Publications by authors named "Stephan Speier"

36 Publications

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells.

Cell Stem Cell 2021 06 28;28(6):1105-1124.e19. Epub 2021 Apr 28.

Institute of Neuroanatomy & Developmental Biology (INDB), Eberhard Karls University Tübingen, Tübingen, Germany.

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.
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http://dx.doi.org/10.1016/j.stem.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461636PMC
June 2021

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices.

J Vis Exp 2021 04 12(170). Epub 2021 Apr 12.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida;

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.
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http://dx.doi.org/10.3791/62207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8314551PMC
April 2021

Image-Based Machine Learning Algorithms for Disease Characterization in the Human Type 1 Diabetes Pancreas.

Am J Pathol 2021 03 8;191(3):454-462. Epub 2020 Dec 8.

Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida Diabetes Institute, Gainesville, Florida; Department of Pediatrics, College of Medicine, University of Florida Diabetes Institute, Gainesville, Florida. Electronic address:

Emerging data suggest that type 1 diabetes affects not only the β-cell-containing islets of Langerhans, but also the surrounding exocrine compartment. Using digital pathology, machine learning algorithms were applied to high-resolution, whole-slide images of human pancreata to determine whether the tissue composition in individuals with or at risk for type 1 diabetes differs from those without diabetes. Transplant-grade pancreata from organ donors were evaluated from 16 nondiabetic autoantibody-negative controls, 8 nondiabetic autoantibody-positive subjects with increased type 1 diabetes risk, and 19 persons with type 1 diabetes (0 to 12 years' duration). HALO image analysis algorithms were implemented to compare architecture of the main pancreatic duct as well as cell size, density, and area of acinar, endocrine, ductal, and other nonendocrine, nonexocrine tissues. Type 1 diabetes was found to affect exocrine area, acinar cell density, and size, whereas the type of difference correlated with the presence or absence of insulin-positive cells remaining in the pancreas. These changes were not observed before disease onset, as indicated by modeling cross-sectional data from pancreata of autoantibody-positive subjects and those diagnosed with type 1 diabetes. These data provide novel insights into anatomic differences in type 1 diabetes pancreata and demonstrate that machine learning can be adapted for the evaluation of disease processes from cross-sectional data sets.
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http://dx.doi.org/10.1016/j.ajpath.2020.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919855PMC
March 2021

Noninvasive Monitoring of Glycemia-Induced Regulation of GLP-1R Expression in Murine and Human Islets of Langerhans.

Diabetes 2020 11 25;69(11):2246-2252. Epub 2020 Aug 25.

Department of Radiology and Nuclear Medicine, Radboudumc, Nijmegen, the Netherlands.

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify β-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and β-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in β-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.
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http://dx.doi.org/10.2337/db20-0616DOI Listing
November 2020

Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.

Nat Commun 2020 06 29;11(1):3265. Epub 2020 Jun 29.

Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, 33136, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-17040-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324563PMC
June 2020

Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis.

JCI Insight 2020 04 23;5(8). Epub 2020 Apr 23.

Paul Langerhans Institute Dresden of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic β 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, β 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 β cell loss, β cell dysfunction, alterations of β 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.
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http://dx.doi.org/10.1172/jci.insight.134525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205437PMC
April 2020

Dysfunction of Persisting β Cells Is a Key Feature of Early Type 2 Diabetes Pathogenesis.

Cell Rep 2020 04;31(1):107469

Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany; Institute of Physiology, Faculty of Medicine, Technische Universität Dresden, Dresden, Germany; German Center for Diabetes Research (DZD), München-Neuherberg, Germany. Electronic address:

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.
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http://dx.doi.org/10.1016/j.celrep.2020.03.033DOI Listing
April 2020

Using Pancreas Tissue Slices for the Study of Islet Physiology.

Methods Mol Biol 2020 ;2128:301-312

Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany.

