Pancreatic Exocrine Cells Research Articles

Localization of trypsin, chymotrypsin and elastase in the digestive tract of amphioxus Branchiostoma japonicum with implications to the origin of vertebrate pancreas

The organ pancreas is characteristic of all vertebrates, but its evolutionary origin remains largely unknown. The serine proteinases trypsin, chymotrypsin and elastase are produced primarily in the pancreas in vertebrates. Here we clearly demonstrate by enzymatic activity assay, histochemical staining and in situ hybridization that trypsin, chymotrypsin and elastase are widely distributed in the digestive tract of the amphioxus Branchiostoma japonicum, especially in the hepatic caecum and mid-gut. This suggests that an extensive region of the amphioxus digestive tract including the hepatic caecum is homologous to the vertebrate exocrine pancreatic cells, providing a new angle for the study of the origin and evolution of vertebrate pancreas.

Paediatric pancreatoduodenectomy – A video vignette

Pancreatoduodenectomy (PD) is rarely required in the paediatric population but is the only curative option for certain pathologies such as pancreatoblastoma of the head of the pancreas. Pancreatoblastoma is a rare childhood tumour originating from pancreatic exocrine epithelial cells. It is the most common malignant pancreatic tumour in children. We describe the case of a 3-year-old girl with Beckwith-Wiedemann (BW) syndrome, with a background of liver resection aged 18 months for hepatoblastoma. On surveillance imaging, a mass lesion was identified in the head of her pancreas, which was further characterised on dedicated MRI pancreas and endoscopic ultrasound. Fine needle aspirate confirmed the diagnosis of pancreatoblastoma. Because of portal and superior mesenteric venous abutment on the initial imaging, a neo-adjuvant approach was taken with good tumour response to cisplatin and doxorubicin, followed by a Whipple's pancreatoduodenectomy (PD). Using intraoperative video footage, we detail the operative technique of pancreatoduodenectomy in the paediatric population. Modifications to standard reconstruction included a dunking pancreatojejunostomy instead of the more standard Blumgart technique. The patient's background of BW renders a lifelong risk of further solid-organ tumour formation. This case demonstrates how each tumour should be individually assessed with a full MDT approach despite any perceived complexity.

Abstract 2454: Imaging pancreatic ductal adenocarcinoma using a zinc-sensitive MRI contrast agent: A novel method to detect early-stage PDAC lesions

Abstract Background: Zinc content and its flux from the exocrine pancreas are both novel candidates for molecular imaging of pancreatic ductal adenocarcinoma (PDAC) due to the markedly dysregulated homeostasis in PDAC lesions. Here, we introduce the use of a Gd-based zinc probe (GdL) to monitor stimulated exocrine function in the healthy and PDAC bearing mouse pancreas by MRI. Methods: Male adult mice were implanted with syngeneic PDAC cells into the pancreas and allowed to form PDAC for 1 and 2 weeks. Healthy mice were used as controls. Mice were fasted overnight and imaged in a 4.7 T scanner. Two 2D T1-weighted gradient echo scans were obtained as baseline (TE/TR = 2.9/125 ms, Avg = 4). Mice then received either 1) 0.07mmol/kg GdL (i.v.) plus 10 ug/kg caerulein (i.p.), 2) 0.07mmol/kg GdL (i.v.) plus Saline (i.p.), or 3) 0.01 mmol/kg Gd-DOTA (i.v.). Immediately following injections, serial 2D T1-weighted gradient echo scans were obtained for 25 minutes. PDAC mice receiving saline were considered as negative controls. Change in contrast to-noise ratio (CNR) of tumor and pancreas was quantified and reported as a function of time. Tumor and pancreas tissue were then fixed, and histological analysis was done to characterize the expression of zinc transporters in healthy pancreas and PDAC tissues. Results: Secretagogue-stimulated zinc secretion (SSZS) MRI of the exocrine pancreas relies on evoking zinc release from intracellular stores from acinar and ductal cells. After i.p. injection of the secretagogue caerulein in fasted healthy animals, we observed only a small enhancement in the pancreas. However, both groups of animals carrying orthotopically implanted PDAC cells (1-wk and 2-wks) responded in hyper-secretory fashion. Negative controls (saline group) did not show significant pancreatic or tumor enhancement. Importantly, the effect of caerulein in PDAC animals and the increase in overall pancreatic enhancement were only found in PDAC tumor bearing animals. Dysregulation of zinc trafficking was confirmed by immunohistochemistry staining for the zinc import transporters ZIP3 and ZIP4. These transporters were upregulated only in PDAC-bearing pancreas and the positive staining was concentrated mostly in the tumor parenchyma and gradually dissipated in the surrounding pancreatic tissue. Conclusions: Zinc flux is markedly upregulated in PDAC. By stimulating the natural zinc secretion from exocrine pancreatic cells using an exogenous secretagogue, caerulein, we can non-invasively monitor early malignant transformations in pancreatic parenchyma and tumor cells by MRI with a Gd-based zinc sensitive probe. Citation Format: Mozhdeh Sojoodi, Ian Ramsay, Eric Gale, Stephen C. Barrett, Motaz Qadan, Peter Caravan, Kenneth K. Tanabe, Veronica Clavijo Jordan. Imaging pancreatic ductal adenocarcinoma using a zinc-sensitive MRI contrast agent: A novel method to detect early-stage PDAC lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2454.

