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HEMATOPOIESIS AND STEM CELLS
Impact of interactions of cellular components of the bone marrow
microenvironment on hematopoietic stem and progenitor cell function
Brahmananda R. Chitteti,1 Ying-Hua Cheng,2 Bradley Poteat,1 Sonia Rodriguez-Rodriguez,3 W. Scott Goebel,3
Nadia Carlesso,3 Melissa A. Kacena,2,4,5 and Edward F. Srour1,3,6
Departments of 1Medicine, 2Orthopaedic Surgery, 3Pediatrics, and 4Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis; 5Department
of Biomedical Engineering, Indiana University Purdue University Indianapolis, IN; and 6Department of Microbiology and Immunology, Indiana University School
of Medicine, Indianapolis
Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic
and extrinsic parameters. We examined
the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on
LineageⴚSca-1ⴙCD117ⴙ (LSK) cells. CFU
expansion and marrow repopulating potential of cultured LineageⴚSca-1ⴙCD117ⴙ
cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the compe-
tence of the hematopoietic niche. OBmediated enhancement of HSC and HPC
function was reduced in cocultures of OBs
and SCs, suggesting that SCs suppressed
the OB-mediated hematopoiesis-enhancing
activity. Although the suppressive effect of
SC was mediated by adipocytes, probably
through up-regulation of neuropilin-1, the
OB-mediated enhanced hematopoiesis
function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and
2, Delta 1 and 4, Hes 1 and 5, and Deltex was
increased in OB cultures and suppressed in
SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the
hematopoiesis-enhancing activity but
demonstrated that this function is common to OBs from different anatomic sites.
These data illustrate that OBs promote in
vitro maintenance of hematopoietic functions, including repopulating potential by
up-regulating Notch-mediated signaling
between HSCs and OBs. (Blood. 2010;
115(16):3239-3248)
Introduction
Hematopoietic stem cells (HSCs) are multipotent progenitor cells
that give rise to all types of mature blood cells. HSCs reside in a
complex cellular microenvironment containing osteoblasts (OBs),
osteoclasts, endothelial cells, stromal cells (SCs), mesenchymal
progenitor cells, and adipocytes as well as multiple components of
the extracellular matrix. Collectively, these cellular elements and
the extracellular matrix constitute the hematopoietic niche, which
most probably regulates the size of the stem cell pool and controls
HSC fate.1
OBs play a critical role in HSC function and self-renewal.
Primitive HSCs that are in association with the endosteal region
have high proliferative and repopulating capacities.2 OBs can
deliver proliferative signals to HSCs during mobilization.3 Human
OBs secrete cytokines, such as granulocyte colony-stimulating
factor, granulocyte-macrophage colony-stimulating factor, and
leukemia inhibitory factor, thereby supporting hematopoietic progenitor cell (HPC) function in vitro.4-6 Furthermore, OBs secrete
angiopoietin, thrombopoietin, and stromal cell–derived factor-1, all
of which regulate HSC maintenance.7-9 Physical and molecular
interactions between HPCs and OBs supported in vitro hematopoiesis5 and survival,10 whereas cotransplantation of OBs with HSCs
improved engraftment.11 However, others questioned whether OBs
contribute to the formation of niches where vascular and perivascular cells play a major role in maintaining HSC function.12
In addition to stem cell–enhancing activity, microenvironment
cells in multiple systems can down-regulate stem cell function.
Endothelial cells in the perivascular niche reduce the adipogenic
potential of adipose stromal cells by up-regulating inhibitors of
adipogenesis.13 In the hematopoietic system, adipocytes inhibit
lineage-specific differentiation14 and engraftment of transplanted
cells.15 These observations suggest that different cells of the
hematopoietic niche mediate both positive and negative effects on
stem and progenitor cells.
Notch signaling is crucial for HSC formation during embryonic
development16 and is critical for HSC maintenance.17 Notch
signaling regulates differentiation and maintenance of HSCs, and
Notch1 activation promotes stem cell self-renewal.18 Calvi19 and
Weber et al20 demonstrated the role of the endosteal niche in
maintaining HSC self-renewal through the activation of Notch
receptors on HSCs by Jagged1 expressed by OBs. However, the
role of Notch signaling in HSC homeostasis has been questioned21,22 because impeding key signaling molecules was ineffective in immediately decreasing HSC numbers or suppressing
hematopoiesis.
At present, we do not know precisely how different cellular
elements of the hematopoietic niche collaborate to promote HSC
self-renewal and to maintain the stem cell pool. Similarly, the
interplay between different cell types of the hematopoietic niche
that promotes or impedes self-renewing signaling pathways is also
not well understood. Herein, we investigated the potential role of
OBs and SCs singularly or together on the in vitro and in vivo HSC
and HPC function. Collectively, our data provide strong evidence
Submitted September 29, 2009; accepted January 23, 2010. Prepublished
online as Blood First Edition paper, February 12, 2010; DOI 10.1182/blood2009-09-246173.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
3239
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3240
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
CHITTETI et al
that OBs and SCs play opposing roles in maintaining and expanding hematopoietic function and illustrate that these activities are
mediated by the regulation of Notch signaling between OBs and
hematopoietic cells.
Methods
Animals
Adult B6.SJL-Pt␥cqPep3b/BoyJ (BoyJ) mice (6- to 8-week-old), C57BL/6
mice (2-day pups and 6- to 8-week-old), C57BL/6 ⫻ BoyJ F1 mice (6- to
8-week-old) were used. Mice were bred and housed in the animal facility at
Indiana University. For transplantation, recipient mice received 1100 cGy
ionizing radiation from a cesium source (700 and 400 cGy split dose). Cells
were infused via the tail vein. All procedures were approved by the
Laboratory Animal Research Facility of the Indiana University School of
Medicine and followed National Institutes of Health guidelines.
Preparation of OBs
Two-day calvariae OBs. Calvarial OBs were prepared after a modification
of published methods.23,24 Calvariae from C57BL/6 mice less than 48 hours
old were dissected, pretreated with ethylenediaminetetraacetic acid in
phosphate-buffered saline (PBS) for 30 minutes, and then subjected to
sequential collagenase digestions (200 U/mL). Fractions 3 to 5 (collected
between 45-60, 60-75, and 75-90 minutes through the digestion) were
collected and used as OBs. These cells are more than 95% OBs or OB
precursors as previously demonstrated.24 Freshly prepared OBs were used
for all studies.
