BACKGROUND & AIMS: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell-sequencing approaches. METHODS: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. RESULTS: We created the first comprehensive atlas of human pancreas cells, to our knowledge Q9 , including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus-sequencing data

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a–c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology.

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The cellular heterogeneity of the human pancreas has not been previously characterized due to the presence of extreme digestive enzymatic activities, causing rapid degradation of cells and RNA upon resection. Therefore, previous cellular mapping studies based on gene expression were focused on pancreatic islets, leading to a vast underrepresentation of the exocrine compartment. By profiling the transcriptome of more than 110,000 cells from human donors, we created the first comprehensive pancreas cell atlas including all the tissue components. We unveiled the existence of four different acinar cell states and suggest a division of labor for enzyme production within the healthy exocrine pancreas, which has so far been considered a homogeneous tissue. This work provides a novel and rich resource for future investigations of the healthy and diseased pancreas. Main textSingle-cell RNA sequencing (scRNA-seq) has tremendously expanded our understanding of heterogeneous human tissues and made the identification of novel functional cell types in the lung, brain and liver possible 1-5 . The development of single-nucleus RNA-seq (sNuc-seq) has further broadened its application to tissues which are difficult to dissociate or already archived, such as clinical samples 6 . Pancreatic exocrine tissues contain among the highest level of digestive enzymatic activities in the human body 7 , hindering the preparation of undegraded RNA from this organ. Therefore, previous scRNA-seq studies of the human pancreas have been restricted to the islets of Langerhans (the endocrine part of the organ) in order to remove the exocrine compartment, namely the acinar and ductal cells responsible for the production and transport of digestive enzymes. Following their isolation, the endocrine islets were cultured in vitro, enzymatically dissociated and processed on microfluidics devices before next-generation sequencing [8][9][10][11][12][13][14] . While this strategy proved to be successful in generating a draft of the endocrine human pancreas cell atlas, it has distinct disadvantages. For example, only a very small number of exocrine cells have been captured and their numbers are largely underrepresented relative to homeostatic physiological conditions (approximately 5% rather than 95%). Moreover, in vitro culture and dissociation steps are known to introduce technical artefacts in gene expression measurements 15 . In this work we opted to use flash-frozen tissue biopsies isolated from pancreata of six human donors followed by sNuc-seq ( Fig. 1a), avoiding in vitro expansion and dissociation procedures, aiming to obtain an unbiased sampling of the organ.

Sirolimus and its derivatives have similar effects on endothelial regrowth and neointimal thickening. The observation of greatest fibrin deposition in the experimental EES group indicates that everolimus may affect vascular healing differently.