Pleiotrophin Regulates the Retention
and Self-Renewal of Hematopoietic Stem Cells
in the Bone Marrow Vascular Niche
Heather A. Himburg,1Jeffrey R. Harris,1Takahiro Ito,4Pamela Daher,1J. Lauren Russell,1Mamle Quarmyne,1
Phuong L. Doan,1Katherine Helms,1Mai Nakamura,1Emma Fixsen,1Gonzalo Herradon,5Tannishtha Reya,4
Nelson J. Chao,1,2Sheila Harroch,6and John P. Chute1,3,*
1Division of Cellular Therapy, Department of Medicine, Duke University Medical Center
2Department of Immunology
3Department of Pharmacology and Cancer Biology
Duke University, Durham, NC 27710, USA
4Department of Pharmacology, University of San Diego, San Diego, CA 92093, USA
5Pharmacology Laboratory, Department of Pharmaceutical and Food Sciences, Facultad de Farmacia, Universidad CEU San Pablo,
28003 Madrid, Spain
6Department of Neuroscience, Pasteur Institute, 75724 Paris, France
The mechanisms through which the bone marrow
(BM) microenvironment regulates hematopoietic
We examined the role of the heparin-binding growth
factor pleiotrophin (PTN) in regulating HSC function
in the niche. PTN?/?mice displayed significantly
decreased BM HSC content and impaired hemato-
poietic regeneration following myelosuppression.
Conversely, mice lacking protein tyrosine phospha-
tase receptor zeta, which is inactivated by PTN, dis-
played significantly increased BM HSC content.
Transplant studies revealed that PTN action was
not HSC autonomous, but rather was mediated by
the BM microenvironment. Interestingly, PTN was
differentially expressed and secreted by BM sinu-
soidal endothelial cells within the vascular niche.
Furthermore, systemic administration of anti-PTN
antibody in mice substantially impaired both the
and the retention of BM HSCs in the niche. PTN is
a secreted component of the BM vascular niche
that regulatesHSCself-renewaland retention in vivo.
Hematopoietic stem cells (HSCs) are capable of self-renewal
HSC fate determination in vivo is regulated by a combination of
intrinsic mechanisms and environmental cues mediated via cell-
cell interactions, cytokines, and secreted growth factors (Blank
et al., 2008; Kiel and Morrison, 2008; Zon, 2008) Although char-
acterization of the cells within the bone marrow (BM) microenvi-
ronment that regulate HSC fate continues to evolve (Butler et al.,
etal.,2005; Me ´ndez-Ferrer etal.,2010; Salter etal.,2009; Zhang
et al., 2003), the mechanisms through which BM-microenviron-
ment cells regulate HSC functions are less well understood.
We previously showed that adult sources of endothelial cells
(ECs) were capable of supporting the expansion of murine and
human HSCs in vitro (Chute et al., 2002, 2004, 2005, 2006a).
Utilizing a genomic screen of primary adult human brain ECs
(HUBECs) that support HSC expansion in noncontact cultures
(Chute et al., 2002, 2005, 2006a), we identified pleiotrophin
(PTN,) a heparin-binding growth factor that is primarily ex-
pressed in the nervous system (Li et al., 1990), to be >100-fold
overexpressed in HUBECs compared with non-HSC-supportive
ECs(Himburg et al.,2010). Wesubsequently showedthat in vitro
treatment of murine BM HSCs with PTN, in combination with
other cytokines, supported the expansion of HSCs with long-
term (LT) repopulating capacity (Himburg et al., 2010). However,
the HSC niche, which regulates HSC function in vivo, or whether
PTN had any physiologically relevant function in regulating HSC
pressed by BM-microenvironment cells within the HSC niche,
and whether modulation of PTN expression within the niche
can affect the maintenance, regeneration, or retention of HSCs
in vivo. Here, we show that PTN is uniquely expressed and
secreted by BM sinusoidal ECs within the HSC vascular niche
and has an important role in regulating HSC self-renewal and
retention in the BM.
PTN Regulates HSC Self-Renewal and Is Necessary
for Hematopoietic Regeneration In Vivo
We first examined the hematologic phenotype of mice bearing
a constitutive deletion of ptn (PTN?/?mice) compared with litter-
mate control PTN+/+mice. Knockout of ptn in the mouse strain
was confirmed by RT-PCR analysis (Figure 1A). Eight-week-old
964 Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors
PTN?/?mice displayed no significant differences in peripheral
blood (PB) complete blood counts or spleen size (Figure S1).
We also observed no differences in BM vascular density
between PTN?/?mice and PTN+/+mice (data not shown).
However, adult PTN?/?mice contained significantly decreased
BM c-kit+sca-1+lineage?(KSL) stem/progenitor cells as well as
BM colony-forming unit (CFU) spleen day 12 cells (CFU-S12;
decreased numbers of BM SLAM-receptor (CD150+CD48?
CD41?)-positive KSL (SLAM+KSL) cells (Kiel et al., 2005)
compared with PTN+/+mice, reflecting a deficit in phenotypic
HSCs (Figure 1B). Importantly, competitive transplantation
assays of BM 34?KSL cells, which are highly enriched for
HSCs (Himburg et al., 2010), into lethally irradiated congenic
micecompared with PTN+/+mice.At 12weeks aftercompetitive
transplantation, donor CD45.2+PB cell engraftment was 7-fold
lower in mice that were transplanted with BM 34?KSL cells
from PTN?/?mice compared with recipients of BM 34?KSL cells
from PTN+/+mice (mean 5% versus 35%; Figure 1C). Multiline-
age engraftment of myeloid cells, erythroid cells, B cells, and
T cells was also significantly lower in mice transplanted with
BM HSCs from PTN?/?mice compared with recipients trans-
planted with BM HSCs from PTN+/+mice (Figure 1D). Analysis
over time revealed that mice transplanted with BM HSCs from
PTN?/?mice had 5- to 20-fold decreased donor cell repopula-
tion between 4 weeks and 20 weeks posttransplant compared
with mice transplanted with HSCs from PTN+/+mice, confirming
a loss of both short-term (ST) and LT HSCs in PTN?/?mice (Fig-
ure 1C). Poisson statistical analysis of a limiting dilution trans-
plant assay demonstrated that the competitive repopulating
unit(CRU) frequencywithin PTN?/?micewas 11-fold decreased
with the CRU frequency in PTN+/+mice (1 in 6; CI: 1/2–1/14;
Figure 1E). Taken together, these results demonstrate that
PTN regulates the maintenance of the BM HSC pool.