Two-day long bone OBs. Neonatal long bones (tibiae and femurs)
were dissected from C57BL/6 mice less than 48 hours old. Bones were cut
into less than 1-mm segments, washed twice with PBS, and then subjected
to 2 consecutive collagenase digestions (30 minutes and 1 hour). Cells were
collected from both cycles, pooled, and used as described.
Six- to 8-week long bone and calvariae OBs. For the long bones, the
epiphyses were removed and saved. After flushing bone marrow (BM) cells
in PBS, diaphyses and epiphyses were combined, cut into less than 1-mm
segments, and washed twice with PBS. Calvariae were washed twice with
PBS, and less than 1-mm segments were prepared. Bone segments were
subjected to 2 consecutive collagenase digestions (30 minutes and 1 hour).
Cells were collected after each cycle, pooled, and used as described.
Preparation of SCs
SCs were prepared in “Dexter” cultures as described.25,26 BM cells were
flushed from femurs and tibias of C57BL/6 mice, and low-density cells
were obtained by Ficoll centrifugation (GE Healthcare). Cells were cultured
in Iscove modified Dulbecco medium supplemented with 10% fetal calf
serum, 1% penicillin/streptomycin, and 1% L-glutamine, 0.2mM ␤-mercaptoethanol, and 0.2␮M methylprednisolone for 4 to 5 weeks until a typical
SC monolayer was formed.
Cell staining and flow cytometry
Cells were washed once with stain wash (PBS, 1% bovine calf serum, and
1% penicillin-streptomycin) followed by antibody staining for 15 minutes
on ice. Cells were washed with cold stain wash after each step.
LSK cell sorting and phenotyping. Low-density BM cells from C57BL/6
(CD45.2) or BoyJ (CD45.1) mice were stained with phycoerythrin (PE)–
conjugated CD3, CD4, CD45R, Ter119, and Gr1; allophycocyanin-conjugated
c-Kit (CD117); fluorescein isothiocyanate-conjugated Sca1 (BD Biosciences).
Lineage⫺Sca-1⫹CD117⫹ (LSK) cells were sorted on BD FACSAria (BD Biosciences). Cells harvested from cocultures were stained with the aforementioned
antibody combinations along with Pacific blue-conjugated CD45.1 and PE-Cy7–
conjugated CD45.2. CD45.1⫹ cells were gated and analyzed for the presence of
Lin⫺Sca1⫹ cells on a BD LSRII (BD Biosciences). Because cultured cells
quickly lose the expression of c-Kit, they were not analyzed for CD117. This is
why persistence of primitive cells in culture was assessed via the presence of
Lin⫺Sca1⫹ cells only.
OB phenotyping and sorting. OBs from 4 different sources, calvariae,
and long bones of 2-day and 6- to 8-week-old mice were stained with
allophycocyanin-conjugated CD45, CD31, and Ter119; PE-Cy7–conjugated
Sca1; PE–conjugated ALCAM (eBiosciences); and purified osteopontin
(OPN; Rockland) followed by AlexaFluor 488–conjugated species and
subclass specific secondary antibody (Invitrogen).
Progenitor cell assay
Cells were plated in duplicate in 3-cm Petri dishes containing 1 mL of
methylcellulose with cytokines (MethoCult GF M3434; StemCell Technologies). Cultures were maintained at 37°C in humidified incubator at 5% CO2,
and colonies were counted on an inverted microscope after 7 days.
Quantitative real-time RT-PCR
cDNA was made on ␮MACS columns (Miltenyi Biotec) using the ␮MACS
one-step cDNA kit (Miltenyi Biotec, catalog no. 130-091-902) following
the manufacturer’s instructions. Quantitative PCR was performed in an
MX3000 detection system using SYBR Green PCR reagents following the
manufacturer’s instructions (Stratagene). PCR amplification was performed
in a 25-␮L final volume containing 12.5 ␮L of 2⫻ SYBR Mastermix,
2.5 ␮L of ROX (300nM, reference dye), 2.5 ␮L of each primer (at desired
concentration), and 5 ␮L of template (cDNA diluted) using 95°C for
10 minutes, followed by 40 cycles at 95°C for 30 seconds and 55°C for
1 minute and 72°C for 30 seconds. For each gene analyzed, a calibration
curve was performed and all the oligonucleotides were tested to ensure
specificity and to determine the optimum concentration. For each sample,
arbitrary units obtained using the standard curve and the expression of
glyceraldehyde-3-phosphate dehydrogenase was used to normalize the
amount of the investigated transcript.
ALP activity
Alkaline phosphatase (ALP) activity was determined by the colorimetric
conversion of p-nitrophenol phosphate to p-nitrophenol (Sigma-Aldrich)
and normalized to total protein (bicinchoninic acid, Pierce Chemical).23
Cells were washed twice with PBS, lysed with 0.1% (vol/vol) Triton X-100
supplemented with a cocktail of broad-range protease inhibitors (Pierce
Chemical), frozen and thawed twice, and cleared via centrifugation. Lysates
were incubated with 3 mg/mL p-nitrophenol phosphate in an alkaline buffer
(pH 8.0) for 30 minutes at 37°C. The reaction was stopped by the addition
of 20mM NaOH and read at 405nM (GENios Plus; Tecan). ALP activity
was determined by comparison with known p-nitrophenol standards.
Quantitative analysis of Ca deposition
Ca deposition was assessed by eluting Alizarin Red S from cell monolayers.23 Monolayers were washed twice with PBS, fixed in ice cold 70%
(vol/vol) ethanol for 1 hour, and then washed 2 times with water.