Studies on islet of Langerhans physiology are crucial to understand the role of the endocrine pancreas in diabetes pathogenesis and the development of new therapeutic approaches. However, so far most research addressing islet of Langerhans biology relies on islets obtained via enzymatic isolation from the pancreas, which is known to cause mechanical and chemical stress, thus having a major impact on islet cell physiology. To circumvent the limitations of islet isolation, we have pioneered a platform for the study of islet physiology using the pancreas tissue slice technique. This approach allows to explore the detailed three-dimensional morphology of intact pancreatic tissue at a cellular level and to investigate islet cell function under near-physiological conditions. The described procedure is less damaging and faster than alternative approaches and particularly advantageous for studying infiltrated and structurally damaged islets. Furthermore, pancreas tissue slices have proven valuable for acute studies of endocrine as well as exocrine cell physiology in their conserved natural environment. We here provide a detailed protocol for the preparation of mouse pancreas tissue slices, the assessment of slice viability, and the study of pancreas cell physiology by hormone secretion and immunofluorescence staining.
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http://dx.doi.org/10.1007/978-1-0716-0385-7_20DOI Listing
March 2021

Transplantation of Islets of Langerhans into the Anterior Chamber of the Eye for Longitudinal In Vivo Imaging.

Methods Mol Biol 2020 ;2128:149-157

Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany.

Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.
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http://dx.doi.org/10.1007/978-1-0716-0385-7_11DOI Listing
March 2021

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency.

J Allergy Clin Immunol 2019 05 17;143(5):1849-1864.e4. Epub 2018 Oct 17.

Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke-University, Magdeburg, Germany. Electronic address:

Background: Mast cells (MCs) are best known as key effector cells of allergic reactions, but they also play an important role in host defense against pathogens. Despite increasing evidence for a critical effect of MCs on adaptive immunity, the underlying mechanisms are poorly understood.

Objective: Here we monitored MC intercellular communication with dendritic cells (DCs), MC activation, and degranulation and tracked the fate of exocytosed mast cell granules (MCGs) during skin inflammation.

Methods: Using a strategy to stain intracellular MCGs in vivo, we tracked the MCG fate after skin inflammation-induced MC degranulation. Furthermore, exogenous MCGs were applied to MC-deficient mice by means of intradermal injection. MCG effects on DC functionality and adaptive immune responses in vivo were assessed by combining intravital multiphoton microscopy with flow cytometry and functional assays.

Results: We demonstrate that dermal DCs engulf the intact granules exocytosed by MCs on skin inflammation. Subsequently, the engulfed MCGs are actively shuttled to skin-draining lymph nodes and finally degraded inside DCs within the lymphoid tissue. Most importantly, MCG uptake promotes DC maturation and migration to skin-draining lymph nodes, partially through MC-derived TNF, and boosts their T-cell priming efficiency. Surprisingly, exogenous MCGs alone are sufficient to induce a prominent DC activation and T-cell response.

Conclusion: Our study highlights a unique feature of peripheral MCs to affect lymphoid tissue-borne adaptive immunity over distance by modifying DC functionality through delivery of granule-stored mediators.
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http://dx.doi.org/10.1016/j.jaci.2018.08.052DOI Listing
May 2019

Mast cells acquire MHCII from dendritic cells during skin inflammation.

J Exp Med 2017 Dec 30;214(12):3791-3811. Epub 2017 Oct 30.

Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University Magdeburg, Magdeburg, Germany

Mast cells (MCs) and dendritic cells (DCs) are essential innate sentinels populating host-environment interfaces. Using longitudinal intravital multiphoton microscopy of DC/MC reporter mice, we herein provide in vivo evidence that migratory DCs execute targeted cell-to-cell interactions with stationary MCs before leaving the inflamed skin to draining lymph nodes. During initial stages of skin inflammation, DCs dynamically scan MCs, whereas at a later stage, long-lasting interactions predominate. These innate-to-innate synapse-like contacts ultimately culminate in DC-to-MC molecule transfers including major histocompatibility complex class II (MHCII) proteins enabling subsequent ex vivo priming of allogeneic T cells with a specific cytokine signature. The extent of MHCII transfer to MCs correlates with their T cell priming efficiency. Importantly, preventing the cross talk by preceding DC depletion decreases MC antigen presenting capacity and T cell-driven inflammation. Consequently, we identify an innate intercellular communication arming resident MCs with key DC functions that might contribute to the acute defense potential during critical periods of migration-based DC absence.
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http://dx.doi.org/10.1084/jem.20160783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716026PMC
December 2017

Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis.

Mol Metab 2017 09 8;6(9):943-957. Epub 2017 Jul 8.