Because deletion ofPTN caused asubstantial reductionin BM
HSC content in vivo, we next sought to determine whether PTN
deletion affected hematopoietic regeneration following myelo-
suppressive injury. We irradiated adult PTN?/?mice and
PTN+/+mice with 700 cGy total body irradiation (TBI), a myelo-
suppressive radiation dose, and compared their survival through
day +30. Sixty-nine percent of the PTN+/+mice (11 of 16)
remained alive and well through day +30 (Figure 1F). In contrast,
none of the PTN?/?mice (0 of 7) survived past day +18 post-TBI,
indicating markedly increased radiosensitivity in PTN?/?mice.
Commensurate with this, PTN?/?mice displayed severely
decreased BM progenitor cell content at day +20, whereas
PTN+/+mice showed evidence of recovery of the BM progenitor
cell compartment (Figure 1G). Taken together, these results
demonstrate that PTN is essential for hematopoietic regenera-
tion and survival following radiation-induced myelosuppression.
PTN Regulates the HSC Pool in a Microenvironment-
To determine whether PTN signaling is HSC autonomous or
dependent on the BM microenvironment, we transplanted BM
cells from CD45.1+Bl6.SJL mice into lethally irradiated PTN?/?
phenotypes of these chimeric mice. At 8 weeks posttransplant,
recipient mice demonstrated >95% donor chimerism (mean
96.4% donor cells in wild-type [WT];PTN+/+mice and 95.2% in
WT;PTN?/?mice, n = 8–9 mice/group). Adult PTN?/?mice that
were transplanted with BM cells from Bl6.SJL mice (WT;PTN?/?
mice) displayed significantly decreased numbers of BM KSL
cells, CFU-S12, and SLAM+KSL HSCs compared with age-
matched WT;PTN+/+mice (Figure 1H). Importantly, mice that
were competitively transplanted with BM from WT;PTN?/?
mice also displayed significantly decreased multilineage donor
cell repopulation between 4 weeks and 30 weeks posttransplant
compared with mice transplanted with the identical dose of
BM cells from WT;PTN+/+mice (Figure 1I). Secondary transplan-
tation of BM cells from the primary transplant recipients demon-
strated that secondary mice in the WT;PTN?/?group had 4-fold
decreased donor cell engraftment at 8 weeks posttransplant
compared with secondary recipients in the WT;PTN+/+group
(mean donor CD45.1+cells: 0.5% ± 0.2 versus 2.0% ± 0.7,
p = 0.04, n = 8–9/group). These results demonstrate that PTN
production by the BM microenvironment is necessary for regen-
eration of the HSC pool following BM transplantation.
Because PTN was necessary for normal HSC reconstitution
in vivo following BM transplantation, we next tested whether
pharmacologic administration of PTN could accelerate HSC
reconstitution in a clinically relevant model of HSC transplanta-
tion. We transplanted limiting doses (0.5–1 3 106cells) of human
cord blood (CB) mononuclear cells intravenously into NOD/SCID
IL2R-g?/?(NSG) mice, and compared human hematopoietic
reconstitution over time in mice that were treated intraperitone-
ally with 2–4 mg PTN or saline on days +7, +10, and +13 post-
transplant. PB was analyzed at 4 and 8 weeks posttransplant
for human CD45+cell engraftment. The PTN-treated mice
demonstrated significantly increased human CD45+cell repopu-
lation compared with saline-treated mice over time (4 weeks:
mean 10.9% huCD45+versus 1.4%; 8 weeks: mean 9.9% ver-
sus 1.9%, n = 11–14 mice/group; Figure 1J). Importantly, NSG
mice that were treated with PTN also had a >10-fold increased
human hematopoietic progenitor cell (HPC) content in the
BM at 8 weeks posttransplant compared with saline-treated
controls (Figure 1J). These results show that PTN promotes
human hematopoietic stem/progenitor cell (HSPC) regeneration
in vivo following transplantation, and illustrate the translational
potential of PTN administration as a means to accelerate human
hematopoietic reconstitution, particularly in settings wherein
the HSC dose is limiting, such as human CB transplantation
(Laughlin et al., 2004; Rocha et al., 2004).
Deletion ofProtein Tyrosine PhosphataseReceptor zeta
Expands the HSC Pool In Vivo
In the nervous system, PTN can mediate proliferative signals via
binding and inhibition of the transmembrane receptor PTPRZ
(Meng et al., 2000; Raulo et al., 1994; Stoica et al., 2001). We
sought to determine whether PTN mediates HSC self-renewal
via inactivation of PTPRZ signaling in HSCs. To that end, we
examined the hematopoietic phenotype of Ptprz1?/?mice
compared with Ptprz1+/+mice. RT-PCR confirmed the deletion
of the full-length messenger RNA transcript for ptprz in the
Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors 965
Figure 1. PTN Regulates HSC Self-Renewal and Is Necessary for Hematopoietic Regeneration In Vivo
(A) Quantitative RT-PCR (qRT-PCR) of ptn expression in the BM of PTN?/?mice and PTN+/+mice.
(B) PTN?/?mice contained significantly decreased BM KSL cells/femur, SLAM+KSL (CD150+CD48?CD41?lin?c-kit+sca-1+) cells/femur, and BM CFU-S12
compared with PTN+/+mice (n = 4 for KSL, n = 3 for SLAM+KSL, n = 5 for CFU-S12; *p = 0.01, *p = 0.0002, *p = 0.003 respectively).
