The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 205, No. 4, April 14, 2008 777-783 www.jem.org/cgi/doi/
BRIEF DEFINITIVE REPORT
Hematopoietic stem cells (HSCs) have robust
proliferative potential, as they can undergo ex-
tensive expansion to quickly restore hematopoie-
sis after transplantation or histological injury.
However, under steady state, HSCs proliferate at
a very low rate and most HSCs are kept in the G 0
phase of the cell cycle ( 1 ). Disruption of HSC
quiescence leads to premature exhaustion of the
stem cell pool and causes hematological failure
under stress conditions ( 2, 3 ). Thus, HSC self-re-
newal and quiescence have to be fi nely balanced
to maintain a stable HSC pool that is capable of
producing blood cells for the lifetime of the or-
ganism. Although numerous transcription factors
and cell cycle molecules have been identifi ed to
regulate HSC self-renewal, it is not understood
how nuclear regulatory factors adjust the HSC
self-renewal rate to accommodate hematopoiesis
under homeostatic and cytopenic conditions. It
has been reported that HSCs are relocated from
the osteoblastic niche to vascular zones in the
BM after myeloablation ( 4 ). The translocation of
HSCs is accompanied with an increase in HSC
proliferation, suggesting that signals emanating
from the BM niche where HSCs reside deter-
mine the balance between quiescence and self-
renewal of HSCs.
The chemokine CXCL12 is the major che-
moattractant for HSCs ( 5 ). It is expressed at
a high level by osteoblasts, endothelial cells, and
by a subset of reticular cells scattered through-
out the BM ( 6, 7 ). Inactivation of CXCL12 or
its receptor CXCR4 impairs the translocation
of HSCs from the fetal liver to the BM dur-
ing embryogenesis ( 8 – 11 ), and direct ablation
of CXCR4 signaling or indirect modulation of
CXCL12 level by proteases results in mobiliza-
tion of primitive hematopoietic cells and com-
promises their engrafting activity ( 4, 12 – 14 ).
This suggests an important role for CXCR4/
CXCL12 in BM retention of primitive hema-
topoietic cells. Additional eff ects of CXCR4 on
HSCs are still not fully understood, and studies
evaluating its regulatory role in the cell cycle
yielded contradictory results ( 7, 15 ). To better
understand the function of CXCR4 in HSCs,
we deleted the Cxcr4 gene during adult hemato-
poiesis. We found that the compartment of
primitive hematopoietic cells (Flt3 ? Lin ? Sca-1 +
c-Kit + cells) was stably maintained in the BM
in the absence of CXCR4 and sustained long-
term hematopoiesis. These CXCR4-defi cient
primitive hematopoietic cells proliferated vig-
orously and outcompeted the coexisting WT
counterpart in the same host. CXCL12 di-
rectly inhibited the cell cycle of WT, but
not Cxcr4 ? / ? , primitive hematopoietic cells.
Thus, our results demonstrate a critical role
of CXCR4 in restraining HSCs in the quies-
The online version of this article contains supplemental material.
CXCR4 is required for the quiescence
of primitive hematopoietic cells
Yuchun Nie, Yoon-Chi Han, and Yong-Rui Zou
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032
The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong
steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is
thought that specialized niches in which HSCs reside control the balance between HSC
quiescence and self-renewal, yet little is known about the extrinsic signals provided by
the niche and how these niche signals regulate such a balance. We report that CXCL12
produced by bone marrow (BM) stromal cells is not only the major chemoattractant
for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoi-
etic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic
cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive
HSC proliferation. Notably, the hyperproliferative Cxcr4 ? / ? HSCs are able to maintain
a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that
CXCR4/CXCL12 signaling is essential to confi ne HSCs in the proper niche and controls
CXCR4 INHIBITS HSC PROLIFERATION | Nie et al.
the absence of CXCR4, even though a substantial amount of
Cxcr4 ? / ? Flt3 ? LSK cells emerged into the periphery.
