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Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis.

ABSTRACT: Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45⁺ cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al.¹.

SUBMITTER: AlAsfoor S

PROVIDER: S-EPMC11357800 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis.

AlAsfoor Shefaa S Bernard Cheryl E CE Jessen Erik E Simon Vernadette A VA Rakhshan Fariborz F Cipriani Gianluca G Hsi Linda C LC Patraw Jack A JA Carothers Kari J KJ Lau Julie S JS McDonough Samantha J SJ Dasari Surendra S Farrugia Gianrico G Grover Madhusudan M

STAR protocols 20240811 3

Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting ...[more]

PMID: 39133613

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Project description:BackgroundUlcerative colitis (UC) is a severe threat to humans worldwide. Single-cell RNA sequencing (scRNA-seq) can be used to screen gene expression patterns of each cell in the intestine, provide new insights into the potential mechanism of UC, and analyze the development of immune cell changes. These findings can provide new ideas for the diagnosis and treatment of intestinal diseases. In this study, bioinformatics analysis combined with experiments applied in dextran sulfate sodium (DSS)-induced colitis mice was used to explore new diagnostic genes for UC and their potential relationship with immune cells.MethodsWe downloaded microarray datasets (GSE75214, GSE87473, GSE92415) from the Gene Expression Omnibus and used these datasets to screen differentially expressed genes (DEGs) and conduct Weighted Gene Co-expression Network Analysis (WGCNA) after quality control. The hub genes were screened, and ROC curves were drawn to verify the reliability of the results in both training set (GSE75214, GSE87473, GSE92415) and validation cohort (GSE87466). Also, we explored the relation of diagnostic genes and immune cells by CIBERSORT algorithm and single-cell analysis. Finally, the expression of hub genes and their relation with immune cells were verified in DSS-induced colitis mice.ResultsDiagnostic genes (ANXA5, MMP7, NR1H4, CYP3A4, ABCG2) were identified. In addition, we found these five genes firmly related to immune infiltration. The DSS-induced colitis mice confirm that the expression of ANXA5 mainly increased in the intestinal macrophages and had a strong negative correlation with M2 macrophages, which indicated its possible influence on the polarization of macrophages in UC patients.ConclusionWe identified ANXA5, MMP7, NR1H4, CYP3A4, and ABCG2 as diagnostic genes of UC that are closely related to immune infiltration and ANXA5 maintains a negative correlation with M2 macrophages which indicated its possible influence on the polarization of macrophage in UC patients.

Project description:Background and study aimLocal resection of early rectal cancer is being increasingly used. With invasion of the muscularis propria layer of the rectal wall, the risk of lymph node metastasis becomes too high to consider this the optimal oncological treatment. Therefore, a diagnosis of muscular invasion is important before attempting local resection; however, endoscopic and magnetic resonance imaging (MRI) images have limitations, such as overstaging (26-31%). We investigated the potential of linear endoscopic ultrasound (L-EUS) in the diagnosis of muscularis propria invasion.Patients and methodsThe study consisted of a development phase, in which linear (L)- EUS features, associated with muscular wall invasion were searched and tested, and a validation phase, during which 30 representative videos were assessed by the author F.t.B. and four experienced endosonographists without experience in rectal L-EUS.ResultsThe development cohort consisted of 91 patients (2019-2023). Overall, six EUS features were found to be significantly associated with muscular wall invasion: tornado sign, blob sign, massive connection, layer split, extramural deposit, and, most importantly impaired shiftability between the lesion and muscularis propria layer. During the development phase, these findings demonstrated excellent diagnostic features (sensitivity, 94.4%; specificity, 97.9%; and overstaging, 4%). In the validation phase, the sensitivity, specificity, and overstaging by F.t.B. were 88%, 85%, and 12%, respectively. Among the four inexperienced reviewers, the percentages were 65%-71%, 46%-54%, and 33%-39%, respectively. When considering the 27 videos that were considered easy or moderately difficult to assess, only 55% were correctly interpreted by the inexperienced reviewers.ConclusionsLinear endoscopic ultrasonography may be a valuable tool for the assessment of ingrowth into the muscularis propria in supposedly early rectal cancer, especially using its dynamic potential to assess fixation to the muscular wall by moving the lesion. However, training will be required to achieve satisfactory results.

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Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis.

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Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis.

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