Paul Langerhans Institute Dresden (PLID) of Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, München-Neuherberg, Germany.

Background: Plasma insulin levels are predominantly the product of the morphological mass of insulin producing beta cells in the pancreatic islets of Langerhans and the functional status of each of these beta cells. Thus, deficiency in either beta cell mass or function, or both, can lead to insufficient levels of insulin, resulting in hyperglycemia and diabetes. Nonetheless, the precise contribution of beta cell mass and function to the pathogenesis of diabetes as well as the underlying mechanisms are still unclear. In the past, this was largely due to the restricted number of technologies suitable for studying the scarcely accessible human beta cells. However, in recent years, a number of new platforms have been established to expand the available techniques and to facilitate deeper insight into the role of human beta cell mass and function as cause for diabetes and as potential treatment targets.

Scope Of Review: This review discusses the current knowledge about contribution of human beta cell mass and function to different stages of type 1 and type 2 diabetes pathogenesis. Furthermore, it highlights standard and newly developed technological platforms for the study of human beta cell biology, which can be used to increase our understanding of beta cell mass and function in human glucose homeostasis.

Major Conclusions: In contrast to early disease models, recent studies suggest that in type 1 and type 2 diabetes impairment of beta cell function is an early feature of disease pathogenesis while a substantial decrease in beta cell mass occurs more closely to clinical manifestation. This suggests that, in addition to beta cell mass replacement for late stage therapies, the development of novel strategies for protection and recovery of beta cell function could be most promising for successful diabetes treatment and prevention. The use of today's developing and wide range of technologies and platforms for the study of human beta cells will allow for a more detailed investigation of the underlying mechanisms and will facilitate development of treatment approaches to specifically target human beta cell mass and function.
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http://dx.doi.org/10.1016/j.molmet.2017.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605733PMC
September 2017

Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity.

Diabetologia 2018 Jan 7;61(1):182-192. Epub 2017 Sep 7.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL, 33136, USA.

Aims/hypothesis: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells.

Methods: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.

Results: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity.

Conclusions/interpretation: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
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http://dx.doi.org/10.1007/s00125-017-4416-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868749PMC
January 2018

Vessel Network Architecture of Adult Human Islets Promotes Distinct Cell-Cell Interactions In Situ and Is Altered After Transplantation.

Endocrinology 2017 05;158(5):1373-1385

Paul Langerhans Institute Dresden of Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, 85764 München-Neuherberg, Germany.

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 β to endothelial cell interactions, facilitates connection of α and β cells, and promotes the formation of independent β-cell clusters within islets. Furthermore, our results show that the endogenous vascular network of islets is significantly altered after transplantation in a donor age-related 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.
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http://dx.doi.org/10.1210/en.2016-1184DOI Listing
May 2017

The biomechanical properties of an epithelial tissue determine the location of its vasculature.

Nat Commun 2016 12 20;7:13560. Epub 2016 Dec 20.

Institute of Metabolic Physiology, Department of Biology, Heinrich Heine University, D-40225 Düsseldorf, Germany.

An important question is how growing tissues establish a blood vessel network. Here we study vascular network formation in pancreatic islets, endocrine tissues derived from pancreatic epithelium. We find that depletion of integrin-linked kinase (ILK) in the pancreatic epithelial cells of mice results in glucose intolerance due to a loss of the intra-islet vasculature. In turn, blood vessels accumulate at the islet periphery. Neither alterations in endothelial cell proliferation, apoptosis, morphology, Vegfa expression and VEGF-A secretion nor 'empty sleeves' of vascular basement membrane are found. Instead, biophysical experiments reveal that the biomechanical properties of pancreatic islet cells, such as their actomyosin-mediated cortex tension and adhesive forces to endothelial cells, are significantly changed. These results suggest that a sorting event is driving the segregation of endothelial and epithelial cells and indicate that the epithelial biomechanical properties determine whether the blood vasculature invades or envelops a growing epithelial tissue.
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http://dx.doi.org/10.1038/ncomms13560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187430PMC
December 2016

Identification of proliferative and mature β-cells in the islets of Langerhans.

Nature 2016 07 11;535(7612):430-4. Epub 2016 Jul 11.

