Search

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease., Heller et al analyze transcriptomic and epigenetic datasets from human PSCs differentiating into pancreatic progenitors to elucidate the regulatory mechanisms behind pancreatic development. The authors report that the transcription factor ONECUT1 is expressed specifically in pancreatic development and associates with other key TFs (such as FOXA2, GATA6, PDX1) during endocrine specification.

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

Personalized invitro 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 invitro. 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 invitro and invivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Despite intensive research and progress in personalized medicine, pancreatic ductal adenocarcinoma remains one of the deadliest cancer entities. Pancreatic duct-like organoids (PDLOs) derived from human pluripotent stem cells (PSCs) or pancreatic cancer patient-derived organoids (PDOs) provide unique tools to study early and late stage dysplasia and to foster personalized medicine. However, such advanced systems are neither rapidly nor easily accessible and require an in vivo niche to study tumor formation and interaction with the stroma. Here, the establishment of the porcine urinary bladder (PUB) is revealed as an advanced organ culture model for shaping an ex vivo pancreatic niche. This model allows pancreatic progenitor cells to enter the ductal and endocrine lineages, while PDLOs further mature into duct-like tissue. Accordingly, the PUB offers an ex vivo platform for earliest pancreatic dysplasia and cancer if PDLOs feature KRASG12D mutations. Finally, it is demonstrated that PDOs-on-PUB i) resemble primary pancreatic cancer, ii) preserve cancer subtypes, iii) enable the study of niche epithelial crosstalk by spiking in pancreatic stellate and immune cells into the grafts, and finally iv) allow drug testing. In summary, the PUB advances the existing pancreatic cancer models by adding feasibility, complexity, and customization at low cost and high flexibility., publishedVersion

Preexisting diabetes increases the risk of a severe course of the pandemic coronavirus disease 2019 (COVID-19). Vice versa, exacerbations of a preexisting diabetes as well as new-onset diabetes have been reported upon SARS-CoV-2 infection. Thus, there is an imperative need to clarify whether human pancreatic endocrine cells organized within an islet of Langerhans are permissive for and affected by SARS-CoV-2 infection, and to elucidate the mechanisms underlying the development of diabetes upon COVID-19. Here, we (i) defined ACE2 and TMPRSS2 expression patterns in human pancreatic endocrine and exocrine cell types, (ii) employed human pancreatic islet cultures to demonstrate susceptibility to SARS-CoV-2 infection and to viral replication in β-cells, (iii) showed that SARS-CoV-2 attenuates glucose-stimulated insulin secretion, and (iv) tested remdesivir as eventually effective to prevent β-cell failure. In addition, we (v) visualized viral particles replicating in endocrine pancreatic cells and define their subcellular localization patterns via transmission electron microscopy, and finally (vi) present examples of cell type specific pancreatic infection patterns of COVID-19 deceased patients. Overall, our data demonstrate that SARS-CoV-2 can infect both the exocrine and endocrine compartments of the pancreas and can perturb β-cell integrity, which might lead to an increased risk for diabetes.

Genes involved in distinct diabetes types suggest shared disease mechanisms. Here we show that One Cut Homeobox 1 (ONECUT1) mutations cause monogenic recessive syndromic diabetes in two unrelated patients, characterized by intrauterine growth retardation, pancreas hypoplasia and gallbladder agenesis/hypoplasia, and early-onset diabetes in heterozygous relatives. Heterozygous carriers of rare coding variants of ONECUT1 define a distinctive subgroup of diabetic patients with early-onset, nonautoimmune diabetes, who respond well to diabetes treatment. In addition, common regulatory ONECUT1 variants are associated with multifactorial type 2 diabetes. Directed differentiation of human pluripotent stem cells revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. Loss of ONECUT1 altered transcription factor binding and enhancer activity and NKX2.2/NKX6.1 expression in pancreatic progenitor cells. Collectively, we demonstrate that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development, involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance. Our findings highlight the broad contribution of ONECUT1 in diabetes pathogenesis, marking an important step toward precision diabetes medicine.