C xcr4 ? / ? primitive hematopoietic cells are multipotent
and sustain hematopoiesis
To examine stem cell function of Cxcr4 ? / ? Flt3 ? LSK cells, we
evaluated their reconstitution effi ciency using a competitive
repopulating assay. Diff erent numbers of Cxcr4 ? / ? BM cells
(CD45.2) were transplanted, along with a constant dose (2 ×
10 5 ) of competitive BM cells (CD45.1), into lethally irradiated
mice. Regeneration of HSCs and blood cells in the recipients
was measured by fl ow cytometry 8 wk after transplantation. In
mice that had received equal numbers (2 × 10 5 ) of Cxcr4 ? / ?
and competitor BM cells, no more than 10% of the Flt3 ? LSK
cells were of Cxcr4 ? / ? donor origin. Even a large dose (10 6 ) of
Cxcr4 ? / ? BM cells produced only 50% chimerism in the
Flt3 ? LSK compartment and provided little contribution to B
and myeloid cells ( Fig. 2 A ). These results are in line with pre-
vious reports showing impaired engrafting capacity of Cxcr4 ? / ?
primitive hematopoietic progenitors ( 12, 18 ).
Compromised reconstitution activity of Cxcr4 ? / ? HSCs
could be attributed to defects in homing, self-renewal, or dif-
ferentiation. To circumvent the requirement for CXCR4 in
HSC homing and to directly assess the diff erentiation potential
of Cxcr4 ? / ? HSCs in vivo, we fi rst transplanted equal numbers
(2.5 × 10 6 ) of Cxcr4 C/C (CD45.2, H-2 b/b ) and WT (CD45.1,
H-2 b/b ) marrow cells into lethally irradiated hosts (H-2 b/d ), and
then deleted Cxcr4 and examined frequencies of donor- derived
hematopoietic cells 14 wk after Cxcr4 ablation. In contrast
to the reduced engraftment observed in mice that received
Cxcr4 ? / ? donor cells, Cxcr4 C/C donor cells yielded a much
higher proportion (71%) of Flt3 ? LSK cells over WT donor
cells (29%), and engrafted the myeloid compartment effi ciently
RESULTS AND DISCUSSION
The population of C xcr4 ? / ? primitive hematopoietic cells
is stably maintained
To investigate the function of CXCR4 at early hematopoietic
developmental stages, we conditionally ablated CXCR4 func-
tion in adult primitive hematopoietic cells. We crossed Cxcr4 -
fl oxed mice ( Cxcr4 f/f ) to tamoxifen-inducible Cre transgenic
mice ( ROSA CRE-ERT2 ) ( 16, 17 ), and we activated Cre by in-
jecting tamoxifen. 6 tamoxifen injections over 9 d led to > 99%
deletion of Cxcr4 in HSCs (Fig. S1, available at http://www
we refer to ROSA CRE-ERT2 Cxcr4 f/f mice before tamoxifen in-
duction as Cxcr4 C/C mice, and after tamoxifen treatment as
Cxcr4 ? / ? mice. Control animals used in the following studies
are Cre ? Cxcr4 f/f mice because ROSA CRE-ERT2 Cxcr4 +/f and
Cre ? Cxcr4 f/f are phenotypically identical (Fig. S2).
The involvement of the chemokine CXCL12 in HSC
function was fi rst documented by a study showing that colo-
nization of HSCs in the fetal BM was abolished in CXCL12 ? / ?
animals ( 8 ). Extensive studies have been conducted ever since
to defi ne the role of CXCL12 and CXCR4 in homing and
retention of HSCs. Early transplantation experiments demon-
strated that primitive hematopoietic cells required CXCR4
and CXCL12 interaction for effi cient engraftment ( 12, 18 ).
Later studies involved transplantation of CXCR4-inactivated
primitive hematopoietic cells into irradiated hosts, and showed
that the recovery of these cells in the BM within 24 h was
quantitatively normal compared with that of WT cells ( 13, 19 ).
