Ebf3⁺ niche-derived CXCL12 is required for the localization and maintenance of hematopoietic stem cells

概要

Title

Ebf3⁺ niche-derived CXCL12 is required for the
localization and maintenance of hematopoietic
stem cells

Author(s)

Nakatani, Taichi; Sugiyama, Tatsuki; Omatsu,
Yoshiki et al.

Citation

Nature Communications. 2023, 14(1), p. 6402

Version Type VoR
URL
rights

https://hdl.handle.net/11094/93240
This article is licensed under a Creative
Commons Attribution 4.0 International License.

Note

Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University

Article

https://doi.org/10.1038/s41467-023-42047-2

Ebf3+ niche-derived CXCL12 is required for
the localization and maintenance of hematopoietic stem cells
Received: 18 January 2023

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Accepted: 25 September 2023

Taichi Nakatani1,2,3, Tatsuki Sugiyama 1,2,3, Yoshiki Omatsu
Hitomi Watanabe4, Gen Kondoh4 & Takashi Nagasawa 1,2,3

1,2,3

,

Lympho-hematopoiesis is regulated by cytokines; however, it remains unclear
how cytokines regulate hematopoietic stem cells (HSCs) to induce production
of lymphoid progenitors. Here, we show that in mice whose CXC chemokine
ligand 12 (CXCL12) is deleted from half HSC niche cells, termed CXC chemokine ligand 12 (CXCL12)-abundant reticular (CAR) cells, HSCs migrate from
CXCL12-deficient niches to CXCL12-intact niches. In mice whose CXCL12 is
deleted from all Ebf3+/leptin receptor (LepR)+ CAR cells, HSCs are markedly
reduced and their ability to generate B cell progenitors is reduced compared
with that to generate myeloid progenitors even when transplanted into wildtype mice. Additionally, CXCL12 enables the maintenance of B lineage repopulating ability of HSCs in vitro. These results demonstrate that CAR cellderived CXCL12 attracts HSCs to CAR cells within bone marrow and plays a
critical role in the maintenance of HSCs, especially lymphoid-biased or
balanced HSCs. This study suggests an additional mechanism by which cytokines act on HSCs to produce B cells.

Most blood cells, including immune cells, are generated from hematopoietic stem cells (HSCs), which are maintained throughout life in
the stromal microenvironment termed niches in the bone marrow1–4.
HSC niches comprise support cells (niche cells) that produce cytokines
essential for survival, proliferation, and/or differentiation of HSCs and
their progeny. Initially, dispersed fibroblastic cells, termed CXC chemokine ligand 12 (CXCL12)-abundant reticular (CAR) cells and NestinGFPhigh periarteriolar mesenchymal stem cells in the bone marrow
cavity have been reported to be important for HSC niches5,6. Among
various types of candidate niche cells, several lines of evidence have
demonstrated that CAR cells, which overlap strongly with leptin
receptor-expressing (LepR+) cells, are the population of mesenchymal
stem cells and the major cellular component of niches essential for the
HSC maintenance5,7,8. CAR cells are defined as a lineage that expresses
much levels of platelet-derived growth factor receptor β (PDGFRβ),

HSC niche factors, including CXCL12 and SCF, and transcription factors, including Foxc1 and Ebf3 than any other types of cells5,7–10.
However, it is not completely understood how HSC niche cells regulate
HSCs to produce a specific lineage of immune cells, including B cells.
Cytokines are known to act on the lineage-committed progenitors
to regulate their survival, proliferation, and/or differentiation. For
example, interleukin-7 (IL-7), which is produced by about 62% of the
CAR/LepR+ cells, is essential for the proliferation and differentiation of
B cell progenitors, including pro-B and pre-B cells in the bone
marrow11. In addition, it has been speculated that the cytokine produced by HSC niche cells may act on HSCs and/or multipotent
hematopoietic progenitors to induce their differentiation into the
lineage-committed progenitors.
The chemokine CXCL12 and its primary receptor CXCR4 are
essential for the maintenance of HSCs and the production of B cells

1

Laboratory of Stem Cell Biology and Developmental Immunology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
Laboratory of Stem Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. 3Laboratory of Stem Cell Biology and
Developmental Immunology, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. 4Center for Animal Experiments,
e-mail: nagasawa.takashi.fbs@osaka-u.ac.jp
Institute for Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.