[Background]: Organotypic cultures derived from pancreatic ductal adenocarcinoma (PDAC) termed pancreatic ductal cancer organoids (PDOs) recapitulate the primary cancer and can be derived from primary or metastatic biopsies. Although isolation and culture of patient-derived pancreatic organoids were established several years ago, pros and cons for individualized medicine have not been comprehensively investigated to date., [Methods]: We conducted a feasibility study, systematically comparing head-to-head patient-derived xenograft tumor (PDX) and PDX-derived organoids by rigorous immunohistochemical and molecular characterization. Subsequently, a drug testing platform was set up and validated in vivo. Patient-derived organoids were investigated as well., [Results]: First, PDOs faithfully recapitulated the morphology and marker protein expression patterns of the PDXs. Second, quantitative proteomes from the PDX as well as from corresponding organoid cultures showed high concordance. Third, genomic alterations, as assessed by array-based comparative genomic hybridization, revealed similar results in both groups. Fourth, we established a small-scale pharmacotyping platform adjusted to operate in parallel considering potential obstacles such as culture conditions, timing, drug dosing, and interpretation of the results. In vitro predictions were successfully validated in an in vivo xenograft trial. Translational proof-of-concept is exemplified in a patient with PDAC receiving palliative chemotherapy., [Conclusion]: Small-scale drug screening in organoids appears to be a feasible, robust and easy-to-handle disease modeling method to allow response predictions in parallel to daily clinical routine. Therefore, our fast and cost-efficient assay is a reasonable approach in a predictive clinical setting., The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Main funding is provided by the German Cancer Aid grant to A. Kleger (111879). Additional funding came from the Deutsche Forschungsgemeinschaft (DFG, K.L. 2544/1-1, and 1-2; GRK 2254/1 to T. Seufferlein), the BIU fund (Böhringer Ingelheim), the NDIMED-Verbund PancChip, and the Else-Kröner-Fresenius Memorial funding to A. Kleger. AK receives also funding from the DFG within the Heisenberg program and from the Baden-Württemberg Foundation via ExPo Chip. This project was also funded by ANR-DFG collaborative research project (ANR-18-CE92-0031, DFG KL 2544/5-1) to CJ and AK and via additional DFG funding KL 2544/6-1, KL 2544/7-1, KL 2544/1-1, and KL 2544/1-2 to AK. L. Perkhofer is funded by Bausteinprogramm of the Ulm University hospital. This work was supported by a project grant for André Lechel (Deutsche Krebshilfe/111264), for Patrick C. Hermann (Max Eder Fellowship 111746, Projektnummer 316249678 – SFB 1279, and Hector Foundation Cancer Research grant M65.1). Reinhild Rösler and parts of proteomics method development were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB 1074. Bruno Sainz Jr was funded by a Ramón y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain and a coordinated grant from the Fundación Asociación Española Contra el Cáncer (AECC).

Pancreatic ductal adenocarcinoma (PDAC) is still the Achilles heel in modern oncology, with an increasing incidence accompanied by a persisting high mortality. The developmental process of PDAC is thought to be stepwise via precursor lesions and sequential accumulation of mutations. Thereby, current sequencing studies recapitulate this genetic heterogeneity in PDAC and show besides a handful of driver mutations (KRAS, TP53) a plethora of passenger mutations that allow to define subtypes. However, modeling the mutations of interest and their effects is still challenging. Interestingly, organoids have the potential to recapitulate in vitro, the in vivo characteristics of the tissue they originate from. Here, we could establish and develop tools allowing us to isolate, culture, and genetically modify ductal mouse organoids. Transferred to known effectors in the IPMN-PDAC sequence, we could reveal significantly increased proliferative and self-renewal capacities for PTEN and RNF43 deficiency in the context of oncogenic KRASG12D in mouse pancreatic organoids. Overall, we were able to obtain promising data centering ductal organoids in the focus of future PDAC research.

Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin (Eomes) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes. We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo.