These results imply that early BM homing of primitive hema-
topoietic cells might be CXCR4 independent, but BM reten-
tion of these cells requires CXCR4. However, it is not clear
from these experiments whether HSCs behave in the same
way as hematopoietic progenitors. Specifi cally, these stud-
ies did not directly examine whether extravasation of CXCR4-
inactivated primitive hematopoietic cells from the circulation
into the BM stroma, indeed, occurred. Moreover, we cannot
conclude from these transfer experiments as to whether BM
homing and retention of HSCs under the steady state requires
CXCR4 because the BM microenvironment of the recipients
used in these studies had been altered by irradiation ( 20 ).
To fully evaluate this issue, we inactivated CXCR4 in
8-wk-old mice in which HSCs had colonized in the BM
and had reached a steady state, and then determined the con-
tent of phenotypically defi ned HSCs in the BM and periph-
ery at diff erent times after the fi nal tamoxifen injection.
Flow cytometry analysis revealed that a population of cells
bearing characteristic markers of hematopoietic primitive
cells, including long-term HSCs (Flt3 ? Lin ? Sca-1 + c-Kit +
[Flt3 ? LSK]), was markedly increased in the peripheral blood
and spleen in mutant but not in WT animals 12 d after ta-
moxifen treatment ( Fig. 1 B ). This population continued to
be high in the periphery, even 32 wk later ( Fig. 1 C ). During
the same period, the cell counts of Cxcr4 ? / ? Flt3 ? LSK cells
in the BM remained stable and were even slightly higher than
that of WT ( Fig. 1 C ). These data showed that the compart-
ment of phenotypic HSCs was stably retained in the BM in
Figure 1. Cxcr4 ? / ? HSCs are retained in the BM. (A) Dot plots represent
the Flt3 ? LSK population in the BM of Cxcr4 ? / ? (KO) and control mice (WT) at
day 12 after tamoxifen treatment. The percentages of gated populations of
Flt3 ? LSK cells ± the SD are shown. (B) Absolute numbers of Flt3 ? LSK cells in
the femur (BM), peripheral blood (PB), and spleen (Sp) of Cxcr4 ? / ? and con-
trol mice at day 12 and week 15 after tamoxifen treatment. Values are the
mean ± the SD ( n = 6). (C) Absolute numbers of Flt3 ? LSK cells in the femur
and tibia (BM) and spleen (SP) 32 wk after tamoxifen treatment. Values are
the mean ± the SD ( n = 4). Open bars represent data of WT mice; fi lled bars
show data of mutant animals in B and C.
JEM VOL. 205, April 14, 2008
BRIEF DEFINITIVE REPORT
C xcr4 ? / ? primitive hematopoietic cells
In the mixed BM chimeras, the cellularity of Cxcr4 ? / ?
Flt3 ? LSK cells was twofold higher than that of the WT ( Fig.
2 B ), suggesting that the expansion of these cells was cell in-
trinsic and could result from enhanced survival or self-renewal
of HSCs. Because we did not observe changes in apoptosis
detected by annexin V staining of freshly isolated Cxcr4 ? / ?
Flt3 ? LSK cells (Fig. S4, available at http://www.jem.org/
cgi/content/full/jem.20072513/DC1), we decided to exam-
ine whether CXCR4 defi ciency promoted HSC proliferation
using a BrdU-uptake assay. In WT mice, we found that 4-d
BrdU exposure yielded 28% of BrdU + LSK cells, and that a
longer labeling period (15 d) raised this population to 63%.
In contrast, the frequency of BrdU + LSK cells had increased
from 64 to 91% during the same interval in mice that had
Cxcr4 deleted ( Fig. 3 B ). The proliferation rate of mutant
primitive hematopoietic cells was ? 3-fold higher than that of
WT 14 wk after Cxcr4 deletion ( Fig. 3 C ). Analysis of the cell
cycling status by measuring RNA and DNA content revealed
that the number of cycling Cxcr4 ? / ? LSK cells remained high
even 32 wk after Cxcr4 deletion ( Fig. 3 D ). In accordance
with enhanced proliferation of Cxcr4 ? / ? HSCs, Cxcr4 ? / ?