296-OR: Single-Cell Multiomic Analysis of the Human Pancreas in Type 1 Diabetes

The cis-regulatory programs of specific cell types in the pancreas that are affected in type 1 diabetes (T1D) are poorly understood. Single cell technology enables detailed interrogation of genomic profiles of cell types within a heterogeneous tissue, and when applied to samples across disease states can reveal cell type-specific changes in disease. In this study we applied single nucleus RNA-seq (snRNA-seq) and single nucleus ATAC-seq (snATAC-seq) , as well as paired multiome (snRNA-seq+snATAC-seq) , to cryopreserved whole pancreas samples from 34 donors including 13 control, 9 T1D autoantibody positive (Aab+) , 7 early T1D (1< yr) and 5 late T1D donors in the nPOD biorepository. Clustering of 313,078 gene expression and 342,863 accessible chromatin profiles after extensive processing and filtering resolved pancreatic endocrine, exocrine, immune, stellate, glial and endothelial cell types, as well as known and novel cell sub-types for example REG1A+ acinar cells and MUC5b+ ductal cells. There were marked changes in the abundance of many cell types and sub-types across disease states, including a significant reduction in beta cells and increase in immune cell populations in T1D compared to control. Differential analysis of gene expression and cis-regulatory element (cCRE) activity in each cell type revealed changes in early and late T1D, as well as in T1D Aab+. In beta cells, 2 and 2,177 genes and 1,080 and 9,177 cCREs had differential activity in early and late T1D, respectively, and early and late T1D genes in beta cells were enriched for distinct molecular processes related to interferon gamma signaling and cell death. Intersecting differential beta cell cCREs with comprehensive T1D genetic fine-mapping revealed 13 T1D signals where candidate variants mapped in a differential beta cell cRE of which were proximal to a differentially expressed gene, such as at the GLIS3 locus. Together these results provide insight into the role of cis-regulatory programs in beta cells and other pancreatic cell types in T1D. Disclosure R.Melton: None. S.Preissl: None. M.Sander: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. E.Beebe: None. C.Zeng: None. M.T.Miller: None. G.Wang: None. C.Mcgrail: None. H.Mummey: None. I.Kusmartseva: None. M.A.Atkinson: None. Funding Ruth Kirschstein Institutional National Research Service Award T32 (GM008666) National Institutes of Health (DK120429)

SARS-CoV-2 Infection Causes Hyperglycemia in Cats.

