1. Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues
- Author
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Simranpreet Kaur, Andy Wu, Katharine M. Irvine, Anuj Sehgal, Jean-Pierre Levesque, Liza J. Raggatt, Graham Magor, Ostyn Heng, David P. Sester, Andrew C. Perkins, Lena Batoon, Andrew C.W. Zannettino, Kim M. Summers, Jacqueline E. Noll, Allison R. Pettit, David A. Hume, Susan M. Millard, Cheyenne J. Sandrock, and Khatora S. Opperman
- Subjects
Cell type ,Chemistry ,Macrophages ,Hematopoietic Tissue ,Cell Separation ,Hematopoietic Stem Cells ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Hematopoiesis ,Haematopoiesis ,Mice ,medicine.anatomical_structure ,Bone Marrow ,medicine ,Macrophage ,Animals ,Homeostasis ,Bone marrow ,Fragmentation (cell biology) ,Progenitor cell ,Single-Cell Analysis ,Ex vivo - 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.
- Published
- 2021