Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues

Susan M Millard; Ostyn Heng; Khatora S Opperman; Anuj Sehgal; Katharine M Irvine; Simranpreet Kaur; Cheyenne J Sandrock; Andy C Wu; Graham W Magor; Lena Batoon; Andrew C Perkins; Jacqueline E Noll; Andrew C W Zannettino; David P Sester; Jean-Pierre Levesque; David A Hume; Liza J Raggatt; Kim M Summers; Allison R Pettit

doi:10.1016/j.celrep.2021.110058

Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues

Cell Rep. 2021 Nov 23;37(8):110058. doi: 10.1016/j.celrep.2021.110058.

Authors

Susan M Millard¹, Ostyn Heng¹, Khatora S Opperman², Anuj Sehgal¹, Katharine M Irvine¹, Simranpreet Kaur³, Cheyenne J Sandrock¹, Andy C Wu⁴, Graham W Magor⁵, Lena Batoon¹, Andrew C Perkins⁵, Jacqueline E Noll², Andrew C W Zannettino⁶, David P Sester⁷, Jean-Pierre Levesque¹, David A Hume¹, Liza J Raggatt¹, Kim M Summers¹, Allison R Pettit⁸

Affiliations

¹ Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia.
² Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia; South Australian Health and Medical Research Institute, PO Box 11060, Adelaide, SA 5001, Australia.
³ Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia; The University of Queensland, UQ Diamantina Institute, Brisbane, QLD 4102, Australia.
⁴ Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia; TRI Flow Cytometry Suite, Translational Research Institute, Woolloongabba, QLD 4102, Australia.
⁵ Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia; Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia.
⁶ Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia; South Australian Health and Medical Research Institute, PO Box 11060, Adelaide, SA 5001, Australia; Central Adelaide Local Health Network, Adelaide, SA 5001, Australia.
⁷ TRI Flow Cytometry Suite, Translational Research Institute, Woolloongabba, QLD 4102, Australia.
⁸ Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia. Electronic address: allison.pettit@mater.uq.edu.au.

PMID: 34818538
DOI: 10.1016/j.celrep.2021.110058

Abstract

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169⁺ macrophages in vivo eliminates F4/80⁺ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.

Keywords: RNA-seq; bone marrow; flow cytometry; hematopoietic stem and progenitor cells; hematopoietic tissues; imaging flow cytometry; lymph node; macrophage; spleen; tissue-resident macrophage.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bone Marrow / metabolism
Cell Separation / methods*
Hematopoiesis
Hematopoietic Stem Cells / cytology
Homeostasis
Macrophages / cytology*
Mice
Single-Cell Analysis / methods*