(53%) in a competitive situation ( Fig. 2, B and C ). Although
competent in myelopoiesis, mutant donor cells were severely
impaired in generating B cells ( Fig. 2 C ). We then determined
whether Cxcr4 ? / ? HSCs were able to generate common
lymphoid progenitors (CLPs; Lin ? Sca lo c-Kit lo IL-7R ? + ). We
found that although Cxcr4 ? / ? CLPs were barely detectable in
the BM, a large number of these cells emerged in the periphery
and persisted for > 3 mo ( Fig. 2 D ). Together, these data reveal
that Cxcr4 ? / ? Flt3 ? LSK cells retained in the BM are multipo-
tential and able to sustain myelopoiesis and lymphopoiesis up
to the CLP stage for > 3 mo.
In the absence of CXCR4, HSCs cannot home to the BM
niche to reconstitute hematopoiesis ( Fig. 2 A ). To carry out a
repopulating assay to confi rm the existence of Cxcr4 ? / ? long-
term HSCs, we isolated BM cells 11 wk after Cxcr4 deletion,
infected them with retroviral vector expressing WT CXCR4,
and then transplanted these infected cells into irradiated recip-
ients. Our data presented in Fig. S3 (available at http://www
.jem.org/cgi/content/full/jem.20072513/DC1) clearly show
that a robust hematopoiesis was restored by Cxcr4 ? / ? BM cells
in which CXCR4 was reexpressed. This result unequivocally
demonstrates that HSCs are maintained for at least 11 wk in
the absence of CXCR4.
Figure 2. Cxcr4 ? / ? HSCs sustain hematopoiesis. Open bars represent data of WT mice; fi lled bars show data of mutant animals. (A) BM cells were
isolated from Cxcr4 C/C and control mice 6 wk after tamoxifen treatment and cotransplanted into recipients at the indicated ratios (2 × 10 5 of WT cells).
Hematopoiesis was analyzed 8 wk after transplantation. Bars with SD show cell counts of HSCs, B cells, and myeloid cells in the BM and spleen (Sp).
(B) Cxcr4 C/C , WT, or an equal number (2.5 × 10 6 ) of Cxcr4 C/C and WT BM cells were transferred into irradiated recipients. 2 mo after transplantation, mice were
treated with tamoxifen to delete Cxcr4 . LSK cells in BM chimeras were enumerated 14 wk after tamoxifen treatment ( n = 3). (C) BM chimeras were gener-
ated as described in B. Bars (mean ± the SD; n = 3) show the frequencies of donor-derived LSK, B cells, and myeloid cells of either mutant or WT origin.
(D) Total cell numbers of CLPs in the BM and spleen 15 wk after tamoxifen treatment.
CXCR4 INHIBITS HSC PROLIFERATION | Nie et al.
that the proportion of WT primitive hematopoietic cells ar-
rested in the G 0 phase was progressively increased in the pres-
ence of increasing doses of CXCL12. In contrast, the percentage
of cycling Cxcr4 ? / ? primitive hematopoietic cells was not af-
fected at all by even the highest dose of CXCL12 ( Fig. 4 A ).
These results thus demonstrate that CXCL12 prevents the
entry of HSCs into the active cell cycle. Furthermore, because
the proliferation of Cxcr4 ? / ? primitive hematopoietic cells
cannot be suppressed by CXCL12, we conclude that this ac-
tion is mediated solely by CXCR4 and not by CXCR7,
which is a newly identifi ed receptor of CXCL12 ( 21 ).