Isolated reports of new-onset diabetes in patients with COVID-19 have led researchers to hypothesise that SARS-CoV-2 infects the human exocrine and endocrine pancreatic cells ex vivo and in vivo. However, existing research lacks experimental evidence indicating that SARS-CoV-2 can infect pancreatic tissue. Here, we found that cats infected with a high dose of SARS-CoV-2 exhibited hyperglycaemia. We also detected SARS-CoV-2 RNA in the pancreatic tissues of these cats, and immunohistochemical staining revealed the presence of SARS-CoV-2 nucleocapsid protein (NP) in the islet cells. SARS-CoV-2 NP and Spike proteins were primarily detected in Glu+ cells, and most Glu+ cells expressed ACE2. Additionally, immune protection experiments conducted on cats showed that the blood glucose levels of immunised cats did not increase post-challenge. Our data indicate the cat pancreas as a SARS-CoV-2 target and suggest that the infection of Glu+ cells could contribute to the metabolic dysregulation observed in SARS-CoV-2-infected cats.

Crocetin alleviates the caerulein-induced apoptosis and inflammation in AR42J cells by activating SIRT1 via NF-κB.

The anti-inflammatory and anti-apoptotic properties of crocetin have been widely demonstrated in numerous diseases. However, the exact role and mechanism of crocetin in acute pancreatitis have not been elucidated. Thus, this paper aims at exploring whether crocetin could be used to alleviate acute pancreatitis and further demonstrating the underlying mechanisms. Cell viability of caerulein-induced pancreatic exocrine cell line AR42J treated with crocetin was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). Apoptosis and inflammation of these treated cells were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), western blot and enzyme linked immunosorbent assay (ELISA). The expression of sirtuin-1 (SIRT1) was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. After knockdown of SIRT1, cell viability, apoptosis and inflammation were measured again by corresponding kits. Finally, the NF-κB nuclear translocation and proteins in the NF-κB signaling were examined. Crocetin remarkably suppressed the apoptosis and inflammation of caerulein-induced AR42J cells. The decreased expression of SIRT1 was increased in caerulein-induced AR42J cells after exposure to crocetin. After knockdown of SIRT1, the alleviative effects of crocetin were found to be canceled in these cells. Furthermore, SIRT1 knockdown promoted the NF-κB signal transduction. On the whole, we presented the first evidence for the importance of SIRT1-NF-κB axis in acute pancreatitis and proposed that crocetin alleviates the caerulein-induced apoptosis and inflammation in AR42J cells by activating SIRT1 via NF-κB.

Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry.

SUMMARYType 2 diabetes mellitus (T2D) is a chronic age-related disorder characterized by hyperglycemia due to the failure of pancreatic beta cells to compensate for increased insulin demand. Despite decades of research, the pathogenic mechanisms underlying T2D remain poorly defined. Here, we use imaging mass cytometry (IMC) with a panel of 34 antibodies to simultaneously quantify markers of pancreatic exocrine, islet, and immune cells and stromal components. We analyze over 2 million cells from 16 pancreata obtained from donors with T2D and 13 pancreata from age-similar non-diabetic controls. In the T2D pancreata, we observe significant alterations in islet architecture, endocrine cell composition, and immune cell constituents. Thus, both HLA-DR-positive CD8 T cells and macrophages are enriched intra-islet in the T2D pancreas. These efforts demonstrate the utility of IMC for investigating complex events at the cellular level in order to provide insights into the pathophysiology of T2D.