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.
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http://dx.doi.org/10.1038/nature18624DOI Listing
July 2016

Alterations in β-Cell Calcium Dynamics and Efficacy Outweigh Islet Mass Adaptation in Compensation of Insulin Resistance and Prediabetes Onset.

Diabetes 2016 09 8;65(9):2676-85. Epub 2016 Apr 8.

Paul Langerhans Institute Dresden, Helmholtz Center Munich, University Clinic Carl Gustav Carus, Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany German Research Foundation-Center for Regenerative Therapies Dresden (CRTD), Faculty of Medicine, Technische Universität Dresden, Dresden, Germany German Center for Diabetes Research (DZD), München-Neuherberg, Germany

Emerging insulin resistance is normally compensated by increased insulin production of pancreatic β-cells, thereby maintaining normoglycemia. However, it is unclear whether this is achieved by adaptation of β-cell function, mass, or both. Most importantly, it is still unknown which of these adaptive mechanisms fail when type 2 diabetes develops. We performed longitudinal in vivo imaging of β-cell calcium dynamics and islet mass of transplanted islets of Langerhans throughout diet-induced progression from normal glucose homeostasis, through compensation of insulin resistance, to prediabetes. The results show that compensation of insulin resistance is predominated by alterations of β-cell function, while islet mass only gradually expands. Hereby, functional adaptation is mediated by increased calcium efficacy, which involves Epac signaling. Prior to prediabetes, β-cell function displays decreased stimulated calcium dynamics, whereas islet mass continues to increase through prediabetes onset. Thus, our data reveal a predominant role of islet function with distinct contributions of triggering and amplifying pathway in the in vivo processes preceding diabetes onset. These findings support protection and recovery of β-cell function as primary goals for prevention and treatment of diabetes and provide insight into potential therapeutic targets.
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http://dx.doi.org/10.2337/db15-1718DOI Listing
September 2016

Aldehyde dehydrogenase activity is necessary for beta cell development and functionality in mice.

Diabetologia 2016 Jan 31;59(1):139-150. Epub 2015 Oct 31.

Paul Langerhans Institute Dresden of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Fetscherstrasse 74, 01307, Dresden, Germany.

Aims/hypothesis: Pancreatic beta cells maintain glucose homeostasis and beta cell dysfunction is a major risk factor in developing diabetes. Therefore, understanding the developmental regulatory networks that define a fully functional beta cell is important for elucidating the genetic origins of the disease. Aldehyde dehydrogenase activity has been associated with stem/progenitor cells and we have previously shown that Aldh1b1 is specifically expressed in pancreas progenitor pools. Here we address the hypothesis that Aldh1b1 may regulate the timing of the appearance and eventual functionality of beta cells.

Methods: We generated an Aldh1b1-knockout mouse line (Aldh1b1 (tm1lacZ)) and used this to study pancreatic development, beta cell functionality and glucose homeostasis in the absence of Aldh1b1 function.

Results: Differentiation in the developing pancreas of Aldh1b1 (tm1lacZ) null mice was accelerated. Transcriptome analyses of newborn and adult islets showed misregulation of key beta cell transcription factors and genes crucial for beta cell function. Functional analyses showed that glucose-stimulated insulin secretion was severely compromised in islets isolated from null mice. Several key features of beta cell functionality were affected, including control of oxidative stress, glucose sensing, stimulus-coupling secretion and secretory granule biogenesis. As a result of beta cell dysfunction, homozygous mice developed glucose intolerance and age-dependent hyperglycaemia.

Conclusions/interpretation: These findings show that Aldh1b1 influences the timing of the transition from the pancreas endocrine progenitor to the committed beta cell and demonstrate that changes in the timing of this transition lead to beta cell dysfunction and thus constitute a diabetes risk factor later in life. Gene Expression Omnibus (GEO) accession: GSE58025.
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http://dx.doi.org/10.1007/s00125-015-3784-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670456PMC
January 2016

Distinct roles of β-cell mass and function during type 1 diabetes onset and remission.

Diabetes 2015 Jun 20;64(6):2148-60. Epub 2015 Jan 20.