To gain further insight into the mechanisms by which
CXCR4 defi ciency aff ected cell cycle regulation, we purifi ed
Flt3 ? LSK cells from WT and Cxcr4 ? / ? BM, and quantifi ed
expression of various cell cycle regulators by quantitative RT-
PCR (qRT-PCR). Consistent with the hyperproliferative sta-
tus of mutant cells, the expression of cyclin D1 was increased
fourfold in Cxcr4 ? / ? Flt3 - LSK cells over control cells. Previ-
ous reports have shown that p21 cip1/waf1 and Gfi -1 were re-
quired to impose G 0 arrest of HSCs. However, these molecules
were expressed at similar levels in WT and Cxcr4 ? / ? primitive
hematopoietic cells. Interestingly, the cyclin-dependent kinase
inhibitor p57 kip2 , which was expressed at a particularly high
mice died more readily from hematological failure after de-
pletion of cycling HSCs by weekly challenge with the cell-
cycle cytotoxic agent 5-fl uorouracil ( Fig. 3 E ).
To address whether hyperproliferation was a cell autono-
mous property of Cxcr4 ? / ? HSCs, we transplanted an equal
number (2.5 × 10 6 ) of Cxcr4 C/C (CD45.2, H2 b/b ) and WT
(CD45.1, H2 b/b ) marrow cells into recipients (H2 b/d ), and
then deleted Cxcr4 4 wk after transplantation. The prolifera-
tion rate of HSCs was examined by a BrdU-uptake assay
14 wk after Cxcr4 deletion. We found that 47% of Cxcr4 ? / ?
LSK cells had incorporated BrdU over a 4-d period, whereas
only 18% of WT LSK cells were BrdU + ( Fig. 3 F ). Because
Cxcr4 ? / ? LSK cells proliferated at a higher rate than the ac-
companied WT LSK cells in the same BM, we conclude that
CXCR4 acts intrinsically in primitive hematopoietic cells to
CXCR4 signaling inhibits cell-cycle progression of primitive
Next, we investigated whether CXCR4 signaling directly in-
hibited cell-cycle progression of HSCs. The cell cycling pro-
fi le of primitive hematopoietic cells were analyzed 24 h after
BM cells cultured with diff erent doses of CXCL12. We found
Figure 3. CXCR4 defi ciency causes hyperproliferation of primitive hematopoietic cells. Open bars represent data of WT mice; fi lled bars show
data of mutant mice. (A) The histogram shows representative profi les of BrdU + LSK cells. The shaded histogram represents background staining using an Ig
isotype-matched control antibody. (B) 2 wk after Cxcr4 deletion, Cxcr4 ? / ? and WT mice were labeled with BrdU for 4 or 15 d. Bars ± the SD ( n = 4) repre-
sent percentages of BrdU + cells within the LSK compartment. (C) Bars ± the SD show percentages of BrdU + LSK cells 15 wk after Cxcr4 deletion. Mice were
given BrdU for 20 h. (D) Cell cycle profi les revealed by pyronin Y/Hoechst staining of Flt3 ? LSK cells in mice 32 wk after Cxcr4 deletion ( n = 4). The percent-
age of cells in the given quadrants represents the means ± the SD. (E) 2 wk after tamoxifen treatment, mice were injected weekly with 5-fl uorouracil
(100 mg/kg bodyweight). The survival rate of WT ( n = 5, open circle) and Cxcr4 ? / ? ( n = 10, fi lled circle) mice was monitored. (F) Equal numbers (2.5 × 10 6 )
of Cxcr4 C/C and WT BM cells were cotransplanted into BDF1 recipients. 4 wk after transplantation, mice were treated with tamoxifen. 14 wk later, mice
were labeled with BrdU for 4 d. LSK frequencies of BrdU + cells are shown ( n = 3).
JEM VOL. 205, April 14, 2008
BRIEF DEFINITIVE REPORT
was aff ected by CXCR4 defi ciency under the steady state.
Our results showed that hematopoiesis was sustained for at
least 8 mo, indicating the persistence of functional HSCs in
the BM after CXCR4 inactivation. Indeed, we observed that
the phenotypic Cxcr4 ? / ? HSCs (Flt3 ? LSK cells) were stably
retained. Remarkably, when both Cxcr4 ? / ? and WT HSCs
were present in the same BM, the cellularity of Cxcr4 ? / ?