A Comparative Endocrine Trans-Differentiation Approach to Pancreatic Ductal Adenocarcinoma Cells with Different EMT Phenotypes Identifies Quasi-Mesenchymal Tumor Cells as Those with Highest Plasticity.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancer types with the quasi-mesenchymal (QM) subtype of PDAC having the worst prognosis. De-differentiation of the ductal tumor cells to a mesenchymal phenotype occurs as a result of epithelial–mesenchymal transition (EMT), a process associated with the acquisition of stem cell traits. While QM tumor cells are highly metastatic and drug-resistant, their increased plasticity opens a window of opportunity for trans-differentiation into non-malignant pancreatic cells. In this study we compared established PDAC-derived cell lines of either epithelial (E) or QM phenotype for their potential to be differentiated to pancreatic endocrine cells. We found that QM cells responded more strongly than E cells with transcriptional activation of a pancreatic progenitor or pancreatic β cell-specific program. Our results bear strong implications for a novel type of targeted therapy, namely EMT-based trans-differentiation of highly metastatic PDAC cells in vivo to non-malignant endocrine cells.Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and therapy-resistant cancer types which is largely due to tumor heterogeneity, cancer cell de-differentiation, and early metastatic spread. The major molecular subtypes of PDAC are designated classical/epithelial (E) and quasi-mesenchymal (QM) subtypes, with the latter having the worst prognosis. Epithelial–mesenchymal transition (EMT) and the reverse process, mesenchymal-epithelial transition (MET), are involved in regulating invasion/metastasis and stem cell generation in cancer cells but also early pancreatic endocrine differentiation or de-differentiation of adult pancreatic islet cells in vitro, suggesting that pancreatic ductal exocrine and endocrine cells share common EMT programs. Using a panel of PDAC-derived cell lines classified by epithelial/mesenchymal expression as either E or QM, we compared their trans-differentiation (TD) potential to endocrine progenitor or β cell-like cells since studies with human pancreatic cancer cells for possible future TD therapy in PDAC patients are not available so far. We observed that QM cell lines responded strongly to TD culture using as inducers 5′-aza-2′-deoxycytidine or growth factors/cytokines, while their E counterparts were refractory or showed only a weak response. Moreover, the gain of plasticity was associated with a decrease in proliferative and migratory activities and was directly related to epigenetic changes acquired during selection of a metastatic phenotype as revealed by TD experiments using the paired isogenic COLO 357-L3.6pl model. Our data indicate that a QM phenotype in PDAC coincides with increased plasticity and heightened trans-differentiation potential to activate a pancreatic β cell-specific transcriptional program. We strongly assume that this specific biological feature has potential to be exploited clinically in TD-based therapy to convert metastatic PDAC cells into less malignant or even benign cells.

Abstract 728: Tissue-specific tumorigenesis in multiple endocrine neoplasia type 1

Abstract Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer syndrome caused by a germline mutation in the MEN1 gene. While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in specific tissues such as the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in mouse pancreatic exocrine tissue does not result in tumor formation, suggesting a tissue-specific mechanism. Loss of menin activates a menin-interacting protein retinoblastoma-binding-protein 5 (RBBP5). Since RBBP5 transcriptionally regulates DNA methyltransferase 1 (DNMT1), this causes global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. We hypothesize that while RBBP5 is ubiquitously expressed, it exclusively binds to the DNMT1 promoter in MEN1-target-tissues through its recruitment by tissue-specific factors. Using ChIP-PCR, we demonstrated that Rbbp5 is bound to the Dnmt1 promoter in MEN1-target-tissues, while not in non-target tissues. In the present study, we set out to identify and validate putative endocrine-specific factors involved in Rbbp5 binding to the Dnmt1 promoter. To investigate this tissue-specific occupancy of Rbbp5 at the Dnmt1 promoter, a ChIP-Seq analysis was carried out on DNA purified from pancreatic endocrine islet cells and pancreatic exocrine cells from wild type (WT) mice. These DNA samples were analyzed to identify differentially occupied DNA binding sites of menin and Rbbp5 by a high-throughput tissue-specific ChIP-seq assay. This analysis yielded a list of endocrine-specific genes regulated by both menin and Rbbp5. In order to further identify the tissue-specific factors that may alter menin-Rbbp5 binding to the Dnmt1 promoter, we performed tissue-specific RNA-Seq on endocrine and exocrine pancreas tissues from WT mice. Integrating tissue-specific ChIP-Seq and RNA-seq data, we identified 20 endocrine tissue-specific factors that may alter the interaction of the menin-Rbbp5 complex with the Dnmt1 promoter. By carrying out this unbiased genome-wide screen, we identified endocrine-specific candidate factors that may interact with the menin-Rbbp5 complex at the Dnmt1 promoter. After screening all candidates, the stand-out candidates were identified for further validation. Criteria to be met to establish stand-out candidates included: MEN1-target-tissue-specific expression through immunohistochemical staining and high mRNA expression by qRT-PCR analysis in WT mouse pancreatic islets on samples extracted by laser capture microscopy. In conclusion, we have identified candidates for Rbbp5 recruitment to the Dnmt1 promoter that must be tested further to determine their role in the observed tissue specificity of MEN1-related tumorigenesis. Citation Format: Juliet C. Gardiner. Tissue-specific tumorigenesis in multiple endocrine neoplasia type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 728.