Monolayers were stained with 40mM Alizarin Red S (pH 4.2) for
10 minutes. Unbound dye was removed by washing with water (5 times)
and PBS (1 time for 15 minutes). Bound Alizarin Red was eluted by
incubating monolayers with 1% (vol/vol) cetylpyridinium chloride in
10mM sodium phosphate (pH 7.0) for 15 minutes. Absorbance from
aliquots was measured at 562 nm (GENios Plus; Tecan), and Alizarin Red
concentrations were calculated from measured standards (Ca/mol of dye in
solution).
Statistical analysis
At least 3 individual experiments were performed unless stated otherwise.
Where applicable, data are presented as mean plus or minus SD and were
analyzed using a 2-tailed Student t test. Differences were considered
statistically significant with a P value of less than .05. All statistical
analyses were performed with the Excel 2003 program (Microsoft). Where
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
applicable (Figure 7C-D), Pearson correlation coefficients (bivariate correlation) were used to determine R2 values. Linear regressions using analysis
of variance model were performed to compare groups. All analyses
involving these methods were performed with the Statistical Package for
Social Sciences (SPSS 17; Norusis/SPSS Inc) software and were 2-tailed
with a level of significance set at .05.
IMPACT OF NICHE COMPONENTS ON HEMATOPOIESIS
3241
(supplemental Figure 2). Different proliferation kinetics were
observed for fresh OBs cocultured with SCs that had been in
culture for 4 to 5 weeks. However, neither cell type was eliminated
from the coculture after 7 or 10 days (supplemental Figure 3),
illustrating that neither cell type was totally purged by the other.
Hematopoietic properties of LSK cells cultured with OBs
and SCs
Results
Kinetics of cell growth in cocultures of OBs and SCs
OBs were freshly isolated from calvariae (C) or long bones (LB) of
2-day-old pups or 6- to 8-week-old C57BL/6 mice.23,24 All OB
preparations were used either directly or sorted then used in
cocultures within 6 to 8 hours of their isolation. SCs were prepared
from murine low-density BM cells in “Dexter” cultures25 and
maintained for 4 to 5 weeks until a typical SC monolayer was
formed. SCs were then harvested and used in cocultures as
described. Phenotypic characterization of SCs maintained in culture for 4 to 5 weeks is shown in supplemental Figure 1 (available
on the Blood Web site; see the Supplemental Materials link at the
top of the online article). Invariably, SC cultures contained a
substantial number of adipocytes as evidenced by Oil Red O
staining (supplemental Figure 2). In general, adipocytes constituted
between 12% and 17% of all cells present in SC cultures at week 4
Figure 1. Impact of OB, SC, and OB ⴙ SC cocultures on stem and
progenitor cell function. (A) Cells were cultured in different combinations
as indicated, and all wells received recombinant murine stem cell factor
and interleukin-3 (10 ng/mL), insulin-like growth factor 1 and thrombopoietin (20 ng/mL), interleukin-6 and Fms-like tyrosine kinase 3 (25 ng/mL),
and OPN (50 ng/mL). Cells were harvested on day 7 and counted. Fold
increase in total cell number from the original 1000 LSK cells was
calculated relative to day 0; n ⫽ 6 to 8 independent experiments. (B) LSK
progeny cells harvested on day 7 were plated in methylcellulose-based
clonogenic assays, and colony formation was assessed 7 days later. CFU
fold increase was calculated relative to that obtained from 250 freshly
isolated LSK cells assayed on day 0; n ⫽ 4 or 5 independent experiments.
(C) LSK progeny harvested on day 7 were stained and analyzed for the
Lin⫺Sca1⫹ content; n ⫽ 3 independent experiments. (D) Lin⫺Sca1⫹ cells
were sorted from each group and analyzed for cell-cycle status with
propidium iodide; n ⫽ 5 independent experiments. (E) BM repopulating
potential of freshly isolated and in vitro expanded LSK cells for 10 days in
cocultures of OBs, SCs, or OBs ⫹ SCs or on plastic. LSK cells from
C57Bl/6 (CD45.2) mice were cotransplanted with 100 000 BoyJ (CD45.1)
competitor cells in lethally irradiated (1100 cGy, split dose) CD45.2 ⫻
CD45.1 F1 recipients. Control mice (Fresh) received 1000 freshly isolated
LSK cells and 100 000 competitor cells. At monthly intervals, chimerism
was assessed as [CD45.2/(CD45.2 ⫹ CD45.1)] ⫻ 100, thus eliminating
the contribution of residual host-derived HSCs. Data are from 1 experiment, 4 or 5 mice per group, except for the LSK cells cultured on plastic
where only 1 mouse survived. (F) Secondary transplantations from
primary recipients. At 4 months after primary transplantation, the BM
content of 1 femur from each primary recipient was transplanted into a
lethally irradiated secondary recipient without competitor cells, and engraftment was assessed at monthly intervals. Each group contained 4 mice,
except the LSK cells group, which had 2 mice transplanted with cells from
a single primary recipient. *Significant at P ⬍ .01 compared with OB ⫹ LSK
group for panels A, B, C, and D and at P ⬍ .05 for panels E and F.
Differences between fresh and OB ⫹ LSK groups for primary and secondary transplantations were not significant.
Cocultures of OBs and SCs from C57BL/6 mice were initiated
24 hours before seeding at time 0 (day 0) with 1000 freshly sorted
B6.SJL-Pt␥cqPep3b/BoyJ (BoyJ)–derived LSK cells per well.
Cultures were supplemented with exogenous cytokines as detailed
in Figure 1. After 7 days, fold increase in total cell number
generated from LSK cells was significantly higher in OB ⫹ LSK
cocultures (1353.4 ⫾ 236.5-fold) compared with SC ⫹ LSK cocultures (474.8 ⫾ 129.8-fold; Figure 1A). When all 3 cell types were
cocultured, the presence of SCs significantly suppressed the
OB-mediated LSK cell expansion (463.8 ⫾ 168.3-fold; equivalent
to 75.6% ⫾ 7.3% suppression). A significant suppressive effect
was still evident when the total cell number of OBs and SCs was
reduced by 50% to minimize crowding (data not shown).