HSCs exceeded that of the WT, and the expansion of mutant
HSCs was at the expense of competitive WT HSCs. In marked
contrast, when Cxcr4 deletion preceded transplantation, even
fi vefold more Cxcr4 ? / ? HSCs could not compete with the
cotransplanted WT HSCs. Together, our fi ndings suggest that
CXCR4 plays a critical role in guiding HSCs into the proper
BM niche. However, after seeding in the stem cell niche,
HSCs can be retained through a CXCR4-independent mech-
anism. We also noted that a prominent fraction of HSCs ap-
peared in the periphery after Cxcr4 deletion, similar to a
previous study showing that the CXCR4 antagonist AMD3100
rapidly mobilized HSCs ( 14 ). It remains to be determined
whether these results refl ect that there are two subsets of HSCs
that have diff erent requirements for BM retention or simply
indicate that overly proliferated HSCs cannot be contained in
the BM niche. Recently, a new CXCL12 receptor, CXCR7,
has been identifi ed, and its binding to CXCL12 is unaff ected
by AMD3100 ( 21 ). It will be interesting to elucidate whether
diff erent HSC subsets diff erentially express CXCR4 and
CXCR7, and whether CXCR7 and CXCR4 have distinct
roles in homing and BM retention of HSCs.
It has been proposed that the stem cell niche in BM regu-
lates self-renewal and diff erentiation of HSCs. However, the
niche signals that restrain HSCs in the quiescent state have
not been identifi ed. Our data demonstrate an inhibitory ef-
fect of CXCR4 signaling on proliferation of primitive hema-
topoietic cells, as increased doses of CXCL12 progressively
inhibit G 0 → G 1 cell cycle progression of LSK cells. Consistent
with the in vitro data, we found many more cycling cells in
the Cxcr4 ? / ? Flt3 ? LSK compartment than in the cotrans-
planted WT population. It is noteworthy that hematopoiesis
remained robust even 8 mo after Cxcr4 deletion, suggesting
that extensive proliferation of Cxcr4 ? / ? HSCs did not ex-
haust the mutant stem cell pool during this period. In this re-
gard, Cxcr4 ? / ? HSCs diff er from those carrying mutations in
cell cycle regulators like p21 cip1/waf1 and Gfi -1, in which hy-
perproliferation causes depletion of HSCs in a competitive
environment ( 2, 3 ). Indeed, both p21 cip1/waf1 and Gfi -1 were
expressed at similar levels in WT and Cxcr4 ? / ? HSCs. Inter-
estingly, our study identifi ed p57 kip2 as one of the putative
targets of CXCR4 signaling pathways. These data suggest
diff erent functions of cell cycle regulators in HSCs. Further
experiments are required to determine whether p57 kip2 spe-
cifi cally inhibits HSC proliferation, whereas p21 cip1/waf1 plays
additional roles in HSC self-renewal.
Recently, results of an independently derived line of
CXCR4 conditional knockout mice were published, which
showed that in the absence of CXCR4 primitive hematopoi-
etic cells (LSK cells) were retained in the BM and became
level in long-term HSCs ( 22 ), was reduced to one third of the
normal level in Cxcr4 ? / ? primitive hematopoietic cells.
To assess whether CXCR4 signaling controls p57 kip2 ex-
pression, sorted HSCs (Flt3 ? CD48 ? LSK cells) were incu-
bated with CXCL12 for 24 h and p57 kip2 expression levels
were determined by qRT-PCR. As shown in Fig. 4 C ,
CXCL12 treatment signifi cantly elevated the p57 kip2 ex-
pression level in HSCs, thus establishing p57 kip2 as one of the
direct targets downstream of CXCR4 signaling pathway.
In this study, we ablated CXCR4 after HSCs had seeded in
the BM and directly assessed whether BM retention of HSCs
Figure 4. CXCL12 inhibits cell cycle progression of HSCs.