Deleterious Effects of SARS-CoV-2 Infection on Human Pancreatic Cells.

COVID-19 pandemic has infected more than 154 million people worldwide and caused more than 3.2 million deaths. It is transmitted by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and affects the respiratory tract as well as extra-pulmonary systems, including the pancreas, that express the virus entry receptor, Angiotensin-Converting Enzyme 2 (ACE2) receptor. Importantly, the endocrine and exocrine pancreas, the latter composed of ductal and acinar cells, express high levels of ACE2, which correlates to impaired functionality characterized as acute pancreatitis observed in some cases presenting with COVID-19. Since acute pancreatitis is already one of the most frequent gastrointestinal causes of hospitalization in the U.S. and the majority of studies investigating the effects of SARS-CoV-2 on the pancreas are clinical and observational, we utilized human iPSC technology to investigate the potential deleterious effects of SARS-CoV-2 infection on iPSC-derived pancreatic cultures containing endocrine and exocrine cells. Interestingly, iPSC-derived pancreatic cultures allow SARS-CoV-2 entry and establish infection, thus perturbing their normal molecular and cellular phenotypes. The infection increased a key cytokine, CXCL12, known to be involved in inflammatory responses in the pancreas. Transcriptome analysis of infected pancreatic cultures confirmed that SARS-CoV-2 hijacks the ribosomal machinery in these cells. Notably, the SARS-CoV-2 infectivity of the pancreas was confirmed in post-mortem tissues from COVID-19 patients, which showed co-localization of SARS-CoV-2 in pancreatic endocrine and exocrine cells and increased the expression of some pancreatic ductal stress response genes. Thus, we demonstrate that SARS-CoV-2 can directly infect human iPSC-derived pancreatic cells with strong supporting evidence of presence of the virus in post-mortem pancreatic tissue of confirmed COVID-19 human cases. This novel model of iPSC-derived pancreatic cultures will open new avenues for the comprehension of the SARS-CoV-2 infection and potentially establish a platform for endocrine and exocrine pancreas-specific antiviral drug screening.

Lack of CFTR alters the ferret pancreatic ductal epithelial secretome and cellular proteome: Implications for exocrine/endocrine signaling

BackgroundCystic fibrosis (CF) related diabetes is the most common comorbidity for CF patients and associated with islet dysfunction. Exocrine pancreas remodeling in CF alters the microenvironment in which islets reside. Since CFTR is mainly expressed in pancreatic ductal epithelium, we hypothesized altered CF ductal secretions could impact islet function through paracrine signals. MethodWe evaluated the secretome and cellular proteome of polarized WT and CF ferret ductal epithelia using quantitative ratiometric mass spectrometry. Differentially secreted proteins (DSPs) or expressed cellular proteins were used to mine pathways, upstream regulators and the CFTR interactome to map candidate CF-associated alterations in ductal signaling and phenotype. Candidate DSPs were evaluated for their in vivo pancreatic expression patterns and their functional impact on islet hormone secretion. ResultsThe secretome and cellular proteome of CF ductal epithelia was significantly altered relative to WT and implicated dysregulated TGFβ, WNT, and BMP signaling pathways. Cognate receptors of DSPs from CF epithelia were equally distributed among endocrine, exocrine, and stromal pancreatic cell types. IGFBP7 was a downregulated DSP in CF ductal epithelia in vitro and exhibited reduced CF ductal expression in vivo. IGFBP7 also altered WT islet insulin secretion in response to glucose. Many CFTR-associated proteins, including SLC9A3R1, were differentially expressed in the CF cellular proteome. Upstream regulators of the differential CF ductal proteome included TGFβ, PDX1, AKT/PTEN, and INSR signaling. Data is available via ProteomeXchange with identifier PXD025126. ConclusionThese findings provide a proteomic roadmap for elucidating disturbances in autocrine and paracrine signals from CF pancreatic ducts and how they may alter islet function and maintenance.