966 Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors
mutant mice (Figure 2A). Interestingly, 8-week-old Ptprz1?/?
mice demonstrated significantly increased white blood cell
(WBC), hemoglobin (Hgb), and platelet counts compared with
age-matched Ptprz1+/+mice (Figure 2B). Ptprz1?/?mice also
displayed significantly increased total BM cells, KSL progenitor
cells, CFU-S12, and SLAM+KSL HSCs compared with Ptprz1+/+
mice (Figures 2C and 2D). CRU assays demonstrated that mice
transplanted with BM 34?KSL cells from Ptprz1?/?mice had
6-fold increased donor CD45.2+cell engraftment at 12 weeks
posttransplant compared with mice transplanted with BM cells
from Ptprz1+/+mice (Figure 2E). Mice transplanted with HSCs
from Ptprz1?/?mice also displayed normal and increased multi-
lineage donor myeloid cell, erythroid cell, T cell, and B cell repo-
pulation compared with recipients of BM from Ptprz1+/+mice,
confirming that deletion of PTPRZ did not alter the normal differ-
entiation capacity of BM HSCs (Figure 2F). Mice transplanted
with BM cells from Ptprz1?/?mice also demonstrated 5-fold
and 10-fold increased donor CD45.2+cell engraftment at
4 weeks and 16 weeks posttransplant, respectively, compared
with mice transplanted with BM cells from Ptprz1+/+mice, con-
firming that deletion of PTPRZ increased both ST- and LT-HSC
content in vivo (Figure 2E). A Poisson statistical analysis of donor
cell engraftment at 12 weeks from a limiting dilution assay
demonstrated a CRU frequency of 1 in 23 in Ptprz1?/?mice
(CI: 1/13–1/42) compared with 1 in 72 in Ptprz1+/+mice (CI:
1/27–1/189; Figure 2G). Therefore, deletion of PTPRZ was suffi-
cient to expand the BM HSC pool in vivo, and implicated PTPRZ
as the receptor that mediates PTN signaling in HSCs.
As evidence that PTPRZ is necessary for PTN-mediated
expansion of HSCs, we found that PTN treatment of BM KSL
cells from PTPRZ+/+mice caused a significant expansion of
KSL cells in vitro, whereas PTN treatment of BM KSL cells
from PTPRZ?/?mice failed to expand KSL cells in culture (Fig-
ure S2). Of note, we followed Ptprz1?/?mice through 12 months
of age and these mice displayed no evidence of splenomegaly,
lymphadenopathy, leukemia, or decreased survival compared
with Ptprz1+/+mice. These data suggest that deletion of PTPRZ
alone does not confer clonal myeloproliferative or lymphoproli-
ferative disease in mice (Figure S3).
PTPRZ Signaling Is HSC Autonomous
To determine whether PTN signaling through PTPRZ is
HSC autonomous or mediated via indirect effects on other BM
cell types, we transplanted lethally irradiated Bl6.SJL mice
(CD45.1+) with BM cells from Ptprz1?/?or Ptprz1+/+mice
(CD45.2+) to create Ptprz1?/?;WT mice and Ptprz1+/+;WT mice.
At 8 weeks posttransplant, the recipient mice were R95%
CD45.2+, confirming full donor chimerism (Figure 2H). At this
time point, we examined the BM stem/progenitor cell content
in both groups of mice. Ptprz1?/?;WT mice demonstrated
significant increases in BM KSL cells, CFU-S12, and SLAM+KSL
HSCs compared with Ptprz1+/+;WT mice (Figure 2I). These
results demonstrate that PTPRZ-mediated regulation of the
HSC pool was HSC autonomous and independent of PTPRZ
signaling in the BM microenvironment.
BM ECs and CXCL12+Reticular Cells Express PTN in the
Our transplant studies suggest that maintenance of the HSC
pool is dependent upon production of PTN by the BM microen-
vironment. We next sought to determine which cells within the
BM microenvironment produce PTN. Immunostaining of adult
mice femurs from PTN green fluorescent protein (PTN-GFP)
mice revealed that a subset of VE-cadherin+BM ECs coex-
pressed PTN, as did vascular endothelial growth factor receptor
Hooper et al., 2009; Salter et al., 2009). Similarly, a subset of
CXCL12 (SDF-1)+cells, which appeared to be perivascular,
(C) CD45.1+mice transplanted competitively with a limiting dose (30 cells) of BM CD34?KSL cells from CD45.2+PTN?/?mice demonstrated significantly
mice/group; 4 weeks, *p = 0.007; 8 weeks, *p = 0.006; 12 weeks, *p = 0.0008; 20 weeks, *p = 0.02).
(D) Twelve-week myeloid cell (Mac-1+), erythroid cell (Ter119+), T cell (Thy 1.2+), and B cell (B220+) engraftment (*p = 0.004, *p = 0.01, *p = 0.002, and *p = 0.02,
respectively, for differences between recipients of BM from PTN+/+and PTN?/?mice).
(E) Poisson statistical analysis after limiting dilution transplant assay. Plots were obtained to allow estimation of the CRU frequency in PTN+/+and PTN?/?mice
(n = 9–10 mice transplanted at each cell dose per condition). The plot shows the percentage of recipient (CD45.1+) mice containing <1% CD45.2+cells (non-
engrafted) in the PB at 12 weeksposttransplantation (y axis) versusthe numberof cells injected per mouse(x axis). The horizontal line indicates the point at which
37% of the mice are nonengrafted, and the vertical lines highlight the CRU frequencies in each mouse (PTN?/?mice [1/66] versus PTN+/+mice [1/6]).
(F) PTN?/?mice displayed increased radiosensitivity compared with PTN+/+mice and failed to regenerate hematopoiesis following TBI. Sixty-nine percent of the
PTN+/+mice (11 of 16) were alive at day +30 following 700 cGy TBI (at left). Conversely, none of the PTN?/?mice (0 of 7) survived beyond day +18 (p < 0.0001, log
(G) PTN?/?mice had >15-fold decreased BM CFCs at day +20 following TBI compared with PTN+/+mice (at right, n = 3/group, *p = 0.01).
(H) WT;PTN?/?mice had decreased BM KSL cells, decreased SLAM+KSL HSCs, and decreased CFU-S12 compared with WT;PTN+/+mice (means ± SEM,
n = 8–9 for KSL and CFU-S12; n = 3 for SLAM analysis; KSL, *p = 0.02; SLAM+KSL, *p = 0.02; CFU-S12, *p = 0.002).