(A) Cxcr4 ? / ? and control BM cells were cultured for 24 h in the presence of
cytokines and with CXCL12 at the indicated concentrations. Dot plots
show cycling CD48 ? Flt3 ? LSK cells detected by pyronin Y/Hoechst stain-
ing. Bars (means ± the SD) show the ratio of LSK cells in the G 0 versus G 1
phase of cell cycle from three independent experiments. (B) Relative ex-
pression levels represent the ratio of each gene transcript in Cxcr4 ? / ?
versus WT Flt3 ? LSK cells. cDNA input was normalized to the level
of ? -actin. Values are the means ± the SD of three experiments.
(C) CD48 ? Flt3 ? LSK BM cells were sorted from WT mice and cultured in the
presence of cytokines with or without 300 ng/ml of CXCL12 for 24 h. Each
value was normalized to ? -actin expression levels and is presented as fold
induction compared with the p57 kip2 expression level (set to 1) detected in
CXCL12-untreated cells. Results (means ± the SD) are obtained from three
experiments using independently sorted cells. P = 0.019.
CXCR4 INHIBITS HSC PROLIFERATION | Nie et al.
conjugated anti-Sca1. Long-term HSCs defi ned as Flt3 ? LSK were visual-
ized by staining BM cells with biotin-Flt3 followed by streptavidin-FITC
with the combination of the aforementioned antibodies. Common lym-
phoid progenitors (Lin ? c-Kit lo and Sca-1 lo IL-7R ? + ) were distinguished
by biotinylated anti – IL-7R ? followed by streptavidin-FITC and anti-
bodies against Lin, c-Kit, and Sca-1, as described. For lineage analyses,
cells were stained with APC-B220, PE-TCR, and PE-Cy5-Gr1. All anti-
bodies were obtained from eBioscience. BM cellularity was calculated
from two femurs.
Proliferation analysis. Cxcr4 ? / ? and WT BM cells were cultured in me-
dium (OptiMEM; Invitrogen) with 7% FCS (HyClone), 50 ng/ml SCF,
10 ng/ml IL-3, and 10 ng/ml IL-6. All cytokines were purchased from
R & D Systems. 24 h after incubation with diff erent doses of CXCL12, cells
were harvested for cell cycle analysis. For BrdU uptake assay, Cxcr4 ? / ? mice
were injected with BrdU (1 mg/mouse, i.p.) and fed with drinking water
containing 1mg/ml BrdU for various periods, as indicated. BM cells were
stained with antibodies against PE-Cy5-Lin, APC-Sca1, and PE-Cy7-c-Kit,
and permeabilized, followed by staining with FITC-BrdU. For cell cycle
analysis, cells were fi xed with 4% paraformaldehyde and labeled with
0.5 μ g/ml pyronin Y (Sigma-Aldrich) and 1 μ g/ml Hoechst 33342 (Fluka),
and analyzed on an LSR II fl ow cytometer (BD Biosciences).
Gene expression analysis. LSK cells were purifi ed from Cxcr4 ? / ? and WT
mice by FACS sorting. After mRNA extraction with TRIzol (Life Technol-
ogies), cDNA was synthesized using Superscript II (Invitrogen). qRT-PCR
was done with the ABI7700 Sequence Detection System (Applied Biosys-
tems). Sequences of the primers used for qRT-PCR are listed in Table S1
(available at http://www.jem.org/cgi/content/full/jem.20072513/DC1).
Transplantation. 8-wk-old BDF1 recipient mice (B6D2F1/J; The Jackson
Laboratory; H2-D d ) received 2 doses of 500 rads within a 6-h interval.
Donor BM cells from WT (CD45.1), Cxcr4 ? / ? , and Cxcr4 C/C mice (CD45.2)
were then transferred into irradiated recipients. Recipient mice were in-
jected with tamoxifen to delete Cxcr4 at various times after transplantation,
as indicated in the text.
Online supplemental material. Fig. S1 shows effi cient deletion of Cxcr4
in primitive hematopoietic cells. Fig. S2 shows a normal HSC compartment
in ROSA CRE-ERT 2 mice. Fig. S3 shows that CXCR4-defi cient primitive he-
matopoietic cells are indeed functional HSCs. Fig. S4 shows that CXCR4
defi ciency does not aff ect HSC survival. Table S1 lists primers for qRT-
PCR. The online version of this article is available at http://www.jem
We thank J. Liao for genotyping and K. Gordon for fl ow cytometry sorting.