Scrt1, a transcriptional regulator of \u03b2-cell proliferation identified by differential chromatin accessibility during islet maturation

Glucose-induced insulin secretion, a hallmark of mature β-cells, is achieved after birth and is preceded by a phase of intense proliferation. These events occurring in the neonatal period are decisive for establishing an appropriate functional β-cell mass that provides the required insulin throughout life. However, key regulators of gene expression involved in functional maturation of β-cells remain to be elucidated. Here, we addressed this issue by mapping open chromatin regions in newborn versus adult rat islets using the ATAC-seq assay. We obtained a genome-wide picture of chromatin accessible sites (~ 100,000) among which 20% were differentially accessible during maturation. An enrichment analysis of transcription factor binding sites identified a group of transcription factors that could explain these changes. Among them, Scrt1 was found to act as a transcriptional repressor and to control β-cell proliferation. Interestingly, Scrt1 expression was controlled by the transcriptional repressor RE-1 silencing transcription factor (REST) and was increased in an in vitro reprogramming system of pancreatic exocrine cells to β-like cells. Overall, this study led to the identification of several known and unforeseen key transcriptional events occurring during β-cell maturation. These findings will help defining new strategies to induce the functional maturation of surrogate insulin-producing cells.

A new hypothetical model for pancreatic development based on change in the cell division orientation.

Although lifelong renewal and additional compensatory growth in response to demand are undeniable facts, so far, no specific stem cells have been found for pancreatic cells. According to the consensus model, the development of pancreas results from the hierarchical differentiation of pluripotent stem cells towards the appearance of the first endocrine and exocrine cells at approximately 7.5 to 8th gestation week (GW) of human embryo. However, the primitive endocrine cells arising from the embryonic phase of development do not appear to be mature or fully functional. Asymmetric localization of cellular components, such as Numb, partition protein complexes (PAR), planar cell polarity components, and certain mRNAs on the apical and basal sides of epithelial cells, causes cellular polarization. According to our model, the equal distribution of cellular components during symmetric cell division yields similar daughter cells that are associated with duct expansion. In contrast, asymmetric cell division is associated with uneven distribution of cellular components among daughter cells, resulting in different fates. Asymmetric cell division leads to duct branching and the development of acinar and stellate cells by a daughter cell, as well as the development of islet progenitor cells through partial epithelial-to-mesenchymal transition (EMT) and delamination of another daughter cell. Recently, we have developed an efficient method to obtain insulin-secreting cells from the transdifferentiation of hESC-derived ductal cells inducing a partial EMT by treatment with Wnt3A and activin A in a hypoxic environment. Similar models can be offered for other tissues and organs such as mammary glands, lungs, prostate, liver, etc. This model may open a new horizon in the field of regenerative medicine and be useful in explaining the cause of certain abnormalities, such as the occurrence of certain cysts and tumors.

MECOM permits pancreatic acinar cell dedifferentiation avoiding cell death under stress conditions

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in “Pathways of cancer” with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

Culture, differentiation, and transduction of mouse E12.5 pancreatic spheres: an in vitro model for the secondary transition of pancreas development

ABSTRACT During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.

Pre-existing beta cells but not progenitors contribute to new beta cells in the adult pancreas.