(I) Mice competitively transplanted with a limiting dose (30 cells) of BM CD34?KSL cells from WT;PTN?/?mice demonstrated significantly lower donor CD45.1+
cell engraftment over 4–30 weeks compared with mice transplanted with the identical dose of CD34?KSL cells from WT;PTN+/+mice (n = 8 mice/group; 8 weeks,
*p = 0.001; 30 weeks, *p = 0.04). Error bars represent SEM for all experiments; Student’s t test was performed for comparisons.
circles, and experiment 2 is represented by open triangles) followed by intraperitoneal injections of 2 or 4 mg PTN or saline on days +7, +10, and +13
posttransplant. PTN-treated mice displayed significantly increased human hematopoietic cell engraftment in the PB over time posttransplant compared with
saline-treated controls (left: n = 11–14 per group, 4 weeks *p = 0.04; 8 weeks *p = 0.03). Horizontal bars represent the mean levels of donor human CD45+cell
engraftment. Transplanted NSG mice that were treated with PTN demonstrated significantly increased human CFCs in the BM at 8 weeks compared with saline-
treated NSG mice (right: n = 4 mice/group, *p = 0.02).
See also Figure S1.
Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors 967
Figure 2. Deletion of PTPRZ Is Sufficient to Expand the BM HSC Pool
(A) qRT-PCR analysis of ptprz expression in Ptprz1+/+and Ptprz1?/?mice.
(B) Scatter plots show the complete blood counts in the PB of Ptprz1+/+mice compared with Ptprz1?/?mice (WBCs,*p = 0.005; neutrophils, *p = 0.006;
lymphocytes, *p = 0.003; Hgb, *p = 0.04; platelets, *p = 0.01). Mean values are represented by horizontal lines; n = 8–13 mice per condition.
968 Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors
also coexpressed PTN, whereas the majority of osterix+cells did
Taken together, these results demonstrate a differential expres-
sion of PTN byBMECs and perhaps byCXCL12-abundant retic-
ular cells (CARs; Sugiyama et al., 2006). We further determined
by ELISA that PTN was concentrated within the BM serum of
C57Bl6 mice but was not detectable in PB serum (Figure S4).
In addition, PTN was highly enriched in the conditioned media
from primary BM ECs from C57Bl6 mice, confirming that BM
ECs secrete PTN (Figure S4). These results show that PTN is
differentially expressed and secreted by principal components
of the BM vascular niche and is a paracrine factor for BM
stem/progenitor cells in vivo.
To further characterize the cells within the BM niche that
expressed PTN, we performed fluorescence-activated cell-
sorting (FACS) analysis on BM CD45?PTN+cells and analyzed
for surface expression of the EC marker VE-cadherin. The FACS
analysis revealed a distinct population of VE-cadherin+PTN+
cells in the BM (Figure 3F). We then performed a gene expres-
sion analysis of FACS-sorted VE-cadherin+PTN+cells, which re-
vealed enrichment for VEGFR2 and VEGFR3, which are markers
of BM sinusoidal endothelium (Hooper et al., 2009; Table 1).
Interestingly, VE-cadherin+PTN+cells were also enriched for
expression of CXCL12 and the leptin receptor (lepR), proteins
that have been shown to be expressed by both perivascular
stromal cells and sinusoidal ECs (Dar et al., 2005; Ding et al.,
2012; Ikejima et al., 2004; Sugiyama et al., 2006). VE-cadherin+
PTN+cells lacked expression of Nestin, a marker of BM mesen-
chymal stromal cells (MSCs) (Me ´ndez-Ferrer et al., 2010).
Lastly, to determine the anatomic relationship between PTN+
cells and HSCs in the BM, we immunostained femurs from
PTN-GFP mice to detect CD150+CD48?CD41?lineage?cells.
As previously described, we found CD150+CD48?CD41?
lineage?cells to be rare in the BM (Kiel et al., 2005; Me ´ndez-
Ferrer et al., 2010). However, the majority of the CD150+CD48?
CD41?lineage?cells (82.3%, 51 of 62) were found to be in
contact with or closely adjacent to PTN+cells (30 images, 5
femur sections; Figure 3G). Taken together with our functional
studies of PTN?/?mice, these results suggest an anatomic
and functional relationship between BM HSCs and PTN+cells
in the vascular niche.
3C–3E;Tang etal., 2011).
PTN Regulates HPC Homing to the BM Niche
Having shown that PTN is expressed by sinusoidal BM ECs
within the vascular niche, we sought to determine whether
PTN+ECs regulate HSPC homing to the niche. Adult C57Bl6
mice were injected intravenously with either 50 mg of a specific,
neutralizing anti-PTN antibody (R&D Systems) or 50 mg immuno-
globulin G (IgG), and after 30 min were infused with 2 3 105BM
Sca-1+lin?progenitor cells from ubiquitin C-GFP (UBC-GFP)
mice. At 18 hr posttransplant, the mice were sacrificed and
BM cells were analyzed by FACS to compare the homing of
GFP+cells to the BM in each group. Mice that were pretreated
with anti-PTN antibody displayed a significant decrease in
donor HSPC homing to the BM compared with IgG-treated
recipient mice (Figures 4A and 4B). These results suggested
that PTN is required for the proper homing of HSPCs to the
BM following transplantation. To determine the specific effect
that anti-PTN administration had on the homing of transplanted
HPCs, we performed intravital imaging using confocal micros-
copy to observe intravenously transplanted BM lin?GFP+cells
homing within the calvarial BM endothelium in dsRed mice, as
previously described (Lo Celso et al., 2011). Mice that were pre-
treated with IgG and then intravenously transplanted with 3 3
106BM lin?GFP+cells demonstrated dynamic transmigration
of GFP+cells from the BM vascular space into the BM paren-
chymal space between 1 and 4 hr posttransplant (Figures 4C
and 4D). In contrast, mice that were pretreated with anti-PTN
and then transplanted with equal doses of BM lin?GFP+cells
demonstrated a substantial defect in lodgment along the BM
endothelium and in transmigration across the BM vasculature
into the HSC niche (Figures 4C and 4D). Coupled with the
demonstration that anti-PTN quantitatively decreased HSPC
homing to the niche, these data suggested an important role
for PTN in regulating the lodgment and/or transmigration of
HSPCs from the BM vasculature into the HSC niche. Of note,
to confirm that systemically administered anti-PTN mediated
effects directly upon the BM vascular endothelium, we also
show that mice injected with 50 mg anti-PTN-DyLight650 anti-
body displayed specific binding of the antibody to the intimal
endothelial layer of the BM vasculature, whereas mice injected
with 50 mg IgG-DyLight650 showed no binding to BM ECs
(C) Ptprz1?/?mice have increased mean BM cell counts, KSL cells/femur, and SLAM+KSL cells/femur compared with Ptprz1+/+mice (n = 3–5 mice/group;
*p = 0.04, *p = 0.003, *p = 0.002, respectively). Error bars represent SEM.
