Immunity 25, 977–988, December 2006 ª2006 Elsevier Inc. DOI 10.1016/j.immuni.2006.10.016
Maintenance of the Hematopoietic Stem Cell Pool
by CXCL12-CXCR4 Chemokine Signaling
in Bone Marrow Stromal Cell Niches
Tatsuki Sugiyama,1Hiroshi Kohara,1Mamiko Noda,1
and Takashi Nagasawa1,*
1Department of Medical Systems Control
Institute for Frontier Medical Sciences
Shogoin, Sakyo-ku, Kyoto 606-8507
In the bone marrow, the special microenvironment
niches nurture a pool of hematopoietic stem cells
(HSCs). Many HSCs reside near the vasculature, but
the molecular regulatory mechanism of niches for
HSC maintenance remains unclear. Here we showed
that the induced deletion of CXCR4, a receptor for
CXC chemokine ligand (CXCL) 12 in adult mice, re-
sulted in severe reduction of HSC numbers and in-
creased sensitivity to myelotoxic injury, although it
did not impair expansion of the more mature progeni-
tors. Most HSCs were found in contact with the cells
expressing high amounts of CXCL12, which we have
called CXCL12-abundant reticular (CAR) cells. CAR
cells surrounded sinusoidal endothelial cells or were
plays an essential role in maintaining the quiescent
of HSC niches, including both vascular and endosteal
niches in adult bone marrow.
Hematopoietic stem cells (HSCs) give rise to all types of
blood cells including lymphocytes and myeloid cells. In
the bone marrow, the niches where HSCs reside are
thought to supply the requisite factors and play an
essential role in maintaining a pool of HSCs to provide
the appropriate numbers of mature blood cells through-
out life (Moore and Lemischka, 2006; Wilson and
Trumpp, 2006). Most primitive hematopoietic cells, in-
cluding HSCs, are thought to be in a quiescent G0state
in these niches, probably to prevent depletion of the
regenerative cell pool or sensitivity to myelotoxic injury
(Cheshier et al., 1999). Therefore, it is important to un-
derstand the molecular regulatory mechanism of niches
for maintenance of the stem cell pool, not only for stem
cells but also for bone marrow transplantation and gene
therapy requiring HSCs. It was reported previously that
osteoblasts lining the bone surface act as HSC niches
tin-1 (Ang-1), observed exclusively in osteoblasts, main-
tains HSC quiescence (Arai et al., 2004). In contrast,
imaging of HSCs reveal that many HSCs are associated
with sinusoidal endothelium although only some HSCs
adjoin the endosteum (Kiel et al., 2005). Nevertheless,
the precise cellular components and niche-derived
factors regulating HSCs in the vascular niches remain
CXC chemokine ligand (CXCL)12 (also known as stro-
mal cell-derived factor [SDF]-1 or pre-B-cell-growth-
stimulating factor [PBSF]) is a member of a large family
of structurally related chemoattractive cytokines and
was first characterized as a growth-stimulating factor
for the B cell precursor clone (Nagasawa et al., 1994).
The primary physiologic receptor for CXCL12 is
CXCR4, a hepta helical receptor coupled to heterotri-
meric guanosine triphosphate (GTP) binding proteins,
which also functions as an entry receptor for the HIV-1
virus (Nagasawa et al., 1996; Tachibana et al., 1998;
Zou et al., 1998). Studies of mutant mice with targeted
gene disruption have revealed that CXCL12-CXCR4
signaling is essential for hematopoiesis, including B cell
topoietic progenitors, including HSCs, during ontogeny
as well as cardiovascular formation and neurogenesis
(Ara et al., 2003; Nagasawa et al., 1996; Nagasawa,
2006; Tachibana et al., 1998; Zou et al., 1998). Lethality
caused by deficiencies of CXCL12 and CXCR4 prevents
immediate analysis of their role in adult hematopoiesis.
Treatment with CXCR4-selective antagonist induces
increase in HSCs in the peripheral blood, suggesting
a role for CXCL12 in retaining HSCs in hematopoietic
organs (Broxmeyer et al., 2005). In addition, treatment
of immature human bone marrow CD34+cells with
CXCR4 antibodies prevents the engraftment of primitive
ficiency (NOD-SCID) mouse repopulating cells (SRCs)
(Peled et al., 1999). However, experiments with radiation
term myeloid reconstitution by Cxcr42/2fetal liver cells
(Kawabata et al., 1999; Ma et al., 1999). Therefore, the
roles of CXCL12-CXCR4 signaling in HSCs within adult
bone marrow remain unclear.
Here we show that CXCL12-CXCR4 signaling is es-
sential in adult bone marrow to maintain the HSC pool
and suggest that many HSCs are in contact with a small
population of reticular cells expressing high amounts of
CXCL12 (Tokoyoda et al., 2004). In addition, almost all
HSCs near the sinusoidal endothelium appear to be in
contact with these reticular cells surrounding endothe-
lial cells in the extravascular spaces, suggesting that
these cells are the key cellular components of HSC vas-
Induced Deletion of CXCR4 in Adult Bone Marrow
To determine the roles of CXCL12-CXCR4 signaling in
hematopoiesis in adult animals, we generated CXCR4
conditionally deficient mice. We crossed mice with a
loxP-CXCR4 conditional targeting allele (CXCR4flox(f)/null
mice) (Tokoyoda et al., 2004) to MxCre mice (Kuhn
et al., 1995) in which Cre was expressed after the
induction of interferon by the administration of poly(I)-
poly(C) (pIpC) to inactivate the CXCR4 gene in the adult
animals. The floxed allele was excised almost com-
pletely in bone marrow c-kit+Sca-1+Lineage (Lin)2prim-
itive hematopoietic cells and myeloid lineage cells of
some pIpC-treated MxCre-CXCR4f/nullmice as analyzed
by quantitative, real-time polymerase chain reaction
with reverse transcription (qRT-PCR) and flow cytome-
try (Figure 1A and data not shown). Flow cytometric
analysis revealed that the bone marrow of pIpC-treated
MxCre-CXCR4f/nullmice contained severely reduced
numbers of B cells but modestly reduced numbers of
myeloid or erythroid lineage cells compared with control
ure 1B). In peripheral blood, most pIpC-treated MxCre-
CXCR4f/nullmice showed increased myeloid lineage
cells (data not shown). These results are consistent
with the known phenotype of mice reconstituted with
CXCR4-deficient fetal liver cells (Kawabata et al., 1999;
Ma et al., 1999).