[Objective]: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment. [Design]: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny. [Results]: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids. [Conclusions]: Taken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures., This study was funded by the Deutsche Forschungsgemeinschaft (DFG, K.L. 2544/1-1 and 1-2), the Forschungskern SyStaR to AK, BIU (Böhringer Ingelheim Ulm to AK), the Fritz-Thyssen Foundation (Az. 10.15.2.040), the German Cancer Aid (111879) and the Else-Kröner-Fresenius-Stiftung (2011_A200). AK is indebted to the Baden-Württemberg Stiftung for the financial support of this research project by the Eliteprogramme for Postdocs. AK is also an Else-Kröner-Fresenius Memorial Fellow. LP is supported by a research fellowship of the Else-Kröner-Fresenius Stiftung. MH was supported by the International Graduate School in Molecular Medicine and the Bausteinprogramme (L.SBN. 110), Ulm University. MM is supported by a grant of Ulm University (Baustein for Senior Clinician Scientists). IGC is funded by the Interdisciplinary Center for Clinical Research (IZKF Aachen) and Start Program, RWTH Aachen University Medical School, Aachen, Germany. AK was supported by an UEG Top Abstract Prize awarded at the United European Gastroenterology Week 2015.

Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression., Summary: Myosin 1b and 1c localize to fused secretory vesicles and influence the compression of the actin coat during exocytosis.

The TanDEM-X mission (TDM) is a spaceborne Radar interferometer which delivers a global digital surface model (DSM) with an unprecedented spatial resolution. This allows resolving objects above ground such as buildings. Extracting and characterizing those objects in an automated manner represents a challenging problem but opens simultaneously a broad range of large-area applications. In this paper, we discuss and evaluate the suitability of morphological filters (MFs) for the derivation of normalized DSMs from the TDM in complex urban Environments and introduce a novel region-growing-based progressive MF procedure. This approach is jointly proposed and can be combined with a postclassification processing scheme to specifically allow for a viable reconstruction of urban morphology in a challenging terrain. The filter approach comprises a multistep procedure using concepts of morphological image filtering, region growing, and interpolation techniques. Therefore, it extends the idea of progressive MFs. The latter aim to identify nonground pixels in the DSM by gradually increasing the size of a structuring element and applying iteratively an elevation difference threshold. After the identification of initial nonground pixels, here, potential nonground pixels are identified within each iteration, and their similarity with respect to neighboring nonground pixels is assessed. Pixels are finally labeled as nonground if a constraint is fulfilled. The postclassification processing scheme adapts techniques of object-based image analyses to further refine regions of classified nonground pixels. Digital terrain models are subsequently generated by interpolating between identified ground pixels. Experimental results are obtained for settlement areas that cover large parts of the cities of Izmir (Turkey) and Wuppertal (Germany). They confirm the capability of the proposed approaches for a reduction of omission errors compared to basic MF-based methods when classifying ground pixels, which is favorable in a mountainous Terrain with steep slopes.

In September 2013 the production of TanDEM-X digital elevation model (DEM) started. As the data acquisition for difficult terrain lasted until April 2014, final DEM production started for flat to moderate terrain regions where two final coverages surfice. This paper focuses on a first validation of moderate terrain to prove the absolute height accuracy. In a detailed comparison three DEM tiles from different continents are chosen to validate the TanDEM-X DEM by computing differences to GPS tracks, ICESat validation points, and SRTM. On a global scale all TanDEM-X DEMs produced so far are compared with ICESat and GPS tracks. Both validations presented here for the first time indicate that the absolute height error for moderate terrain for TanDEM-X is below 2m and therefore much better than the specified 10m/LE90.

For the global TanDEM-X DEM the whole world will be acquired by at least two coverages. Thereby on the one hand phase unwrapping errors are reduced by applying the dual-baseline method and on the other hand a low noise level is ensured even for difficult areas like forests and steep terrain. During DEM mosaicking, the single interferometric DEMs are merged together. This paper focuses on the combination of heights in overlapping areas with significant height differences. The challenge here is to choose the most reliable height value. The improvement applying this strategy in contrast to simple averaging and the general benefit of using more than one acquisition is shown by means of some example mosaics.