Progeny of LSK cells were assayed on day 7 for their
colony-forming unit (CFU) content and corrected for the total
number contained in each culture (Figure 1B). Although the
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3242
CHITTETI et al
number of CFU increased in OB cultures by more than 80.7 plus or
minus 13.0-fold, CFU expansion was significantly lower in cultures supported by SCs (30.1 ⫾ 7.3-fold) and in cultures of LSK
only (24.5 ⫾ 5.6-fold). More importantly, the OB-mediated enhanced HPC function was significantly suppressed in the presence
of SCs (Figure 1B). SC conditioned medium did not suppress
OB-mediated enhancement of hematopoietic properties (supplemental Figure 4), illustrating the need for cell-cell contact for the
transmission of the suppressive effect of SCs on OB-mediated
enhancement of hematopoiesis.
Cells harvested on day 7 were analyzed for the expression of
Sca1 and lineage markers (Figure 1C) and for cell-cycle status
(Figure 1D). Because the expression of CD117 is quickly downregulated via the internalization of the receptor in cultures supplemented with exogenous stem cell factor,27,28 we did not use CD117
to track the phenotypic makeup of cultured cells on day 7.
A significantly higher percentage of Lin⫺Sca1⫹ cells
(34.5% ⫾ 8.3%) was present in OB ⫹ LSK cultures relative to
SC ⫹ LSK and OB ⫹ SC ⫹ LSK cultures, suggesting that maintenance of Lin⫺Sca1⫹ cells may be responsible for the fold increase
in CFU in these cultures (Figure 1B). The significant increase in
LSK progeny in OB ⫹ LSK cultures on day 7 (Figure 1A)
probably resulted from the low percentage (75%) of Lin⫺Sca1⫹
cells in the G0/G1 phase of cell cycle (Figure 1D). It should be noted
that analysis of apoptosis (with annexin V staining) of cells in
cultures shown in Figure 1C and D (and in other culture conditions;
Figures 2,4,5,7) showed a very small percentage of apoptotic cells
and no significant differences between cells in any culture condition (data not shown). Taken together, a higher number of LSK
progeny (in the absence of changes in apoptosis), increased number
of clonogenic cells, higher percentage of Lin⫺Sca1⫹ cells, and
decreased percentage of cells in G0/G1 in OB ⫹ LSK cultures
suggest that OBs promote cell proliferation and expansion while
maintaining primitive hematopoietic cell properties.
These results suggested that LSK progeny may retain a high
level of BM repopulating potential. To examine this, the expansion
equivalent of 1000 LSK cells (CD45.2) maintained under different
culture conditions for 10 days was used in a competitive repopulation assay. Figure 1E illustrates that LSK cells maintained in OB
cultures retained a high level of repopulating activity relative to
freshly isolated cells (P ⬎ .05 at all time points). In contrast, LSK
cells maintained with SCs only failed to repopulate lethally
irradiated recipients. Furthermore, SCs significantly suppressed the
OB-mediated maintenance of LSK function when all 3 cell types
were cocultured together (Figure 1E). Only 1 mouse survived in the
group of LSK cells maintained on plastic, probably resulting from
the small number of competitor cells used in these studies. As
expected, cultures not seeded with LSK cells did not sustain
chimerism (Figure 1E). BM repopulating ability of LSK cells
maintained in OB cultures was also evident in secondary recipients
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
that received BM cells from primary recipients 4 months after
transplantation (Figure 1F). Levels of chimerism in secondary
recipients of cells maintained with OBs were significantly higher
than those in mice receiving cells cocultured with SCs under any
condition (each secondary recipient received the cell content of
1 femur from a primary recipient). Results from secondary
transplantations illustrate the ability of OBs to maintain HSC
function and demonstrate the suppressive effect of SCs on the
OB-mediated hematopoiesis-enhancing activity.
Mechanism of hematopoiesis-enhancing activity in
OB cocultures
We investigated whether observations made in cocultures of OBs
and HPCs were mediated by the HSC receptor Notch and its
OB-expressed ligand Jagged. To that effect, cocultures of OB, SC,
and LSK cells were treated with ␥-secretase inhibitor (GSI). In
3 independent experiments, the addition of GSI suppressed both
proliferation of LSK cells and their progeny (Figure 2A) and
generation of CFU (Figure 2B), especially in cocultures of
OB ⫹ LSK and OB ⫹ SC ⫹ LSK, suggesting that the hematopoiesis-enhancing activity of OBs is mediated in part through Notch
signaling. Cocultures of SCs and LSK, where Notch signaling is
not expected to play a major role in sustaining hematopoietic cells,
were not impacted by GSI. Interestingly, autonomous Notch
signaling in LSK cells29 was also inhibited by GSI.
To verify the involvement of Notch in mediating the effects
seen in Figure 2, we monitored Notch activation in LSK cells
cocultured with OBs or SCs and the potential role of intrinsic
Notch signaling in regulating OBs. First, we examined the expression of Notch receptors, ligands, and downstream Notch targets in
LSK, OBs, and SCs. OBs and LSK expressed high levels of N1,
whereas SCs expressed N1 at relatively low levels (Figure 3A).
Both Notch ligands J1 and J2 were highly expressed on OBs
compared with LSK and SCs. Coexpression of Notch receptors and
their ligands, in various combinations, is found in all cell types in
the BM microenvironment30 and accounts for basal Notch signaling through homotypic cell-cell interactions. Analysis of Notch
signaling activation by expression of downstream transcriptional
targets Hes1 and Deltex revealed a basal activation of Notch
signaling in all cell types, with the strongest signaling in OBs
(Figure 3A).
Next, we determined whether basal activation of Notch on LSK
cells was modulated by coculture with OBs or SCs. As is evident by
the expression of Notch target genes Hes1 and Deltex, Notch
signaling was up-regulated in LSK cells when cocultured with OBs
and was significantly inhibited in the presence of SCs (Figure 3B).
The presence of SCs in LSK ⫹ OB cocultures resulted in a
dominant inhibitory effect on Notch, suggesting that SCs suppress
the OB-mediated regulation and maintenance of HSC function.