HSCs Are Severely Reduced in Adult Bone Marrow
of CXCR4 Conditionally Deficient Mice
HSCs can be highly purified as CD342c-kit+Sca-1+Lin2
differential ability to efflux Hoechst dye, which defines
a small subset of side population (SP) cells (Goodell
et al., 1996, 1997). SP cells with higher amounts of
Hoechst efflux (lower SP) are a nearly homogeneous
subset of bone marrow HSCs (Camargo et al., 2006),
and they can reconstitute myeloablated mice with abso-
lute efficiency (Matsuzaki et al., 2004). Consistent with
these, the numbers of CD342c-kit+Sca-1+Lin2cells
were similar to those of lower SP (SPlow) cells and in-
creased with age in wild-type bone marrow (data not
shown; Sudo et al., 2000).
topoietic cells, probably reflecting a transient antiproli-
ferative and/or apoptotic effect of interferon induced
by pIpC treatment (Gidali et al., 1981). In addition, the
numbers of CD342c-kit+Sca-1+Lin2and SPlowcells
were reduced in pIpC-treated wild-type and control
MxCre-CXCR4f/wtmice as well as MxCre-CXCR4f/null
mice at early time points after injection of pIpC (see
Figure S1 in the Supplemental Data available online
and data not shown). Therefore, we analyzed the role of
CXCR4 in HSCs via pIpC-treated MxCre-CXCR4f/null
and control mice at the late stages after pIpC injection
when the mice recovered from the effects of pIpC in
terms of the HSC numbers. Multiparameter flow cyto-
metric analysis revealed that the numbers of CD342
c-kit+Sca-1+Lin2and SPlowcells in bone marrow were
comparable in pIpC-treated MxCre-CXCR4f/wtmice but
severely reduced in pIpC-treated MxCre-CXCR4f/null
mice compared with pIpC-treated MxCre-CXCR4wt/wt
mice and untreated animals at 16 to 36 weeks after the
final pIpC injection (Figures 2A and 2B, and data not
shown), indicating that CXCL12-CXCR4 signaling is
required to maintain the HSC pool within bone marrow.
Next we compared the expression of genes that were
highly expressed in HSCs in the bone marrow c-kit+
Sca-1+Lin2cell population in pIpC-treated MxCre-
CXCR4f/nulland control mice. Among the genes involved
in HSC regulation, the expressions of Tek (encoding
Tie2), which is a physiologic receptor for Ang-1 (Arai
et al., 2004), Vegfa (Gerber et al., 2002), and Junb
(Passegue et al., 2004) increased in CD342c-kit+Sca-1+
Lin2HSCs compared with CD34+c-kit+Sca-1+Lin2cells,
which were enriched for non-self-renewing primitive
hematopoietic progeniters including multipotent pro-
genitors (MPPs) (Nakauchi et al., 1999; Osawa et al.,
1996) in wild-type mice via qRT-PCR (Figure 2C, left).
Gene expression analysis revealed that the expressions
of Tek, Vegfa, and Junb were markedly reduced in pIpC-
treated MxCre-CXCR4f/nullmice compared with control
right; and data not shown). These results support the
idea that the reduction of CD342c-kit+Sca-1+Lin2cell
numbers in pIpC-treated MxCre-CXCR4f/nullmice re-
flected the decreased numbers of HSCs, but not merely
as a consequence of upregulated CD34 expression in
HSCs based on the amount of cell activation (Sato
et al., 1999) in these mutants.
The Numbers of LTC-ICs and CRUs in Adult Bone
Marrow of CXCR4 Conditionally Deficient Mice
We performed long-term in vitro cultures (LTCs) on pri-
mary bone marrow stromal cells via limiting dilution
LTC-initiating cell (IC) assays (Ploemacher et al., 1989;
Stier et al., 2005). The frequency of LTC-ICs has been
shown to correlate with in vivo repopulating potential
(Ploemacher et al., 1989). A marked reduction of LTC-
ICs was observed in bone marrow from pIpC-treated
MxCre-CXCR4f/nullmice compared with control animals
(Figure 2D). This suggests that the numbers of HSCs
were reduced in CXCR4 conditionally deficient mice
Figure 1. Hematopoiesis in Adult Bone Mar-
rowfrom CXCR4 Conditionally Deficient Mice
(A) QRT-PCR analysis of mRNA expression of
CXCR4 in c-kit+Sca-1+Lin2cells in the bone
(f/null) and MxCre-CXCR4f/wt(f/wt) mice at 16
weeks after the final pIpC treatment (n = 3,
p = 0.009).
(B) Flow cytometric analysis of the numbers
of B220+IgM2B cell precursors, B220+IgM+
B cells, Gr-1+myeloid lineage cells and
Ter119+CD71+erythroblasts in bone marrow
from pIpC-treated MxCre-CXCR4f/nulland
MxCre-CXCR4f/wtmice at 16 weeks after final
pIpC treatment (n = 3; *p < 0.02; **p < 0.05).
Error bars represent SD of the mean.
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