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Isolation of human cutaneous immune cells for single-cell RNA sequencing.

ABSTRACT: Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data. For complete details on the use and execution of this protocol, please refer to Cook et al. (2022)¹ and Liu et al. (2022).².

SUBMITTER: Hailer AA

PROVIDER: S-EPMC10173011 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Isolation of human cutaneous immune cells for single-cell RNA sequencing.

Hailer Ashley A AA Wu David D El Kurdi Abdullah A Yuan Michelle M Cho Raymond J RJ Cheng Jeffrey B JB

STAR protocols 20230428 2

Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytom ...[more]

PMID: 37120815

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Project description:BackgroundSevere acute pancreatitis (SAP) is characterized by inflammation, with inflammatory immune cells playing a pivotal role in disease progression. This study aims to understand variations in specific immune cell subtypes in SAP, uncover their mechanisms of action, and identify potential biological markers for predicting Acute Pancreatitis (AP) severity.MethodsWe collected peripheral blood from 7 untreated SAP patients and employed single-cell RNA sequencing for the first time to construct a transcriptome atlas of peripheral blood mononuclear cells (PBMCs) in SAP. Integrating SAP transcriptomic data with 6 healthy controls from the GEO database facilitated the analysis of immune cell roles in SAP. We obtained comprehensive transcriptomic datasets from AP samples in the GEO database and identified potential biomarkers associated with AP severity using the "Scissor" tool in single-cell transcriptomic data.ResultsThis study presents the inaugural construction of a peripheral blood single-cell atlas for SAP patients, identifying 20 cell subtypes. Notably, there was a significant decrease in effector T cell subsets and a noteworthy increase in monocytes compared to healthy controls. Moreover, we identified a novel monocyte subpopulation expressing high levels of PPBP and PF4 which was significantly elevated in SAP. The proportion of monocyte subpopulations with high CCL3 expression was also markedly increased compared to healthy controls, as verified by flow cytometry. Additionally, cell communication analysis revealed insights into immune and inflammation-related signaling pathways in SAP patient monocytes. Finally, our findings suggest that the subpopulation with high CCL3 expression, along with upregulated pro-inflammatory genes such as S100A12, IL1B, and CCL3, holds promise as biomarkers for predicting AP severity.ConclusionThis study reveals monocytes' crucial role in SAP initiation and progression, characterized by distinct pro-inflammatory features intricately linked to AP severity. A monocyte subpopulation with elevated PPBP and CCL3 levels emerges as a potential biomarker and therapeutic target.

Dataset Information

Isolation of human cutaneous immune cells for single-cell RNA sequencing.

Publications

Isolation of human cutaneous immune cells for single-cell RNA sequencing.

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