(D) Ptprz1?/?mice contain increased BM CFU-S12 compared with Ptprz1+/+mice (n = 7–9/group, p < 0.0001).
(E) Mice transplanted with a limiting dose (30 cells) of BM CD34?KSL cells from Ptprz1?/?mice demonstrated significantly higher donor CD45.2+donor cell
engraftment over time compared with recipients of the identical dose of CD34?KSL cells from Ptprz1+/+mice (means ± SEM are shown, n = 7–10 mice/group;
4 weeks, *p = 0.01; 8 weeks, *p = 0.03; 12 weeks, *p = 0.03; 16 weeks, *p = 0.04; Student’s t test for all comparisons).
(F)Multilineageengraftment ofmyeloid cells,erythroidcells,Tcells, andBcellsis shownat12weeks posttransplantation; n=7–8mice/group; myeloid, *p=0.03;
T cell, *p = 0.02.
(G) Poisson statistical analysis after limiting dilution transplant assay; plots were obtained to allow estimation of CRU frequency in Ptprz1+/+mice (1 in 72) and
Ptprz1?/?mice (1 in 23); n = 7–8 mice transplanted at each cell dose per condition.
(H) The mean percent donor chimerism is shown at 8 weeks posttransplantation of BM cells from Ptprz1+/+or Ptprz1?/?(CD45.2+) mice into lethally irradiated
Bl6.SJL recipient (CD45.1+) mice (WT) to generate chimeric Ptprz1?/?;WT mice and Ptprz1+/+;WT mice. Mean donor cell chimerism was R 95% in Ptprz1?/?;WT
mice and Ptprz1+/+;WT mice (n = 4–5 mice/group, means ± SEM).
(I) Ptprz1?/?;WT mice contained significantly increased BM KSL cells/femur, SLAM+KSL cells/femur and CFU-S12 compared with Ptprz1+/+;WT mice (n =
3–6/group, *p = 0.004, *p < 0.0001, and *p = 0.002, respectively; data represent means ± SEM; Student’s t test for comparisons).
See also Figures S2 and S3.
Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors 969
HSPC homing to the BM niche is regulated by numerous
cooperative mechanisms, including HSPC rolling, lodgment,
and transmigration through BM sinusoidal vasculature, medi-
ated by VLA4–VCAM and CD44–hyaluronic-acid interactions
between HSPCs and BM ECs, and via the CXCR4-CXCL12
axis (Avigdor et al., 2004; Kahn et al., 2004; Papayannopoulou
et al., 1995). We performed in vitro migration assays in which
2 3 105BM ckit+lin?cells were placed in the upper chamber
of transwell cultures, and 200 ng PTN, 200 ng SDF-1
(CXCL12), or media were placed in the lower chamber. At 4 hr
Figure 3. PTN Is Expressed by BM ECs in the HSC Niche
(A) PTN-GFP reporter mice stained with VE-cadherin antibody (red) demonstrate that VE-cadherin+ECs express PTN (green). White arrows indicate cells that
express both PTN and VE-cadherin. Inset box is shown magnified on the right; white scale bar represents 10 mm.
(B) VEGFR3+sinusoidal vessels (red) that coexpress PTN (green). White arrows indicate cells expressing both VEGFR3 and PTN. Right: High-power image.
(C) Staining for osterix+cells (red) indicates that very few osterix+cells express PTN (green). Right: High-power image.
(D) CARs (red) that coexpress PTN (green) are identified. White arrows indicate cells expressing both PTN and CXCL12.
(E) Quantification of the percentage of VE-cadherin+, VEGFR3+, osterix (Osx)+, and CXCL12+cells that coexpress PTN. Osterix+cells had the lowest amount of
sections/stain; bars represent means ± SEM).
(F) Representative FACS analysis of BM cells from PTN-GFP reporter mice costained with CD45 and VE-cadherin antibodies, demonstrating that PTN is
expressed by VE-cadherin+cells.
(G) Immunohistochemical staining for CD150+CD48?CD41?lineage?cells in the femurs of PTN-GFP mice was performed. A representative image is shown;
82.3% of the CD150+CD48?CD41?lineage?cells (51 of 62, magenta) were within a 5 mm distance of PTN+cells (green) in the BM (30 images analyzed from
5 femur sections).
See also Figure S4.
970 Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors
of culture, we observed no migration of HSPCs toward PTN,
whereas 35% of HSPCs migrated toward SDF1 (Figure 4F).
These data suggested that PTN alone did not provide a gradient
for HSPC migration. When HSPCs were preincubated for 1 hr
with PTN, we observed a significant increase in HSPC migration
toward SDF1, suggesting that PTN augments HSPC migration
toward an SDF1 gradient (Figure 4F). However, incubation with
PTN did not upregulate CXCR4 or VLA4 expression on BM
ckit+lin?cells, suggesting that PTN regulates HSPC homing
through an alternative mechanism (Figure 4F).
