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Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells

Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, b...

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Autores principales: Luo, Shufeng, Lin, Huiling, Zhu, Lan, Chen, Hai-Tian, Yang, Siqian, Li, Jinheng, Liu, Mingyu, Zheng, Limin, Wu, Chong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079767/
https://www.ncbi.nlm.nih.gov/pubmed/33936078
http://dx.doi.org/10.3389/fimmu.2021.654094
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author Luo, Shufeng
Lin, Huiling
Zhu, Lan
Chen, Hai-Tian
Yang, Siqian
Li, Jinheng
Liu, Mingyu
Zheng, Limin
Wu, Chong
author_facet Luo, Shufeng
Lin, Huiling
Zhu, Lan
Chen, Hai-Tian
Yang, Siqian
Li, Jinheng
Liu, Mingyu
Zheng, Limin
Wu, Chong
author_sort Luo, Shufeng
collection PubMed
description Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production.
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spelling pubmed-80797672021-04-29 Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells Luo, Shufeng Lin, Huiling Zhu, Lan Chen, Hai-Tian Yang, Siqian Li, Jinheng Liu, Mingyu Zheng, Limin Wu, Chong Front Immunol Immunology Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production. Frontiers Media S.A. 2021-04-14 /pmc/articles/PMC8079767/ /pubmed/33936078 http://dx.doi.org/10.3389/fimmu.2021.654094 Text en Copyright © 2021 Luo, Lin, Zhu, Chen, Yang, Li, Liu, Zheng and Wu https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Luo, Shufeng
Lin, Huiling
Zhu, Lan
Chen, Hai-Tian
Yang, Siqian
Li, Jinheng
Liu, Mingyu
Zheng, Limin
Wu, Chong
Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title_full Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title_fullStr Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title_full_unstemmed Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title_short Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells
title_sort optimized intracellular staining reveals heterogeneous cytokine production ability of murine and human hematopoietic stem and progenitor cells
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079767/
https://www.ncbi.nlm.nih.gov/pubmed/33936078
http://dx.doi.org/10.3389/fimmu.2021.654094
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