It has been suggested that new beta cells can arise from specific populations of adult pancreatic progenitors or facultative stem cells. However, their existence remains controversial, and the conditions under which they would contribute to new beta-cell formation are not clear. Here, we use a suite of mouse models enabling dual-recombinase-mediated genetic tracing to simultaneously fate map insulin-positive and insulin-negative cells in the adult pancreas. We find that the insulin-negative cells, of both endocrine and exocrine origin, do not generate new beta cells in the adult pancreas during homeostasis, pregnancy or injury, including partial pancreatectomy, pancreatic duct ligation or beta-cell ablation with streptozotocin. However, non-beta cells can give rise to insulin-positive cells after extreme genetic ablation of beta cells, consistent with transdifferentiation. Together, our data indicate that pancreatic endocrine and exocrine progenitor cells do not contribute to new beta-cell formation in the adult mouse pancreas under physiological conditions.

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

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.

Exocrine Pancreatic Cell NF-κB/AP-1 Crosstalk Downstream of ERK MAPK

Aim: To test the hypothesis that cooperative interactions between transcription factors Nuclear Factor-kappa B (NF-κB) and Activator Protein-1 (AP1) augment transcriptional activity in exocrine pancreatic cells.

Rb and p53 Execute Distinct Roles in the Development of Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PanNET) were classified into grades (G) 1 to 3 by the World Health Organization in 2017, but the precise mechanisms of PanNET initiation and progression have remained unclear. In this study, we used a genetically engineered mouse model to investigate the mechanisms of PanNET formation. Although pancreas-specific deletion of the Rb gene (Pdx1-Cre;Rbf/f ) in mice did not affect pancreatic exocrine cells, the α-cell/β-cell ratio of islet cells was decreased at 8 months of age. During long-term observation (18-20 months), mice formed well-differentiated PanNET with a Ki67-labeling index of 2.7%. In contrast, pancreas-specific induction of a p53 mutation (Pdx1-Cre;Trp53R172H ) had no effect on pancreatic exocrine and endocrine tissues, but simultaneous induction of a p53 mutation with Rb gene deletion (Pdx1-Cre;Trp53R172H;Rb f/f ) resulted in the formation of aggressive PanNET with a Ki67-labeling index of 24.7% over the short-term (4 months). In Pdx1-Cre;Trp53R172H;Rbf/f mice, mRNA expression of Pten and Tsc2, negative regulators of the mTOR pathway, significantly decreased in the islet cells, and activation of the mTOR pathway was confirmed in subsequently formed PanNET. Thus, by manipulating Rb and p53 genes, we established a multistep progression model from dysplastic islet to indolent PanNET and aggressive metastatic PanNET in mice. These observations suggest that Rb and p53 have distinct roles in the development of PanNET. SIGNIFICANCE: Pancreas-specific manipulation of Rb and p53 genes induced malignant transformation of islet cells, reproducing stepwise progression from microadenomas to indolent (grade 1) and subsequent aggressive PanNETs (grade 2-3).

REST Inhibits Direct Reprogramming of Pancreatic Exocrine to Endocrine Cells by Preventing PDX1-Mediated Activation of Endocrine Genes

The emerging appreciation of plasticity among pancreatic lineages has created interest in harnessing cellular reprogramming for β cell replacement therapy of diabetes. Current reprogramming methodologies are inefficient, largely because of a limited understanding of the underlying mechanisms. Using an invitro reprogramming system, we reveal the transcriptional repressor RE-1 silencing transcription factor (REST) as a barrier for β cell gene expression in the reprogramming of pancreatic exocrine cells. We observe that REST-bound loci lie adjacent to the binding sites of multiple key β cell transcription factors, including PDX1. Accordingly, a loss of REST function combined with PDX1 expression results in the synergistic activation of endocrine genes. This is accompanied by increased histone acetylation and PDX1 binding at endocrine gene loci. Collectively, our data identify a mechanism for REST activity involving the prevention of PDX1-mediated activation of endocrine genes and uncover REST downregulation and the resulting chromatin alterations as key events in β cell reprogramming.