Our results suggest that PTN has an important role in regulating
HSPC homing to the BM niche. We further hypothesized that
PTN might also regulate HSC retention in the niche, and that
systemic administration of anti-PTN might promote HSPC mobi-
lization. To test this hypothesis, we treated adult C57Bl6 mice
with either 50 mg IgG, 50 mg anti-PTN, or the CXCR4 antagonist
PB was collected and analyzed for mobilization of ckit+sca-
1+lin?cells (KSL cells), which are enriched for HSPCs. Interest-
ingly, treatment with anti-PTN alone significantly increased the
number of KSL cells in the PB at 1 hr posttreatment compared
with IgG-treated control mice (Figures 4G and 4H). As expected,
AMD3100, which is used clinically to mobilize HSPCs (Malard
et al., 2012), also promoted HSPC mobilization. Importantly,
the combination of AMD3100 and anti-PTN caused a 2-fold
increase the mobilization of BM KSL cells compared with the
effect of AMD3100 treatment alone (Figures 4G and 4H). Taken
together, these data suggest that PTN also regulates the reten-
tion of HSPCs in the BM niche and cooperates with the
CXCR4-SDF1 axis in this regard.
Recent studies have implicated several different cell types within
theBMmicroenvironment ashaving importantroles inregulating
HSC self-renewal and retention in vivo (Butler et al., 2010; Calvi
Me ´ndez-Ferrer et al., 2010; Salter et al., 2009; Zhang et al.,
2003). However, the mechanisms through which BM-microenvi-
ronment cells regulate HSC functions in vivo remain incom-
pletely understood. Here we show that PTN, a heparin-binding
growth factor, is expressed by sinusoidal ECs within the BM
vascular niche and regulates the maintenance of the HSC pool
in vivo. Furthermore, genetic deletion of PTPRZ, a receptor for
PTN that is expressed by HSCs, caused a significant expansion
of the HSC pool in vivo. This observed effect is consistent with
the established function of PTN as inactivating PTPZ phospha-
tase activity upon receptor binding. The observed deficit in
HSC numbers coupled with only slight reductions in PB
complete blood counts in PTN?/?mice suggests the possibility
of compensation by other factors (Herradon et al., 2005) in
PTN?/?mice. However, PTN appears to be indispensable for
hematopoietic regeneration to occur following myelosuppres-
sion, since PTN?/?mice had significantly increased mortality
following a myelosuppressive dose of TBI (700 cGy), coupled
with a severe deficit in the recovery of BM progenitor cells
compared with PTN+/+mice. These results suggest an essential
role for PTN in regulating hematopoietic regeneration following
Previous studies identified cellular components of a BM
vascular niche for HSCs, including VEGFR2+VEGFR3+sinu-
for maintenance ofthe HSC pool duringhomeostasis (Ding etal.,
2012; Hooper et al., 2009; Sugiyama et al., 2006). Nestin+MSCs
were also shown to contribute to both the vascular and endos-
teal niches for HSCs in vivo (Me ´ndez-Ferrer et al., 2010).
However, the signaling mechanisms through which cells within
the BM vascular niche regulate HSC homeostasis or regenera-
tion are not well understood. Here we demonstrate via immuno-
histochemical and FACS analyses that PTN is expressed
uniquely by VE-cadherin+ECs that coexpress VEGFR2 and
VEGFR3+, consistent with BM sinusoidal ECs (Hooper et al.,
2009). Interestingly, PTN+ECs also express CXCL12 and lepR,
reticular cells (Dar et al., 2005; Ding et al., 2012; Ikejima et al.,
2004; Sugiyama et al., 2006). Of note, a prior study (Tezuka
et al., 1990) suggested that calvarial bone osteoblasts express
PTN, but we found little evidence that osterix+bone lineage cells
expressed PTN in the BM. Commensurate with our findings that
PTN was highly expressed by BM sinusoidal ECs, PTN was
highly concentrated in BM supernatants and in the conditioned
media of primary BM sinusoidal ECs in culture, but was unde-
tectable in the PB of WT mice. These results, coupled with the
observed deficit in HSC repopulating cell content in WT;PTN?/?
within the vascular niche.
In addition to their role in regulating the maintenance of
the HSC pool in vivo, BM sinusoidal ECs regulate the rolling,
lodgment, and transmigration of transplanted HSPCs from the
vascular space into HSC niches (Avigdor et al., 2004; Papayan-
nopoulou et al., 1995). For example, VLA4–VCAM1 and CD44–
hyaluronic-acid interactions between HSPCs and BM ECs
control the initial steps in the homing of HSPCs across the
vascular endothelium (Avigdor et al., 2004; Papayannopoulou
et al., 1995). Because PTN is strongly expressed by BM
Table 1. Gene Expression of VE-cadherin+PTN+Cells
VEGFR2 Vascular endothelial growth
factor receptor 2
VEGFR3Vascular endothelial growth
factor receptor 3
CXCL12 Chemokine CXC ligand 1248.0 0.003
LepR Leptin receptor136.2 0.02
BM cells were collected from PTN-GFP reporter mice and stained with
anti-VE-cadherin or isotype antibody. FACS was performed to collect
VE-cad+PTN+cells, and RNA was isolated for qRT-PCR analysis for the
genes identified on the left. The fold differences in gene expression
between VE-cad+PTN+cells versus CD45+PTN?BM cells are shown.
n = 3 replicates/group; t test; ND, not detected.
Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors 971
Figure 4. PTN Regulates the Homing and Retention of BM HSPCs
(A) Systemic administration of anti-PTN significantly decreased HSPC homing to the BM niche. Shown is a representative FACS analysis of the percentage GFP+
donor hematopoietic cells in the BM of recipient C57Bl6 mice at 18 hr following intravenous infusion of BM Sca-1+lin?GFP+cells after pretreatment of recipient
mice with either anti-PTN or IgG.
972 Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors
sinusoidal ECs, we sought to determine whether PTN also regu-
lates HSPC migration and homing to the niche. Interestingly,
administration of a single dose of anti-PTN antibody sub-
stantially inhibited HSPC homing to the niche. Furthermore,
intravital imaging revealed that transplanted HSPCs displayed
impaired lodgment along the BM endothelium and deficient
transmigration from the vascular space into the HSC niche.