This work was supported by the Irene Diamond Fund.
The authors have no confl icting fi nancial interest.
Submitted: 27 November 2007
Accepted: 12 March 2008
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hyperproliferative, a phenotype nearly identical to that de-
scribed herein ( 7 ). However, this report noted a drastically
reduced HSC compartment (CD34 ? LSK cells) in the BM
accompanied by impaired hematopoiesis 4 mo after Cxcr4 de-
letion. In this mouse model, the Cxcr4 gene was deleted by
poly I/poly C – activated Mx-Cre. It should be pointed out
that the poly I/poly C treatment used in this study is deleteri-
ous to HSCs, because injections of poly I/poly C (300 μ g/
mouse, 4 times) wiped out WT HSCs in 2 d and HSCs in the
BM did not recover even 3 wk after the treatment. On the
contrary, tamoxifen administration used in our system did not
cause noticeable toxic eff ect on the WT HSC compartment.
Under these diff erent conditions, although the Cxcr4 ? / ? HSC
compartment was preserved in mice treated with tamoxifen, it
was “ lost ” in mice that received poly I/poly C. At present, the
precise reason for this discrepancy cannot be ascertained, but
might be related to a distinct role of CXCR4 in HSC survival
in homeostatic state or under hematologic stress caused by
poly I/poly C treatment. CXCL12 has been reported to en-
hance survival of primitive hematopoietic cells ( 23, 24 ), and of
myeloid progenitor cells after cytokine withdrawal ( 25 ). Al-
though we did not observe any changes in apoptosis of freshly
isolated Cxcr4 ? / ? HSCs as compared with the WT HSCs, it is
possible that the survival of Cxcr4 ? / ? HSCs is impaired under
stress conditions, thereby compromising hematologic recov-
ery from chemoablation such as poly I/poly C treatment. It is
also conceivable that both WT and Cxcr4 ? / ? HSCs could no
longer reside in the stem cell niche under stress condition after
poly I/poly C treatment. Although WT HSCs may repopu-
late the BM niche, Cxcr4 ? / ? HCSs could not, thereby result-
ing in depletion of the mutant HSCs.
In summary, we demonstrate that the CXCL12 – CXCR4
axis is essential for HSCs homing into the BM, but less
critical for the BM retention. Our results also indicate that
CXCR4 signaling restricts HSCs in quiescence, and it prob-
ably does so through up-regulating the cell cycle inhibitor
p57 kip2 . Interestingly, HIV/gp120, which is a viral ligand of
CXCR4, has been shown to inhibit neural progenitor cell
proliferation ( 26 ). This result thus suggests a universal role of
CXCR4 signaling in the control of the quiescence of other
somatic stem cells. Further investigation will be required to
determine whether this inhibition also involves the same cell
cycle regulators. It is also worthwhile to examine whether
this mechanism may also contribute to HIV-1 – associated de-
mentia and immunodefi ciency.
MATERIALS AND METHODS
Mice. Cxcr4 fl /fl mice were crossed to Cxcr4 fl /fl ROSA CRE-ERT2/+ mice (pro-
vided by T. Ludwig, Columbia University, New York, NY) to generate
Cxcr4 C/C mice. To delete Cxcr4 , two sets of three consecutive administra-
tions of tamoxifen (5 mg/mouse, i.p.) were delivered 3 d apart. Mice were
maintained under specifi c pathogen – free conditions, and used according to
the protocol approved by the Columbia University Institutional Animal
Care and Use Committee.
Flow cytometry. BM cells were stained with PE-Cy5 – conjugated anti-
bodies against lineage markers (Lin), including B220, CD3, CD4, CD8,
CD11b, Gr1, and Ter119; PE-Cy7 – conjugated anti – c-Kit; and APC-
JEM VOL. 205, April 14, 2008
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