Deutsche Forschungsgemeinschaft (DFG)-Research Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Germany Paul Langerhans Institute Dresden of Helmholtz Centre Munich, University Clinic Carl Gustav Carus of Technische Universität Dresden, German Centre for Diabetes Research (DZD), Dresden, Germany

Cure of type 1 diabetes (T1D) by immune intervention at disease onset depends on the restoration of insulin secretion by endogenous β-cells. However, little is known about the potential of β-cell mass and function to recover after autoimmune attack ablation. Using a longitudinal in vivo imaging approach, we show how functional status and mass of β-cells adapt in response to the onset and remission of T1D. We demonstrate that infiltration reduces β-cell mass prior to onset and, together with emerging hyperglycemia, affects β-cell function. After immune intervention, persisting hyperglycemia prevents functional recovery but promotes β-cell mass increase in mouse islets. When blood glucose levels return to normoglycemia β-cell mass expansion stops, and subsequently glucose tolerance recovers in combination with β-cell function. Similar to mouse islets, human islets exhibit cell exhaustion and recovery in response to transient hyperglycemia. However, the effect of hyperglycemia on human islet mass increase is minor and transient. Our data demonstrate a major role of functional exhaustion and recovery of β-cells during T1D onset and remission. Therefore, these findings support early intervention therapy for individuals with T1D.
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http://dx.doi.org/10.2337/db14-1055DOI Listing
June 2015

Mast cell promotion of T cell-driven antigen-induced arthritis despite being dispensable for antibody-induced arthritis in which T cells are bypassed.

Arthritis Rheumatol 2015 Apr;67(4):903-13

Technische Universität Dresden, Dresden, Germany.

Objective: The function of mast cells (MCs) in autoimmune disorders has been a subject of controversy recently. MC-deficient Kit(W/W-v) mice were found to be resistant to K/BxN serum-transfer arthritis, whereas Kit(W-sh/W-sh) mice and a genetic model of MC deficiency independent of the Kit mutation were found to be fully susceptible. This debate might lead to the assumption that MCs are dispensable in autoimmunity in general. Thus, the purpose of this study was to examine the relevance of MCs to arthritis using a genetic model of inducible MC deficiency without compromised Kit signaling.

Methods: We compared MC functions in K/BxN serum-induced arthritis and in collagen-induced arthritis (CIA) in a mouse model of inducible MC deficiency by analyzing joint inflammation, parameters of cartilage degradation and bone erosion, and the autoreactive adaptive immune response.

Results: We observed a redundant role of MCs in K/BxN serum-induced arthritis, where joint inflammation is triggered by cartilage-bound immune complexes independently of T cells. In contrast, we found MCs to be critically relevant in CIA, which is provoked by two arms of autoimmune attack: autoreactive antibodies and effector T cells. In addition to diminished joint inflammation in the absence of MCs, we found a dramatic loss of T cell expansion upon immunization, accompanied by reduced T cell cytokine responses.

Conclusion: In this analysis of an arthritis model in which the cellular arm of adaptive immunity was not bypassed, we identified MCs as important promoters of T cell-conditioned autoimmune disorders and, consequently, as potential therapeutic targets in rheumatoid arthritis.
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http://dx.doi.org/10.1002/art.38996DOI Listing
April 2015

Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

Nat Protoc 2014 Dec 13;9(12):2809-22. Epub 2014 Nov 13.

1] Deutsche Forschungsgemeinschaft (DFG) Research Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Germany. [2] Paul Langerhans Institute Dresden, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. [3] German Center for Diabetes Research (DZD), Dresden, Germany.

Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.
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http://dx.doi.org/10.1038/nprot.2014.195DOI Listing
December 2014

Mesenchymal stromal cells improve transplanted islet survival and islet function in a syngeneic mouse model.

Diabetologia 2014 Mar 20;57(3):522-31. Epub 2013 Nov 20.

DFG-Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstrasse 105, 01307, Dresden, Germany.

Aims/hypothesis: Islet transplantation is used therapeutically in a minority of patients with type 1 diabetes. Successful outcomes are hampered by early islet beta cell loss. The adjuvant co-transplantation of mesenchymal stromal cells (MSCs) has the promise to improve islet transplant outcome.