Figure 2. Impact of Notch signaling inhibition on OB-mediated
enhancement of HPC function. Increase in total cell number from the
original 1000 LSK cells (A) and production of CFU (B) in cocultures of OB,
SC, and LSK cells with and without the Notch inhibitor, GSI. Cell numbers
and CFU content were assayed, and fold increase was calculated to day
0 values. Data shown are from 1 of 3 independent experiments with similar
results. GSI was added at 10nM on day 0 and replenished twice during the
next 7-day culture period. #P ⬍ .01, comparisons within each group
between treatments with and without GSI. ⫹P ⬍ .01 compared with
OB ⫹ LSK group without GSI. *P ⬍ .05 compared with OB ⫹ LSK
with GSI.
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
IMPACT OF NICHE COMPONENTS ON HEMATOPOIESIS
3243
Figure 3. Notch activation in OB, SC, and LSK cocultures. (A) Endogenous expression of components of the Notch pathway. Quantitative RT-PCR was performed on cDNA
generated from mRNA derived from sorted LSK cells, cultured SCs, and freshly isolated 2-day C OBs (performed in triplicate for each sample). Bar graphs represent ratio of
each specific transcript to glyceraldehyde-3-phosphate dehydrogenase. Expression of each transcript in OBs and SCs was expressed as fold change relative to the expression
of that transcript in LSK cells, which was normalized to 1. (B) Quantitative RT-PCR data from cells isolated from cocultures. Cocultures were established with SCs from C57Bl/6
GFP mice (CD45.2), OBs (2-day C) from C57Bl/6 mice (CD45.2), and LSK cells from BoyJ mice (CD45.1). On day 7, cells were harvested and stained with PE-CD45.1. mRNA
was prepared from GFP-CD45.1⫹ cells (LSK progeny only) and analyzed. Bar graphs show fold increase in the expression of the indicated genes in LSK cultured alone
(normalized to 1 in cultures without GSI) or with other cell types shown for each condition in parentheses. Quantitative RT-PCR was performed in triplicates for each sample and
each condition. Data are representative of 2 independent experiments with similar results. GSI was added at 10nM on day 0 and replenished twice during the next 7-day culture
period. The legend shown in the plot of Deltex in panel B applies to all other plots in the figure (N1, N2, J1, J2, and Hes1).
Expression of Notch receptors and ligands mirrored the pattern of
Notch activation on LSK cells: they were up-regulated by OBs and
dominantly inhibited by SCs. Addition of GSI abrogated the
stimulatory effect of OBs on hematopoietic cells but did not rescue
the suppressive effect of SCs, thus excluding the possibility of a
negative feedback loop triggered by Notch signaling itself. We also
investigated whether LSK cells had a reciprocal effect on OBs, thus
altering their intrinsic Notch activity. PCR analysis of sorted OBs
cocultured with LSK cells showed a decrease in the expression of
N1, N2, J1, and Hes1 and a significant up-regulation of J2 (data not
shown). Overall, activation of Notch signaling in LSK cells highly
correlated with their functional activity in colony assays and BM
transplantation (Figure 1), suggesting a major role of Notch
signaling in HSC maintenance.
Impact of different cell types within SCs on in vitro
hematopoietic function
Recently, it was shown that BM adipocytes suppress granulopoiesis14 and may prevent hematopoietic expansion in homeostasis
and after transplantation.15 Because SCs contain various numbers
of adipocytes, we examined the impact of an established mesenchymal stromal cell line, GZL,31 with various numbers of adipocytes
on the in vitro maintenance of hematopoietic cells. GZL cells with
a higher content of adipocytes (GZL/Adi) were less efficient than
their counterparts with normal adipocyte content (GZL) in supporting both LSK proliferation and CFU production (Figure 4A). That
GZL/Adi contained a higher proportion of adipocytes compared
with GZL was confirmed by the expression of adiponectin and
FABP4 (Figure 4B). Belaid-Choucair et al14 recently demonstrated
that BM adipocytes block granulopoiesis through neuropilin-1, a
coreceptor to a tyrosine kinase receptor. We assessed the expression
of this receptor on both variants of GZL cells used in this assay
relative to primary SCs. Neuropilin-1 was up-regulated on GZL/
Adi relative to GZL cells and SCs (Figure 4C). These data suggest
that the suppressive effect of SCs on the hematopoiesis-enhancing
activity of OBs is partially mediated by adipocytes within SCs,
possibly via the production of neuropilin-1. Given that adipocytes
are a critical component of the hematopoietic microenvironment
and that the BM content of adipocytes increases with age (concomitant with a decline in hematopoietic activity), these data suggest
that adipocytes may play a significant role in down modulating
HSC function.
Phenotypic makeup of OBs with hematopoiesis-enhancing
activity
OBs used in our studies were not phenotypically defined. We
therefore attempted to prospectively identify OB flow cytometrically, arguing that this will not only identify OBs capable of
enhancing HSC function but will also begin to establish a model for
the osteoblastic developmental hierarchy. We first examined the
expression of ALCAM (CD166) on 2-day calvarial OBs, which
was recently identified by Arai et al32 as a marker capable of
distinguishing mature and immature OBs from long bones of adult
mice when combined with Sca1 and a CD45, CD31, and Ter119
Figure 4. Impact of adipocytes on the maintenance of HPCs in vitro. Without frequent media changes and at high cell density, GZL, an established MSC cell line,
differentiates preferentially into adipocytes (GZL/Adi). The number of adipocytes is greatly reduced when the cells are propagated under more favorable conditions (GZL). Both
GZL and GZL/Adi were used along with primary SCs to sustain LSK cells for 7 days. (A) Cells were harvested on day 7, counted, and used in clonogenic assays. (B) SC, GZL,
and GZL/Adi were assayed by quantitative RT-PCR for the expression of adiponectin and FABP4 as indicators of adipogenic differentiation. Data were normalized to primary
SCs. (C) SCs, GZL, and GZL/Adi were separated from LSK progeny on day 7 by cell sorting and assayed by quantitative RT-PCR for the expression of neuropilin-1. Expression
of Np1 was normalized to primary SCs. P ⬍ .05 between SC and GZL/Adi groups in panel A.
