond Pancreatlas to meet countless imaging or other structured data-management needs., Graphical Abstract, Highlights • Human organ phenotyping databases benefit from intuitive user interfaces • Pancreatlas resource enables exploration of bioimaging data from human pancreas • The front-end framework of Pancreatlas, FFIND, is modular and easily adaptable • FFIND provides structured data-exploration capabilities across countless domains, The Bigger Picture Scientists need cost-effective yet fully featured database solutions that facilitate large dataset sharing in a structured and easily digestible manner. Flexible Framework for Integrating and Navigating Data (FFIND) is a data-agnostic web application that is designed to easily connect existing databases with data-browsing clients. We used FFIND to build Pancreatlas, an online imaging resource containing datasets linking imaging data with clinical data to facilitate advances in the understanding of diabetes, pancreatitis, and pancreatic cancer. FFIND architecture, which is available as open-source software, can be easily adapted to meet other field- or project-specific needs; we hope it will help data scientists reach a broader audience by reducing the development life cycle and providing familiar interactivity in communicating data and underlying stories., Human tissue phenotyping generates complex imaging data that is difficult to share in publications, and many organ-specific databases lack intuitive user interfaces or have limited support for multiplexed imaging. Therefore, we built a Pancreatlas resource (https://www.pancreatlas.org) that integrates several technologies into a unique interface, allowing users to access richly annotated web pages. To create this imaging resource, we developed a data-agnostic, React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND; https://github.com/Powers-Brissova-Research-Group/FFIND).

Summary The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β cells has recently been questioned as small amounts of C-peptide are detectable in patients with long-standing disease. We analyzed protein and gene expression levels for proinsulin, insulin, C-peptide, and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+), and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, proinsulin and INS mRNA were detected in the majority of T1D pancreata. Interestingly, heterogeneous nuclear RNA (hnRNA) for insulin and INS-IGF2, both originating from the INS promoter, were essentially undetectable in T1D pancreata, arguing for a silent INS promoter. Expression of PCSK1, a convertase responsible for proinsulin processing, was reduced in T1D pancreata, supportive of persistent proinsulin. These data implicate the existence of β cells enriched for inefficient insulin/C-peptide production in T1D patients, potentially less susceptible to autoimmune destruction.