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.

Deleterious effects of SARS-CoV-2 infection on human pancreatic cells

SUMMARYCOVID-19 pandemic has infected more than 46 million people worldwide and caused more than 1.2 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, SARS-CoV-2 is capable of infecting iPSC-derived pancreatic cells, thus perturbing their normal molecular and cellular phenotypes. The infection increased a key inflammatory cytokine, CXCL12, known to be involved in pancreas dysfunction. 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 is 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 for the first time that SARS-CoV-2 can directly infect human iPSC-derived pancreatic cells with 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.

Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into β-Like Cells and the Identification of Key miRNAs for Differentiation

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into β-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into β-like cells. The differentiated β-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into β-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into β-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes

CURRENT VIEWS ON HISTOPHYSIOLOGY OF BRONCHIOLAR EXOCRINE CELLS

В обзоре приводятся новые данные о структуре и функции бронхиолярных экзокриноцитов. Впервые нереснитчатые клетки в бронхиолах были описаны ещё А. фон Кёлликером в 1881 г. Детальное изучение этих клеток в бронхиолах человека и кроликов было проведено М. Клара в 1937 г., в честь которого они были названы. В обзоре обсуждаются следующие функции клеток Клара (КК), или бронхиолярных экзокриноцитов: защитная функция, обусловленная секрецией специфических белков, а также жидкого субстрата, располагающегося на поверхности слизистой оболочки; участие в восстановлении повреждённых реснитчатых клеток в качестве своеобразных стволовых (прогениторных) клеток; функция детоксикации вредных веществ, попадающих в лёгкие, а именно: метаболизация ксенобиотиков и канцерогенных веществ; участие в развитии многих форм рака лёгких, источником формирования которых являются бронхиолярные экзокриноциты, включая аденокарциному - наиболее часто диагностируемую опухоль лёгкого. This review provides new data on the structure and function of bronchiolar exocrine cells. The nonciliary cells in the bronchioles were first described by Kolliker as early as in 1881. The detailed study of these cells in human and rabbit bronchioles was carried out by M. Clara in 1973, and the cells were named after him. The review discusses the following functions of Clara cells or bronchiolar exocrine cells: a protective function due to the secretion of specific proteins, as well as a liquid substrate located on the surface of the mucous membrane; participation in the restoration of damaged ciliary cells as a kind of stem (progenitor) cells; the function of detoxification of harmful substances that enter the lungs, namely: the metabolism of xenobiotics and carcinogens; participation in the development of many forms of lung cancer, the source of the formation of which are bronchiolar exocrine cells, including adenocarcinoma, the most commonly diagnosed lung tumor.

Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies.

Pancreas-specific miR-216a regulates proliferation and endocrine and exocrine cell function in vivo

Pancreas is a vital organ composed of exocrine and endocrine cells that aid digestion of food and regulate blood glucose levels. Perturbations in the function of pancreatic cells leads to the development of life-burdening and/or threatening diseases such as diabetes and pancreatic cancer. Thus, it is critical to understand the molecular check-points that maintain normal pancreas physiology. MicroRNAs (miRNAs) are small non-coding RNAs involved in regulating gene expression in normal and diseased tissues. Several miRNAs have tissue-specific patterns consistent with crucial functions in many biological processes. Yet, there is limited knowledge about the role of pancreas-specific miRNAs in pancreatic pathologies. Here, we report that miR-216a is a conserved, pancreas-specific miRNA that is expressed in both endocrine and exocrine cells. Deletion of miR-216a in mice leads to reduced β-cell mass and a reduction in islet size under both chow and high-fat diet feeding conditions. We show that inhibition of miR-216a increases apoptosis and decreases cell proliferation in β- and exocrine cells. Smad7 is upregulated in miR-216a deficient islets and cell cycle and proliferation are among the most significantly regulated biological processes in miR-216 knockout pancreata. Re-introduction of miR-216a in the pancreatic cancer line, PANC-1, increases cell migration more than 2-fold. In vivo, deletion of miR-216a in the pancreatic cancer prone mouse line KrasG12D;Ptf1aCreER inhibits the propensity of pancreatic cancer precursor lesions. Our study identifies miR-216a as an important pancreas-specific miRNA which may have implications for both diabetes and pancreatic cancer.

