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Spatial and temporal heterogeneity of mouse and human
microglia at single-cell resolution
Citation for published version:
Masuda, T, Sankowski, R, Staszewski, O, Böttcher, C, Amann, L, Scheiwe, C, Nessler, S, Kunz, P, van
Loo, G, Arnd Coenen, V, Reinacher, PC, Michel, A, Sure, U, Gold, R, Grün, D, Priller, J, Stadelmann, C &
Prinz, M 2019, 'Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution',
Nature, vol. 566, no. 7744, pp. 388-392. https://doi.org/10.1038/s41586-019-0924-x
Digital Object Identifier (DOI):
10.1038/s41586-019-0924-x
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Peer reviewed version
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Nature
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Download date: 09. Aug. 2022
Microglial heterogeneity by single-cell RNA-seq
1
1
Spatial and developmental heterogeneity of 2
mouse and human microglia at single-cell resolution 3
4
5
Takahiro Masuda
1,14
, Roman Sankowski
1,14
, Ori Staszewski
1,14
, Chotima 6
Böttcher
2
, Lukas Amann
1,15
, Christian Scheiwe
3
, Stefan Nessler
4
, Patrik Kunz
4
, 7
Geert van Loo
5,6
, Volker Arnd Coenen
7
, Peter C. Reinacher
7
, Anna Michel
8
, 8
Ulrich Sure
8
, Ralf Gold
9
, Josef Priller
2,10,11
, Christine Stadelmann
4
9
& Marco Prinz
1,12,13
10
11
1
Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany 12
2
Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité – 13
Universitätsmedizin Berlin, Berlin, Germany, 14
3
Clinic for Neurosurgery, Faculty of Medicine, University of Freiburg, Freiburg, Germany 15
4
Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany 16
5
VIB Center for Inflammation Research, Ghent, Belgium 17
6
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 18
7
Department of Stereotactic and Functional Neurosurgery, Medical Faculty, University of 19
Freiburg, Freiburg, Germany 20
8
Department of Neurosurgery, University Hospital Essen, Germany 21
9
Department of Neurology, St. Josef-Hospital, Ruhr University Bochum, Bochum, Germany 22
10
DZNE and BIH, Berlin, Germany 23
11
University of Edinburgh and UK DRI, Edinburgh, UK 24
12
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany 25
13
CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany 26
14
These authors contributed equally to this work 27
15
Faculty of Biology, University of Freiburg, Freiburg, Germany. 28
29
30
Correspondence to: 31
Marco Prinz, M.D. 32
Institute of Neuropathology 33
University of Freiburg 34
Breisacher Str. 64 35
D-79106 Freiburg, Germany 36
Phone: +49-761-270-51050 37
E-mail: marco.prinz@uniklinik-freiburg.de 38
39
Microglial heterogeneity by single-cell RNA-seq
2
ABSTRACT
40
41
42
Microglia play critical roles in neural development and homeostasis. They are also implicated 43
in neurodegenerative and neuroinflammatory diseases of the central nervous system (CNS). 44
However, little is known about the presence of spatially and temporally restricted subclasses 45
of microglia during CNS development and disease. Here, we combined massively parallel 46
single-cell analysis, single-molecule FISH, advanced immunohistochemistry and 47
computational modelling to comprehensively characterize novel microglia subclasses in up to 48
six different regions during development and disease. Single-cell analysis of mouse CNS 49
tissues revealed specific time- and region-dependent microglia subtypes, which were 50
transcriptionally distinct from perivascular macrophages, during homeostasis. Demyelinating 51
and neurodegenerative diseases evoked context-dependent microglia subtypes with distinct 52
molecular hallmarks and diverse cellular kinetics. Diverse microglia clusters were also 53
identified in normal and diseased human brains. Our data provide new insights into the 54
endogenous immune system of the CNS during development, health and disease. 55
56
Key words: microglia, perivascular macrophages, single-cell analysis, immune system, 57
human, mouse 58
59
60
61
Microglial heterogeneity by single-cell RNA-seq
3
INTRODUCTION
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63
Tissue-resident myeloid cells in the central nervous system (CNS) represent a 64
heterogeneous class of innate immune cells that are essential for the maintenance of tissue 65
homeostasis (1). Parenchymal microglia and the CNS-associated macrophages (CAMs), 66
including leptomeningeal (mMΦ), perivascular (pvMΦ) and choroid plexus macrophages 67