Methods: We used a syngeneic marginal islet mass transplantation model in a mouse model of diabetes. Mice received islets or islets plus 250,000 MSCs. Kidney subcapsule, intra-hepatic and intra-ocular islet transplantation sites were used. Apoptosis, vascularisation, beta cell proliferation, MSC differentiation and laminin levels were determined by immunohistochemical analysis and image quantification post-transplant.

Results: Glucose homeostasis after the transplantation of syngeneic islets was improved by the co-transplantation of MSCs together with islets under the kidney capsule (p = 0.01) and by intravenous infusion of MSCs after intra-hepatic islet transplantation (p = 0.05). MSC co-transplantation resulted in reduced islet apoptosis, with reduced numbers of islet cells positive for cleaved caspase 3 being observed 14 days post-transplant. In kidney subcapsule, but not in intra-ocular islet transplant models, we observed increased re-vascularisation rates, but not increased blood vessel density in and around islets co-transplanted with MSCs compared with islets that were transplanted alone. Co-transplantation of MSCs did not increase beta cell proliferation, extracellular matrix protein laminin production or alpha cell numbers, and there was negligible MSC transdifferentiation into beta cells.

Conclusions/interpretation: Co-transplantation of MSCs may lead to improved islet function and survival in the early post-transplantation period in humans receiving islet transplantation.
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http://dx.doi.org/10.1007/s00125-013-3109-4DOI Listing
March 2014

Reporter islets in the eye reveal the plasticity of the endocrine pancreas.

Proc Natl Acad Sci U S A 2013 Dec 18;110(51):20581-6. Epub 2013 Nov 18.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, Stockholm SE-171 76, Sweden.

The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo.
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http://dx.doi.org/10.1073/pnas.1313696110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870708PMC
December 2013

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PLoS One 2013 4;8(11):e78706. Epub 2013 Nov 4.

CRTD - DFG Research Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany ; Paul Langerhans Institute Dresden, German Center for Diabetes Research (DZD), Dresden, Germany.

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078706PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817072PMC
August 2014

Age-dependent labeling and imaging of insulin secretory granules.

Diabetes 2013 Nov 8;62(11):3687-96. Epub 2013 Aug 8.

Molecular Diabetology, Paul Langerhans Institute Dresden, Dresden University of Technology, Dresden, Germany.

Insulin is stored within the secretory granules of pancreatic β-cells, and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here, we illustrate a technology that enables the study of granule aging in insulinoma cells and β-cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic β-cells. Furthermore, the method is of general interest for the study of membrane traffic and aging.
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http://dx.doi.org/10.2337/db12-1819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806613PMC
November 2013

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function.

Proc Natl Acad Sci U S A 2012 Dec 10;109(52):21456-61. Epub 2012 Dec 10.

Diabetes Research Institute, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.
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http://dx.doi.org/10.1073/pnas.1211659110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535593PMC
December 2012

Donor islet endothelial cells in pancreatic islet revascularization.

Diabetes 2011 Oct 26;60(10):2571-7. Epub 2011 Aug 26.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden.

Objective: Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets.

Research Design And Methods: Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts' metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy.

Results: DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets.

Conclusions: In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.
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http://dx.doi.org/10.2337/db10-1711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178280PMC
October 2011

Experimental approaches for high-resolution in vivo imaging of islet of Langerhans biology.

Authors:
Stephan Speier

Curr Diab Rep 2011 Oct;11(5):420-5

Center for Regenerative Therapies Dresden and Paul Langerhans Institute Dresden, School of Medicine, Dresden University of Technology, 01307 Dresden, Germany.

Under physiological conditions and in the pathogenesis of diabetes mellitus systemic influences play a substantial role for function and survival of cells of the islet of Langerhans. Therefore, in vivo studies to understand islet biology are indispensible and imaging techniques are increasingly used for this purpose. Among the diverse imaging modalities currently only laser scanning microscopy (LSM) allows resolution and visualization of individual cells and cellular processes. To overcome limited tissue penetration and working distance of LSM and enable in vivo investigations of islet cell physiology, various experimental approaches have been developed. Especially, the recently developed imaging platforms have significantly improved the possibility to study islets at a cellular level in vivo, and provided novel insight into islet biology in health and disease. The various approaches, their applications, and reported results, as well as their limitations are reviewed in this article.
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http://dx.doi.org/10.1007/s11892-011-0207-xDOI Listing
October 2011
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