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3244
CHITTETI et al
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
Figure 5. Effect of phenotypically defined groups of
2-day C OBs on HPC function. (A) Two-day C OBs
were stained as described in “Methods.” Gated
CD45⫺CD31⫺Ter119⫺ cells were separated into
Sca1⫺ALCAM⫹ and Sca1⫺ALCAM⫺ cells, which were
used in assays shown in panel B. (B) Increase in cell
number from the original LSK cells and production of
clonogenic progenitors in the presence of phenotypically
defined groups of OBs. ⫹P ⬍ .01 compared with fold
increase in cell number in calvarial OB cultures. *P ⬍ .01
compared with fold increase in CFU in calvarial OB
cultures.
“lineage” cocktail. These markers defined Lin⫺Sca1⫺ALCAM⫺
cells as immature OBs and Lin⫺Sca1⫺ALCAM⫹ cells as mature
OBs.32 Using this approach, we isolated 2 distinct phenotypes of
OBs from 2-day calvariae (Figure 5A) and examined their hematopoiesis-enhancing activities (Figure 5B). Interestingly, the hematopoiesis-enhancing activity could not be segregated into either
Lin⫺Sca1⫺ALCAM⫹ or Lin⫺Sca1⫺ALCAM⫺ cells, suggesting
that these markers are not sufficient to fully define OBs or to
segregate the observed hematopoiesis-enhancing activity.
We next examined OPN, which has been previously used to
identify OBs and characterize their function.3 Because OB function
varies considerably with age and anatomic location,33 we examined
OBs from 2 distinct anatomic sites, namely, the calvariae (C) and
LB and from 2 different ages; either newborn mice (2-day-old) or
adult (6- to 8-week-old mice). All 4 sources of OBs (2-day C, 2-day
LB, 6- to 8-week C, 6- to 8-week LB) contained cells that lacked
the expression of the lineage markers CD45/CD31/Ter119 and
Sca1 (Lin⫺Sca1⫺; Figure 6). Arai et al32 defined Lin⫺Sca1⫹ cells in
bone preparations as mesenchymal progenitors. We therefore
focused on Lin⫺Sca1⫺ cells (Figure 6) as candidate OBs. Lin⫺Sca1⫺
cells were analyzed for OPN and ALCAM expression to identify
11 distinct groups of cells from the 4 sources of OBs as shown in
sort windows 1 through 11 in the lower row of dot plots in Figure 6.
Lin⫺Sca1⫺ cells decreased from 2-day C to 6- to 8-week LB,
suggesting that the percentage of total OBs decreased with age and
anatomic site. All 11 groups of OBs and the 4 parent populations
were examined for 2 fundamental functional osteoblastic properties34: Ca deposition (mineralization) and ALP activity (Figure 7A).
ALP is the major OB enzymatic activity and Ca deposition is a
surrogate marker for mineralization.34 The highest amount of Ca
deposition was among 2-day C OB and the lowest was in 6- to
8-week C, although these cells displayed similar activities as those
observed with 6- to 8-week LB, suggesting that Ca deposition
Figure 6. Phenotypic analysis of OBs from calvariae
(C) or long bones (LB) of newborn (2-day) and 6- to
8-week-old mice. OBs were stained as described in “Cell
staining and flow cytometry.” Cells were analyzed for
Sca1 versus CD45/CD31/Ter119 (top row), and doublenegative cells were then analyzed for OPN versus ALCAM (bottom row). Different numbers of events were
collected and are displayed for each file. Sort gates
defining groups 1 through 11 were established based on
fluorescence levels of control samples. Cells in these
gates were sorted and used in different assays depending on the number of cells recovered for each fraction.
Dot plots are from 1 representative experiment of 3.
diminished with location and age. ALP activity was lowest in 2-day
LB with very high activity in 6- to 8-week LB. The importance of
ALP⫹ SCs in supporting ex vivo and in vivo hematopoiesis was
recognized many years ago.35
Most of these OB fractions and their parent populations were
also examined for their hematopoiesis-enhancing activities (Figure
7B). All sources of OBs, except the 2-day LB, supported hematopoietic properties beyond those observed for LSK cells cultured on
plastic. Data in Figure 7B illustrate that (1) OPN, as previously
observed for ALCAM (Figure 5), does not fractionate the OBenhancing activity of HSC function; hematopoiesis-enhancing
activities were detected for example in OPN⫺ (groups 1 and 4) and
OPN⫹ (groups 3 and 6) cells; (2) even together, OPN and ALCAM
fail to completely segregate the hematopoiesis-enhancing activity
into 1 group of cells; activity was detected in OPN⫹ALCAM⫹
(group 2) and OPN⫹ALCAM⫺ (group 3) cells; and (3) Ca and ALP
activities were significantly correlated with anatomic location and
age. As illustrated in Figure 7C and D, a significant association was
observed between the source of OB and both Ca deposition
(P ⬍ .001, R2 ⫽ 0.746) and ALP activity (P ⫽ .016, R2 ⫽ 0.486).
Surprisingly, however, CFU content was not significantly correlated with either one of these primary OB functional properties
regardless of whether the analysis focused on anatomic location
and age or on the subfractions of these sources of OBs. This
observation illustrates that the OB-mediated enhancement of
hematopoiesis, which was investigated here predominantly with
2-day C OBs, is not a unique characteristic of these OBs, but
instead, is a functional property of OBs from other anatomic sites.
Furthermore, although our data in Figure 7B examined the impact
of OBs on progenitor and not stem cell function, these data suggest
that the OB-mediated hematopoiesis supportive activity is not age
dependent. Consequently, these data most probably describe an
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
IMPACT OF NICHE COMPONENTS ON HEMATOPOIESIS
3245
Figure 7. Osteogenic and hematopoietic functional studies of
isolated fractions of OBs. (A) Ca deposition and enzymatic ALP
activity of parental populations and groups 1 through 11 shown in
Figure 6. Cells were cultured in osteogenic media (␣minimal essential
medium supplemented with 10% fetal calf serum, 50 ␮g/mL ascorbic
acid, 2 times per week). Starting on day 7, cultures were supplemented with 5mM ␤-glycerophosphate to induce mineralization and
were assayed on day 14. ALP activity and Ca deposition were
assessed as described in “ALP activity” and “Quantitative analysis of
Ca deposition.” With the exception of the unsorted 2-day C and 2-day
LB data (n ⫽ 3 in duplicate), results are from 2 experiments each
performed in duplicate. (B) Parental populations and cell fractions
collected in sufficient numbers were used in cocultures with LSK cells.
