pancreatic-tissue-dissection-to-isolate-viable-single-cells

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

The Hebrew University of Jerusalem

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens.

Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.

Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpesvirus. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure patients from the virus. In addition, there is an urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well as for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV.

Targeting the Kaposi's sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells.

While the existence of an indigenous placental microbiota remains controversial, several pathogens are known to be involved in adverse pregnancy outcomes. Fusobacterium nucleatum is an oral bacterium that is one of several bacteria associated with preterm birth. Oral fusobacteria translocate to the placenta hematogenously; however, the mechanisms localizing them to the placenta remain unclear. Here, using peanut agglutinin, we demonstrate that the level of Gal-GalNAc (Galβ1-3GalNAc; Thomsen Friedenreich antigen) found on trophoblasts facing entering maternal blood rises during gestation and is recognized by the fusobacterial Fap2 Gal-GalNAc lectin. F. nucleatum binding to human and mouse placenta correlates with Gal-GalNAc levels and is reduced upon O-glycanase treatment or with soluble Gal-GalNAc. Fap2-inactivated F. nucleatum shows reduced binding to Gal-GalNAc-displaying placental sections. In a mouse model, intravenously injected Fap2-expressing F. nucleatum, but not a Fap2 mutant, reduces mouse fetal survival by 70%.

Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc.

Recent advances in single-cell RNA sequencing and bioinformatics have drastically increased our ability to interrogate the cellular composition of traditionally difficult to study organs, such as the pancreas. With the advent of these technologies and approaches, the field has grown, in just a few years, from profiling pancreas disease states to identifying molecular mechanisms of therapy resistance in pancreatic ductal adenocarcinoma, a particularly deadly cancer. Single-cell transcriptomics and related spatial approaches have identified previously undescribed epithelial and stromal cell types and states, how these populations change with disease progression, and potential mechanisms of action which will serve as the basis for designing new therapeutic strategies. Here, we review the recent literature on how single-cell transcriptomic approaches have changed our understanding of pancreas biology and disease progression.

Article	Year
Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell\| PubMed ID: 27984732	2016
Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity. Nature communications\| PubMed ID: 32908137	2020
Targeting the Kaposi's sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells. Virology journal\| PubMed ID: 33731154	2021
Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc. Cell reports\| PubMed ID: 35320712	2022
It is better to light a candle than to curse the darkness: single-cell transcriptomics sheds new light on pancreas biology and disease. Gut\| PubMed ID: 36997301	2023

Oren Parnas

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