2

Nature Communications | (2023)14:6402

1

Article
and their progenitors as well as plasmacytoid dendritic cells (pDCs)
and NK cells5,12–15 and CAR/LepR+ cells are the major producer of
CXCL12 within bone marrow7. However, it remains unclear how
CXCL12 regulates both HSC maintenance and B cell production. It was
reported that the number of HSCs was unaltered in bone marrow but
increased in peripheral blood when CXCL12 was conditionally deleted
from LepR+ cells using LepR-Cre;CXCL12f/- mice. This suggests that
LepR+ cell-derived CXCL12 is essential to inhibit egress of HSCs from
bone marrow but not to maintain HSCs16,17. However, since CXCL12 was
deleted from about 70% of the CAR cells in the mutants probably due
to lower LepR expression and recombination efficiency of LepR-Cre in
early postnatal bone marrow16,18, there is a possibility that CXCL12
production from a small subset of CXCL12-intact CAR cells contributes
to the HSC maintenance.
In this study, we examine the role of CAR cell-derived CXCL12
regarding the behavior and ability of HSCs to generate B cell progenitors. HSCs migrated to CXCL12-intact CAR cells within bone marrow in Ebf3-CreERT2;CXCL12f/- mice, in which CXCL12 was deleted in
about half of the CAR cells. In Ebf3-CreERT2;CXCL12f/- mice, in which
CXCL12 was deleted in almost all the CAR cells, HSC numbers were
reduced and the ability of HSCs to generate B cell progenitors was
markedly reduced. It was reported that HSCs comprise several populations, including lymphoid-biased or balanced and myeloid-biased
subsets of HSCs19–24. Thus, our findings suggest that CAR cell-derived
CXCL12 supports the localization of HSCs and the maintenance of
HSCs, especially lymphoid-biased HSCs to produce the required
number of B cell progenitors.

Results
CXCL12 attracts HSCs to CAR cells within bone marrow
We generated CXCL12f/- mice (Supplementary Fig. 1) and crossed
them with mice expressing the CreERT2 transgene under the
control of the Ebf3 gene, in which Cre recombinase can be activated in CAR cells but not in other bone marrow cell populations
when injected with tamoxifen (Ebf3-CreERT2 mice)10. CXCL12 was
deleted in a portion of CAR cells when Ebf3-CreERT2;CXCL12f/mice were injected with tamoxifen once and analyzed 10–14 weeks
after tamoxifen treatment. Quantitative real-time polymerase
chain reaction with reverse transcription (qRT-PCR) analysis of
sorted PDGFRβ+Sca-1-CD31-CD45-Ter119- CAR cells9 revealed that
CXCL12 was deleted from about 70% of the CAR cells in the mutant
mice (Fig. 1a). Flow cytometric analysis revealed that the bone
marrow from the mutant mice contained normal total hematopoietic cell counts, normal numbers of c-kit+CD19+IgM- pro-B cells
and c-kit-CD19+IgM- pre-B cells, and only modestly reduced numbers of the CD34-CD150+CD48- or CD150+CD48- subset of Lin-Sca1+c-kit+ (LSK) cells, which are highly enriched for long-term repopulating HSCs (LT-HSCs) compared with control animals
(Fig. 1b–d). These results raise the possibility that CXCL12 production from a subset of CXCL12-intact CAR cells contributes to
HSC maintenance in the mutants.
To address this possibility, we analyzed the localization of HSCs
relative to CXCL12-intact CAR cells in these HSC-intact CXCL12 conditionally deficient mice. To visualize HSCs, we generated HSCreporter mice, in which three EGFP genes were knocked into the
HSC-specific Evi1 gene (Evi1-GFP mice) (Supplementary Fig. 2a). Evi1 is
a transcription factor, which is specifically expressed in HSCs in the
hematopoietic system and essential for the maintenance of HSCs25,26.
In these mice, the majority of phenotypic and functional HSCs were
Evi1-GFPhic-kit+ (Supplementary Fig. 2b, c). Conversely, the majority of
Evi1-GFPhic-kit+ cells comprised CD150+CD48- LSK (LSK-SLAM) HSCs
(Supplementary Fig. 2d). Limiting dilution analysis revealed that the
frequency of long-term in vivo competitive repopulating units (CRU)
of the Evi1-GFPhic-kit+ cells was 1 in 3.8 cells (Supplementary Fig. 2e).
Flow cytometric analysis revealed that Lin-Sca-1+c-kit+CD150-CD48-

Nature Communications | (2023)14:6402

https://doi.org/10.1038/s41467-023-42047-2

multipotent progenitors (MPPs) expressed lower levels of Evi1-GFP
compared to HSCs and other primitive hematopoietic progenitors,
including Lin-Sca-1-c-kit+CD34+FcγRII/IIIhi granulocyte/macrophage
progenitors (GMPs) and Lin-Sca-1-c-kit+CD34-FcγRII/IIIlo megakaryocyte/erythrocyte progenitors (MEPs), did not express Evi1-GFP
(Supplementary Fig. 2b). We could detect the fluorescence signals in
fixed HSCs but not in fixed MPPs isolated from Evi1-GFP mice by histology (Supplementary Fig. 3). Flow cytometric analysis revealed HSC
numbers were unaltered in Evi1-GFP mice (Supplementary Fig. 4). In
addition, the mRNA levels of Evi1 in HSCs were unaltered in the
absence of CXCL12 in CAR cells as described later. Together, Evi1-GFP
mice allowed visualization of HSCs in the bone marrow sections of
control and CXCL12 conditionally deficient mice. To visualize CXCL12intact and CXCL12-deficient CAR cells, we generated another conditional CXCL12-targeted mouse line by inserting loxP sites that flank
exons 2 and 3 of the Cxcl12 gene and a linked tandem dimer Tomato
(tdTomato) gene to act as a CXCL12-specific reporter (CXCL12tdTomatof/f mice) (Fig. 1e), and we crossed them with Ebf3-CreERT2
mice. ...

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