Cultured cells were harvested on day 7, counted, and assayed for
HPC content performed in triplicate. Data shown were collected from
2 sorting experiments. (C-D) Results from Ca deposition (C) and
enzymatic ALP activity (D) from 2-day C, 2-day LB, 6- to 8-week C,
and 6- to 8-week LB (n ⫽ 6-8 for all groups) are reported as box plots
depicting the range of data points and the mean (line). Error bars
represent SD associated with the mean. Data points more than 2 SD
from the mean are identified on the plot and were still used in statistical
determinations. Statistical analysis methods used to analyze data
shown in panels C and D are described in “Statistical analysis.”
OB-mediated activity that is representative of the impact of OBs
from different anatomic sites and different ages on HSC function.
Discussion
Self-renewal and differentiation divisions of HSCs are tightly
regulated to achieve a balance between homeostasis and maintenance of the stem cell pool. This balance is regulated through a
complex signaling network involving a large number of soluble
factors and multiple cell-cell interactions.36 Interactions between
HSCs and OBs37 and between HSCs and SCs38 are very prominent
in determining HSC fate. Based on these interactions, the hematopoietic microenvironment is considered to be composed of
2 specialized niches, the endosteal and the vascular niche.38 At
present, it is thought that primitive long-term repopulating HSCs
associate with cells of the OB lineage that line bone surfaces,
whereas more mature hematopoietic cells (such as short-term
HSCs and HPCs) associate more with endothelial (and other) cells
in the vascular niche.7,39-42 We therefore sought to examine the
impact of single or multiple interactions between HSCs, OBs, and
SCs on hematopoietic function. Our results demonstrate that,
although OBs mediate a positive regulatory effect on stem cell
function, most probably through the up-regulation of Notch
signaling, SCs suppress the OB-mediated hematopoiesis-enhancing activity via the down-regulation of Notch signaling, possibly
through mechanisms involving adipocytes.
In the presence of OBs, LSK cells had a higher rate of
proliferation, produced more CFU, and maintained a higher
percentage of Lin⫺Sca1⫹ cells than when cocultured with SCs
alone or a mixture of OBs and SCs. Although the OB-enhancing
activity was not significantly higher for some datasets than what
was observed with LSK cells cultured alone (Figure 1), it is critical
to point out that the novel observation of our studies is
the significant suppressive effect of SCs on the OB-mediated
hematopoiesis-enhancing activity, which was demonstrated in all
of our studies presented here. Furthermore, it should be noted that
interexperimental variability probably contributed to lack of significance and that, when normalized, significant differences were
observed between datasets, including, for example, the fold
increase in cell number between OB ⫹ LSK and LSK cultured on
plastic (data not shown). In particular, a significantly higher
repopulating potential was noted in primary and secondary recipients of LSK cells expanded with OBs only compared with cells
cultured with SCs or in an OB ⫹ SC coculture. At 1 month after
transplantation, chimerism in primary recipients of LSK cells
cultured with OBs only was higher than that observed for fresh
LSK cells, suggesting that OBs may have preferentially expanded
the number of short-term repopulating cells. These results may
have a profound impact on the design of culture conditions
intended for the expansion of short- and long-term repopulating
cells. These data suggest that, although SCs support hematopoiesis
as previously reported by several laboratories,43-46 the hematopoiesis-enhancing activity mediated by OBs is superior. Furthermore,
our data demonstrate that the hematopoiesis-enhancing activity of
OBs is suppressed by SCs, even when these cells are present in
small numbers relative to the number of cocultured OBs.
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3246
CHITTETI et al
The Notch pathway is a highly conserved signaling cascade
present in all vertebrates studied so far.30 It plays a crucial role in
determining cell fate decisions and HSC self-renewal. The Notchspecific inhibitor GSI significantly suppressed the OB-mediated
hematopoiesis-enhancing activity, illustrating that Notch signaling
is critical for the support of HSC function via their direct
interaction with OBs. Because GSI did not affect the behavior of
LSK cultured with SCs, Notch signaling is probably not involved
in the interaction of these 2 cell types. That Notch signaling is
central to the OB-induced support of HSCs was further demonstrated by the expression of different ligands, receptors, and
intermediate molecules involved in this pathway. Endogenous
expression of these moieties was highest in OBs. When cocultured
for 7 days and then separated by flow cytometric cell sorting, LSK
cells cultured in the presence of OBs demonstrated significant
up-regulation of Notch signaling components compared with their
counterparts cultured with SCs. The relative expression of these
molecules was substantially lower in LSK cells cocultured with
OBs and SCs, simultaneously demonstrating the presence of a
correlative relationship between Notch signaling and the hematopoiesis-enhancing versus hematopoiesis-suppressing activities of OBs
and SCs, respectively. The specificity of these observations vis-àvis the involvement of Notch signaling in our results was corroborated by the fact that GSI significantly interfered with the expression of all these intermediate molecules.
Further investigation revealed that adipocytes are most
probably responsible for the suppressive effect of SCs on HPC
function. This speculation is in agreement with the recent data
from Naveiras et al15 demonstrating the negative regulatory role
of adipocytes in the BM microenvironment. It has been previously demonstrated that BM adipocytes can block granulopoiesis via a coreceptor to a tyrosine kinase receptor, neuropilin1.14 In our studies, the expression of neuropilin-1 in GZL/Adi
was much higher than in the parental cell line, GZL. Furthermore, expression of neuropilin-1 in primary SCs used throughout these studies was almost equal to that in GZL/Adi. Together,
these data illustrate that the negative impact of SCs on
hematopoiesis is at least partially the result of adipocytes and
that neuropilin-1 may be responsible for this suppressive
activity. Whether adipocytes also impact Notch signaling to
suppress hematopoiesis remains to be determined.