Dynamics of distribution WGA+ receptors in pancreas in norm and after intranatal action of antigens

Study of features of expression of carbohydrate bits (receptors to lectins) on the surface of cells, in the contests of intracellular granules and on components of extracellular matrix allows to widen the understanding of processes of organs morphogenesis and fabrics and understand the features of reactivity in reply to the action of various factors in the antenatal period of development. Research aim — to set the features of distribution and dynamics of expression of receptors to the wheat germ agglutinin (WGA) in the structures of pancreas in a norm and after the intranatal action of antigens. 126 pancreases of white laboratory rats from a moment birth to ninetieth time of postnatal life were examined in the research work. Rats were retained in a vivarium according to corresponding recommendations. The investigated animals were divided into three groups. The first group is intact rats; the second are control rats, the third are rats, what were undergone intranatal injection of Vaxygrip vaccine. During work with experimental animals followed European convention for defense of vertebrates that are used in experimental and other scientific aims (Strasbourg, 18.03.86). Exposure of carbohydrate bits and pieces of N-acetyl-D-glucosamine in microsections was conducted with application of wheat germ agglutinin (WGA), drawing on the standard set of lectins of SEC “Lectintest” (Lviv). Intensity of deposit of benzidine mark was estimated semi-quantitative. It is set that for experimental animals regardless of way of introduction to the antigen is observed increase of closeness of distribution of carbohydrate bits and pieces of N-acetyl-D-glucosamine in all investigated structures, most expressive in the wall of channels and vessels. Fastening of zymogenic granules of pancreatic exocrine cells acinus with bits and pieces of carbohydrate of N-acetyl-D-glucosamine testifies, that converting of proenzyme into an active form takes place at participation of glycopolymers. As carbohydrate determinants enter in the complement of zymogenic granules of pancreatic exocrine cells, the educed increase in an amount receptor to the lectin of embryos of wheat can influence on composition and properties of enzymes that is synthesized in acinar cells.

Cell Type-Selective Expression of Circular RNAs in Human Pancreatic Islets

Understanding distinct cell-type specific gene expression in human pancreatic islets is important for developing islet regeneration strategies and therapies to improve β-cell function in type 1 diabetes (T1D). While numerous transcriptome-wide studies on human islet cell-types have focused on protein-coding genes, the non-coding repertoire, such as long non-coding RNA, including circular RNAs, remains mostly unexplored. Here, we explored transcriptional landscape of human α-, β-, and exocrine cells from published total RNA sequencing (RNA-seq) datasets to identify circular RNAs (circRNAs). Our analysis revealed that circRNAs are highly abundant in both α- and β-cells. We identified 10,830 high-confidence circRNAs expressed in human α-, β-, and exocrine cells. The most highly expressed candidates were MAN1A2, RMST, and HIPK3 across the three cell-types. Alternate circular isoforms were observed for circRNAs in the three cell-types, indicative of potential distinct functions. Highly selective α- and β-cell circRNAs were identified, which is suggestive of their potential role in regulating β-cell function.

Localization of microRNA-375 in perinatal rat pancreas

MicroRNA (miRNA)-375 is highly expressed in the pancreatic endocrine islets. Maintaining appropriate miR-375 levels is very important for beta cell development, function and proliferation. The aim of the current study was to investigate the regulation and localization of miR-375 in rat perinatal pancreas at embryonic day 20 (E20), postnatal day 0 (D0) and day 2 (D2). Expression levels of miR-375 were measured by in situ hybridization on fixed neonatal rat pancreas. Interestingly, while miR-375 was detectable at robust levels at all three time points, the major site of expression of miR-375 at D0 and D2 was in pancreatic exocrine cells. Our data show that miR-375 has a dynamic change of expression in pancreatic exocrine tissue during the perinatal period. Moreover, these findings indicate that pancreatic endocrine cells may not always be the major source of expression of miR-375 in pancreas. We suggest that the marked change of miR-375 levels in exocrine cells following birth could regulate processes involved in the adaptation of the exocrine pancreas to digestion of external nutrients derived from milk.