(cpMΦ), are the organ-specific macrophages of the CNS with pivotal roles in health and 68
disease (2-4). 69
Despite of the similarities that microglia and CAMs share with various other tissue-resident 70
macrophages, the parenchymal and non-parenchymal CNS macrophages have two 71
distinctive properties, namely a restricted prenatal origin and a remarkable longevity (4, 5). It 72
is now generally believed that microglia and CAMs are derived from early yolk sac 73
erythromyeloid precursors in a c-myb- and chemokine receptor (CCR)2-independent fashion 74
(6-8). These specific developmental pathways and anatomical niches make CNS-75
endogenous macrophages distinct from other tissue macrophages, such as those in the 76
aorta, skin, heart, liver, spleen and other organs (9-12). 77
When compared to other hematopoietic cells, microglia and CAMs persist over a very long 78
period of time with low but constant rates of self-renewal (13, 14) coupled to cell apoptosis 79
(15). This longevity necessitates adaptivity of microglia towards environmental challenges 80
(16, 17) and cell perturbations (18). Since microglia act as guardians of the CNS, 81
continuously scavenging for dying cells, pathogens, and molecules through microbial-82
associated molecular pattern receptor-dependent and -independent mechanisms (1), these 83
highly diverse and specialized functions may be executed by microglia subsets that already 84
pre-exist in situ, or alternatively, by specific development of microglia subsets from a 85
homogeneous pool of cells upon demand. To date, the spatiotemporal heterogeneity of 86
microglia during development, homeostasis and disease has not been studied at the single-87
cell level. 88
Previous approaches used to analyse microglial diversity have largely relied on 89
immunophenotyping by flow cytometry complemented with histological analysis of RNA and 90
Microglial heterogeneity by single-cell RNA-seq
4
proteins in situ (19, 20). More recently, comprehensive transcriptomic (21) and proteomic
91
(22) profiling of bulk populations of large numbers of microglia helped to reveal microglial 92
heterogeneity in the mouse brain. Indeed, different microglia states were identified during 93
development (7, 12, 23-25), homeostasis (26) and disease (27). Although these approaches 94
provided important insights, they have notable limitations. Earlier single-cell analyses of 95
microglia, for instance via flow cytometry, in situ hybridization or immunohistochemistry, were 96
limited to probing a few selected proteins or RNAs. Due to a bias toward candidate 97
genes/proteins, these approaches allow neither analysis of comprehensive expression 98
landscapes nor discovery of previously unrecognized molecules (28). In contrast, 99
transcriptomic analysis of bulk preparations of microglial RNA may conceal the diversity of 100
microglia across different brain regions by relying on ensemble averages (21, 29, 30). 101
During the last few years, the revolution in single-cell genomics has enabled an unbiased 102
genome-wide quantification and multiplex spatial analysis of RNA in single microglia in situ 103
as well as in vitro (31). However, recent single-cell RNA-sequencing (scRNA-seq) studies of 104
microglia either only used pre-sorted myeloid cell populations (32), or whole brain 105
approaches (33) without addressing the question of spatially and temporally restricted 106
subtypes of microglia in several regions of the CNS. Importantly, single-microglia profiling 107
data from humans is not yet available at all, although this knowledge may greatly improve 108
our understanding of the pathogenesis of neuropsychiatric diseases. 109
By combining massively parallel scRNA-seq with single-molecule FISH (smFISH), advanced 110
triple immunohistochemistry, high-resolution microscopy, and computational modelling, we 111
were able to comprehensively characterize microglial diversity in different regions of the 112
mouse and human brain during development and health. We identify molecules that 113
characterize microglial populations involved in neuroinflammatory and neurodegenerative 114
conditions in mice and humans, and highlight context- and time-dependent microglia subsets 115
and their distinct signals. The data provide new potential therapeutic targets and a valuable 116
resource for the study of disease mechanisms in the CNS. 117
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