The developmental hierarchy of OBs is not well defined nor
is it clear whether OBs from different anatomic sites and
chronologic stages share the same phenotypic makeup. We
therefore phenotypically examined OBs from different aged
mice and from different sites to better define the developmental
hierarchy of OBs and to establish which stage of OB maturation
supports this observed hematopoiesis-enhancing activity. Contrary to the classification of Arai et al 32 in which
Lin⫺Sca1⫺ALCAM⫺ cells were defined as immature OBs
whereas Lin⫺Sca1⫺ALCAM⫹ cells were identified as mature
OBs, our data suggest that ALCAM⫺ cells may represent mature
OBs compared with ALCAM⫹ cells. We base this argument on
observations made between groups 1 and 3 versus group 2 in
Figure 7A, for example, in which both Ca deposition and ALP
production were higher in ALCAM⫺ cells (groups 1 and 3) than
in ALCAM⫹ cells (group 2), suggesting that the former is more
mature than the latter. On the other hand, Mayack and Wagers3
identified Lin⫺OPN⫹ cells as OBs. Because ALCAM can be
used to classify OBs based on their maturational stage, it may be
argued that OPN⫹ALCAM⫺ cells are mature OBs, whereas
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
OPN⫹ALCAM⫹ cells are immature OBs. Hence, data shown in
the bottom row in Figure 6 suggest that, although the percentage
of OPN⫹ cells remains relatively constant within the Lin⫺Sca1⫺
group of cells, the percentage of ALCAM⫹ cells (immature
OBs) decreases with age and varies with anatomic location (left
to right in Figure 6).
Proliferation and expansion of HPC in cocultures with
fractionated OB groups indicated that the markers we used so far
are not sufficient to fully segregate the hematopoiesis-enhancing
capacity of OBs into 1 phenotypically defined group (Figure
7B). Similarly, all sorted fractions displayed different levels of
Ca deposition and ALP activity (Figure 7A). However, Ca and
ALP activities were significantly correlated with anatomic
location and age, but neither one was associated with LSK
proliferation and CFU expansion. Obviously, additional markers
are still required to compartmentalize the hematopoiesisenhancing activity of OBs into a phenotypically defined group
of cells. Such studies will also help defining why, for example,
OBs from 2-day long bones have a reduced hematopoiesisenhancing activity and possibly identify more precisely the
maturational stage of OBs responsible for this activity. Many
studies that examined the impact of OBs on hematopoiesis or
made direct observations between HSCs and the endosteal
region, used or focused on calvariae OBs.47 As such, the validity
of using calvariae OBs or interpreting microscopic observations
made in the calvariae as true representatives of HSC-OB
interactions have been questioned.48 Our present studies do not
show any significant correlation between the hematopoiesisenhancing activity of OBs and their anatomic location. In
addition, these data do not support an age-dependent impact on
the ability of OBs to support progenitor cell function. Therefore,
collectively, these results strongly suggest that the calvariae are
a valid choice for the collection of OBs suitable for the type of
investigations presented here.
In these studies, measurement of Ca deposition is a marker
for the mineralization occurring in newly formed osteoid bone
matrix. Ca and phosphate form hydroxyapatite crystals, which is
the mineral content of bone. Therefore, our data are not
inconsistent with those of Adams et al49 in which the authors
suggest that preferential localization of HSCs in the endosteal
niche is probably the result of high Ca concentration given that
HSCs express a Ca-sensing receptor.49 One of the functions of
the skeleton is to provide a reservoir of minerals, including Ca
and phosphate,50 and through bone remodeling, osteoclasts
release these minerals under the control of various stimuli to
maintain homeostasis. Ca that is freed by bone resorption
through osteoclastic activity is bioavailable; however, Ca measured in these studies is not free Ca as described by Adams et
al49 and, as such, is not bioavailable but rather is a component of
the mineral matrix of bone.
Taken together, our data demonstrate that hematopoiesis is
most probably maintained in the hematopoietic niche through
opposing functions of OBs and SCs. Furthermore, the activities
of these cell types on the fate of HSCs appear to be mediated via
Notch signaling and its powerful impact on self-renewal and the
maintenance of the HSC pool. Our data also suggest that the
observed negative activity of SCs on hematopoiesis most
probably involves multiple cellular and soluble mediators,
including adipocytes and neuropilin-1, the role of which requires further investigations.
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
IMPACT OF NICHE COMPONENTS ON HEMATOPOIESIS
Acknowledgments
The authors thank the operators of the Indiana University Melvin
and Bren Simon Cancer Center Flow Cytometry Resource Facility
for their outstanding technical help and support.
This work was supported in part by grant NHLBI HL55716 (E.F.S.).
The Flow Cytometry Research Facility is partially funded by NCI P30
CA082709.
Authorship
Contribution: B.R.C. performed the majority of the experimental work and assisted in writing the manuscript; Y.-H.C.
performed all the experimental work involving preparation and
3247
culturing of osteoblasts; B.P. contributed to the culture and
animal work; S.R.-R. performed all the PCR work involving
Notch signaling; W.S.G. helped in the design of experiments
involving adipocytes; N.C. designed and interpreted the work
focusing on Notch signaling; M.A.K. designed all the work
related to osteoblast preparation and culturing, helped in the
design of other experiments, and assisted in writing the manuscript; and E.F.S. designed the research, interpreted data, and
wrote the manuscript.
Conflict-of-interest disclosure: W.S.G. is a medical director of
General BioTechnology LLC, Indianapolis, IN. The remaining
authors declare no competing financial interests.
Correspondence: Edward F. Srour, Department of Medicine,
Indiana University School of Medicine, 980 W Walnut St, R3C312, Indianapolis, IN 46202; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2010 115: 3239-3248
doi:10.1182/blood-2009-09-246173 originally published
online February 12, 2010
Impact of interactions of cellular components of the bone marrow
microenvironment on hematopoietic stem and progenitor cell function
Brahmananda R. Chitteti, Ying-Hua Cheng, Bradley Poteat, Sonia Rodriguez-Rodriguez, W. Scott
Goebel, Nadia Carlesso, Melissa A. Kacena and Edward F. Srour
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