The German SAR interferometry mission TanDEM-X performed on two TerraSAR-X satellites flying in close formation will provide a global Digital Elevation Model (DEM). A by-product is so-called the Water Indication Mask (WAM). The purpose of this supplementary information layer is to support the DEM editing process. Water surfaces usually show lower coherence in an interferometric data set due to temporal de-correlation and low backscattering. Consequently the corresponding elevation values derived from the interferogram are random and produce a virtual relief. This paper introduces the operational water body detection workflow that synergistically evaluates amplitude and coherence information. The presented results of two test sites reveal that the methodology is globally applicable, classifications are highly accurate and the algorithm is appropriate for operational image processing. The water body detection consists of two steps: the Water Body Detection (WBD) derived of one single DEM scene and the mosaicking of multiple WBD to a single Water Indication Mask (WAM). The fusion strategy for the final TanDEM-X WAM considers all WBD acquired at different times in two global coverages and bases on a fusion by union containing the results of the amplitude and the coherence.

The global process of urbanization is associated with various ecological, social and economic changes in both the built-up area and the adjacent natural or cultivated landscape. To manage the effects and impacts of this development, effective urban and regional planning requires accurate and up to date information on the urban dynamics. This paper introduces a methodology to automatically detect human settlements and then further characterize the identified built-up areas in terms of the building density based on VHR SAR data. The SAR imagery is acquired by the German satellite system TerraSAR-X. Regarding the delineation of the built-up area in the region of Munich we achieved an overall accuracy of 94 % and a Kappa of 0.86. The estimation of building density showed a coefficient of determination (r2) of up to 0.74. The mean absolute error of the modeled building densities was 5 %.

The main product of the TanDEM-X mission is an interferometric DEM product that is finally calibrated due to residual systematic offsets and tilts and where different, generally two coverages, are mosaicked. Above this, a water mask is provided to support later editing of rough water areas. In this presentation the commissioning phase work to set the DEM production chain into operation is described and the first commissioning phase products are shown.

Additionally to the main product of the TanDEM-X mission - the global Digital Elevation Model (DEM) - a global water body mask will be produced. The main goal of this water mask is to deliver an information layer to support a subsequent DEM editing process. It is derived from the SAR amplitude and the single pass coherence. In this paper, the concept of the global water body detection is explained and a first evaluation of the single coverage water body detection is presented.

The aimed accuracies for the final TanDEM-X DEM of 10m absolute and 2m relative height error will be ensured by calibration data. One crucial data set for the relative accuracy is tie-points that connect adjacent DEM acquisitions in the approximately 4km-overlap-area with each other. In this paper an improved concept for tie-point candidates is presented that is based on averaging a larger region instead of comparing single points. This concept should be more robust against noise. It is validated by applying the DEM calibration on a simulated test area, as real TanDEM-X data was not yet available. Also, the DEM calibration will be validated for the first time on a larger “real” test site by applying the TanDEM-X processing scenario.

The current process of megacity development and urban sprawl are unique in human history. More and more so-called megacities with more than 10 million inhabitants are evolving throughout the world. The presented study is focusing on the earthquake-prone megacity Istanbul officially counting 12 million inhabitants in 2007. During the past decades, the megacity has undergone an enormous suburbanization into its outskirts. Recent urban developments, however, seem to indicate changing housing trends respectively types of urbanization in Istanbul. In our study we focus on a multi-temporal and multisensoral analysis using Landsat and TerraSAR-X data. By implementing an object-oriented classification approach settlement masks for 1975, 1987, 2000, and 2008 have been created. Furthermore, post-classification change detection is displaying medium and large scale urban developments of the megacity for the past decades. The results are conforming to current social studies focusing on urbanity and lifestyle: Istanbul is facing new types and factors of urban development. The study demonstrates both the synergistic usage of multi-temporal and multi-sensoral remotely sensed data. Additionally, the synergistic potential of remote sensing and applied urban studies to work out useful information for urban planners is presented.

Infection-related diabetes can arise as a result of virus-associated β-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.