These data suggest that HSPC anchoring to PTN on sinusoidal
ECs may be a critical step in HSPC homing to the BM. In vitro
migration assays revealed that HSPCs did not migrate toward
a PTN gradient, but pretreatment with PTN did augment
HSPC migration toward SDF1. Since PTN treatment did not
upregulate CXCR4 or VLA4 expression on HSPCs, PTN may
facilitate HSPC homing toward SDF1 via an as yet unidentified
In addition to its evident role in regulating the homing of
HSPCs to the BM, PTN also regulates the retention of HSPCs
in the niche. Administration of a single dose of anti-PTN caused
a 2-fold increase in PB HSPCs at 1 hr post-exposure compared
with isotype-treated mice, and a similar increase in HSPC mobi-
lization was observed when anti-PTN was combined with
AMD3100, a CXCR4 antagonist that is used in clinical practice
to mobilize human HSPCs. The rapidity of the effect of anti-
PTN administration on HSPC mobilization suggests that PTN
plays an important role in the retention of HSPCs in the niche.
Moreover, the doubling of HSPC mobilization when anti-
PTN was combined with AMD3100 suggests an additive or
synergistic role for PTN in modulating HSPC retention via the
CXCR4-SDF1 axis. These results also suggest an important
potential clinical application for anti-PTN in mobilizing HSPCs
in patients undergoing stem cell transplantation.
Istvanffy et al. (2011) recently reported that deletion of PTN in
the BMmicroenvironment was associated with a gain of LT-HSC
function compared with mice that retained PTN in the marrow.
Important differences between the mouse models used in that
study and the one presented here may explain the apparently
divergent results: First, we used PTN?/?mice (C57Bl6 back-
ground; Jackson Laboratory) and syngeneic (B6.SJL) recipient
mice for CRU transplantation studies and for the generation of
WT;PTN?/?mice to assess effects of PTN on HSC content.
Because these donor and recipient mice were genetically iden-
tical, there were no immunological factors that could confound
estimates of HSC content. Conversely, in the study by Istvanffy
et al. (2011), the CRU assays did not involve syngeneic mice,
but rather allogeneic mice. Therefore, immunologic processes
such as graft rejection and graft-versus-host reaction, or the
effects of PTN on these immune processes, could have affected
estimates of HSC content in that model independently of any
direct effects of PTN on the HSC pool. Secondly, in this study
we used purified HSCs (CD34?KSL cells) for competitive trans-
plantation assays to allow precise determination of the effects
of PTN deletion on HSC content and function, whereas Istvanffy
et al. (2011) used whole BM cells, and thus it is possible that
effects on adventitious cells in the graft could have affected their
Much remains unknown regarding the mechanisms through
which BM-microenvironment cells regulate HSC functions
in vivo. Here, we provide evidence that BM sinusoidal ECs
uniquely express a secreted protein, PTN, that regulates
the maintenance, regeneration, and retention of HSCs in the
vascular niche. PTN represents a unique target for pharmaco-
logic approaches to modulate HSC function in vivo.
All animal procedures were performed in accordance with a Duke University
IACUC-approved animal use protocol. Embryos from mice bearing a constitu-
tivedeletionofPTN(Ochiaietal.,2004)wereobtained fromtheRIKEN Institute
(Tsukuba,Japan) by theJackson Laboratory (BarHarbor, ME) andrederivedin
a C57BL6 background. Mice bearing a constitutive deletion of the PTN
(B) Anti-PTN treated mice had >2-fold decreased donor GFP+cells in the BM at 18 hr postinfusion compared with IgG-treated control mice (n = 5–6/group,
means ± SEM, *p = 0.0004).
(C and D) Administration of anti-PTN inhibits the lodgment and transmigration of HPCs from the BM vasculature into the stem cell niche. Representative intravital
images of the calvarial BM space in living dsRed mice during the first 4 hr postintravenous infusion of 3 3 106BM lin?GFP+cells are shown. Mice that were
pretreated with IgG antibody (C) demonstrated abundant homing of transplanted cells (green) from the BM sinusoidal vasculature (gray) into the niche, whereas
micepretreated withanti-PTN displayed amarkedly decreasednumber of donorHPCswithin theextravascular BM space (D).A single GFP+cell (green) is shown
within the BM vasculature in the anti-PTN-treated mice.
mice at 30 min after intravenous injection of either IgG-DyLight650 antibody (left: 203, scale bar 50 mm) or anti-PTN-DyLight650 (middle: 203, scale bar 50 mm).
Binding and illumination of the BM sinusoidal vasculature by anti-PTN antibody (gray outline, white arrows) is shown in high power at right (43 zoom of the white
box area in the previous image; scale bar 20 mm).
(F) Preincubation with PTN augments HPC migration to an SDF1 gradient. BM ckit+lin?cells demonstrated no migration to media alone or PTN in a 4 hr transwell
assay (left, n = 4/group). A percentage of BM ckit+lin?cells migrated to an SDF1 gradient in the lower chamber of transwell cultures. Left: BM ckit+lin?cells that
werepreincubatedwithPTN31hrdemonstratedsignificantly increased migration toSDF1compared withcontrol culturesat4hrintranswellassay(n=6/group,
means ± SEM, *p = 0.002). Incubation of BM ckit+lin?cells with PTN had no effect on cell-surface expression of CXCR4 (middle) or VLA4 (right); n = 6/group,
means ± SEM.
(G) Administration of anti-PTN promoted the rapid mobilization of HSPCs in WT mice. Shown are representative FACS plots of the percentage of KSL cells in the
PB of adult C57Bl6 mice at 1 hr following intravenous administration of IgG, anti-PTN, AMD3100, or AMD3100 + anti-PTN. Both anti-PTN and AMD3100
increased the mobilization of KSL cells to the PB compared with IgG-treated control mice. Mice treated with AMD3100 + anti-PTN displayed increased KSL cell
mobilization compared with AMD3100 treatment alone.
(H) The bar graphs show the means ± SEM of KSL cells in the PB of IgG-treated mice, anti-PTN-treated mice, AMD3100-treated mice, and mice treated with
AMD3100+anti-PTN.Treatmentwithanti-PTN aloneor incombination withAMD3100significantly increased KSLcellmobilization compared withIgG-treatedor
AMD3100-treated mice, respectively. *p = 0.02 for anti-PTN versus IgG (n = 8/group, means ± SEM); *p = 0.03 for AMD3100 + anti-PTN versus AMD3100 alone
(n = 5/group, means ± SEM).
Cell Reports 2, 964–975, October 25, 2012 ª2012 The Authors 973
receptor PTPRZ were a generous gift from Dr. Sheila Harroch of L’Institut
Pasteur, Paris, France (Harroch et al., 2000, 2002). Sperm from PTN-GFP
mice developed as part of the GENSAT Project (Rockefeller University) was
obtained from the Mutant Mouse Regional Resource Center and the strain
was rederived in a C57BL6 background.
Isolation of Murine BM HSCs
BM HSCswerecollected from allmice aspreviouslydescribed(Himburg etal.,
2010). Briefly, collected BM was first treated with red blood cell (RBC)-lysis
buffer (Sigma Aldrich), and lineage committed cells were removed using
a lineage depletion column (Miltenyi Biotec, Auburn CA). Lin?cells were
stained with fluorescein isothiocyanate-conjugated anti-CD34 (eBioscience,
San Diego, CA), PE-conjugated anti-sca-1, and APC-conjugated anti-ckit
(Becton Dickinson [BD], San Jose, CA), or isotype controls. Sterile cell sorting
was conducted on aBDFACS-Aria cytometer. PurifiedCD34?c-kit+sca-1+lin?
(34?KSL) subsets were collected into Iscove’s modified Dulbecco’s medium
(IMDM) + 10% fetal bovine serum (FBS) + 1% pcn/strp.
Colony-Forming-Cell and CFU-S12 Assays
Colony-forming-cell (CFC) assays (CFU-granulocyte monocyte [CFU-GM],
burst-forming unit-erythroid [BFU-E], and CFU-mix [CFU-GEMM]) were per-
formed in triplicate as previously described (Chute et al., 2002, 2005). Briefly,
5,000 cells from each condition were placed in MethoCult (StemCell
Technologies, Vancouver, Canada) for 14 days, and the total colonies were
calculated. The BM HSC content in the mutant mice was assayed by
CFU-S12 assay. RBC-depleted BM was transplanted at a dose of 1 3 105
cells/mouse into lethally irradiated (950 cGy) C57Bl6 mice. At day 12 post-
transplant, the mice were euthanized and the spleens were collected. The
numbers of hematopoietic colonies on each spleen were counted.
BM 34?KSL cells from the PTN?/?and Ptprz1?/?mice, and PTN+/+and
Ptprz1+/+mice carrying the CD45.2 allele were isolated by FACS (Himburg
et al., 2010). Recipient B6.SJL animals expressing the CD45.1 allele received
950 cGy TBI via a Cs137 irradiator and were then transplanted via tail vein
injectionwith 5,10, 30, or 100BM 34?KSL cells. Nonirradiated host BM mono-
nuclear cells (MNCs; 1 3 105cells/mouse) were injected as competitor cells.
Multilineage hematologic reconstitution was monitored in the PB by flow
cytometry, as previously described (Chute et al., 2007; Himburg et al., 2010),
at 4, 8, 12, 16, and 20 weeks posttransplant. PB was collected via submandib-
ular puncture and stained with antilineage marker antibodies as previously
described (Himburg et al., 2010). Animals were considered to be engrafted if
donor CD45.2 cells were present at R1%. CRU calculations were performed
with the use of L-Calc software (Stem Cell Technologies; Chute et al., 2005,
PTN Reporter Mice
PTN-GFP reporter mice (Jackson Laboratory) were given an intravenous
injection of 200 mg of rat anti-mouse Alexa Fluor 647 VE-cadherin antibody
in PBS. The mice were sacrificed within 1 hr of injection and the BM was
flushed through a 30 mm filter in Hanks’ balanced salt solution with Ca2+and
Mg2+. The portion of the BM retained in the 30 mm filter was collected and
mechanically disrupted by pipetting. The filter-retained cells were then stained
with PerCP-conjugated CD45 and FACs sorted to obtain CD45?VE-cadherin+
PTN+and CD45?VE-cadherin?PTN+cells. These populations were compared
with the CD45+PTN?cell population for RT-PCR analysis.
PTN Treatment of Ptprz1?/?Cells In Vitro
CD34?KSL cells were isolated from Ptprz1?/?and Ptprz+/+mice and cultured
for 7 days in IMDM containing 10% FBS, 1% pen-strep, 125 ng/ml stem cell
factor, 50 ng/ml Flt-3 ligand, and 20 ng/ml thrombopoietin either with or
without 100 ng/ml PTN. Following culture, the progeny were analyzed for total
KSL cell expansion.
Human CB Transplant Model
Human CB units were obtained according to a protocol approved by the
Institutional Review Board of Duke University. The units were purified for
MNCs using a density gradient separation in Ficoll-HyPaque followed by RBC
lysis. Then 0.5–1 3 106human CB MNCs were transplanted into 6-week-old
NSG mice conditioned with 250 cGy radiation on a Cesium source. Following
transplantation, the mice were treated intraperitoneally on days +7, +10,
and+13posttransplantwith2–4 mgPTN orsaline.PBwasdrawnretro-orbitally
at 4 and 8 weeks posttransplant to assess human CD45+cell engraftment.
Supplemental Information includes Extended Experimental Procedures and
four figures and can be found with this article online at http://dx.doi.org/10.
This is an open-access article distributed under the terms of the Creative
Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported
License (CC-BY-NC-ND; http://creativecommons.org/licenses/by-nc-nd/3.0/
The authors thank Dr. Joel Ross for assistance with graphical art. This work
was supported in part by National Institute for Allergy and Infectious Diseases
grant AI067798-06 (J.P.C.) and National Heart, Lung and Blood Institute grant
Received: March 25, 2011
Revised: February 10, 2012
Accepted: September 6, 2012
Published online: October 18, 2012
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