Lysophosphatidic Acid Mediates Myeloid Differentiation
within the Human Bone Marrow Microenvironment
Denis Evseenko1,2*., Brooke Latour3., Wade Richardson4, Mirko Corselli1, Arineh Sahaghian1,
Sofie Cardinal5, Yuhua Zhu1, Rebecca Chan1, Bruce Dunn2,4, Gay M. Crooks2,3
1University of California Los Angeles (UCLA), Department of Orthopaedic Surgery, Los Angeles, California, United States of America, 2Eli and Edythe Broad Institute for
Regenerative Medicine and Stem Cell Research, Los Angeles, California, United States of America, 3University of California Los Angeles (UCLA), David Geffen School of
Medicine, Department of Pathology and Laboratory Medicine, Los Angeles, California, United States of America, 4University of California Los Angeles (UCLA), Department
of Engineering, Los Angeles, California, United States of America, 5University of California Los Angeles (UCLA), David Geffen School of Medicine, Department of Pathology
and Laboratory Medicine, Los Angeles, California, United States of America
Lysophosphatidic acid (LPA) is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its
diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if
LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human
hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA
receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP) than either multipotent
Hematopoietic Stem/Progenitor Cells (HSPC) or Common Lymphoid Progenitors (CLP) suggesting that LPA acts on
committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation
and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone
marrow sections showed that PPAP2A, (the enzyme which degrades LPA) was highly expressed in the osteoblastic niche but
not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA) was expressed in perivascular regions
of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the
endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow
Citation: Evseenko D, Latour B, Richardson W, Corselli M, Sahaghian A, et al. (2013) Lysophosphatidic Acid Mediates Myeloid Differentiation within the Human
Bone Marrow Microenvironment. PLoS ONE 8(5): e63718. doi:10.1371/journal.pone.0063718
Editor: Spencer B. Gibson, University of Manitoba, Canada
Received September 4, 2012; Accepted April 5, 2013; Published May 16, 2013
Copyright: ? 2013 Evseenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Institutes of Health (NIH) K01AR061415 and by NIH/National Center for Advancing Translational
Science (NCATS) UCLA CTSI Grant Number UL1TR000124 (DE), the California Institute of Regenerative Medicine RB3-05217 (GMC), and from the Broad Stem Cell
Research Center (BSCRC) at UCLA.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Lysophosphatidic acid (LPA) is a phospholipid that mediates a
myriad of biological actions, including cell proliferation, migra-
tion, and survival. LPA species are detectable in biological samples
such as plasma and saliva and are secreted by activated platelets as
a major growth factor in serum . Albumin binds LPA and
protects it from degradation ; thus high levels of LPA in serum
create a challenge when testing the effect of LPA on hematopoiesis
using either in vitro or in vivo assays. Autotaxin (ATX) is the key
LPA producing enzyme in plasma and eukaryotic tissues,
mediating removal of choline from lysophosphatidylcholine .
Cell membrane lipid phosphate phosphatases (PPAP), most
importantly PPAP2A, attenuate the activity of LPA by dephos-
phorylation . The pleiotropic effects described for LPA are in
part due to differential expression patterns of LPA receptors
(LPAR1-LPAR6) within different tissues .
Several studies have demonstrated a role for sphingosine-1
phosphate (S1P), a lipid structurally related to LPA, in increasing
engraftment by augmenting signaling through CXCR4 in
response to stromal derived growth factor-1 (SDF-1) . However,
little is known about the role of LPA signaling during hematopoi-
etic differentiation. A recent study demonstrated LPAR3 is
essential for the induction of erythropoiesis , and another
showed that LPA enhances migration of murine lin-sca-1+ckit+
cells, a population that includes hematopoietic stem cells and early
progenitors . Our goal was to investigate the role of LPA during
lineage commitment of human hematopoietic progenitors.
Materials and Methods
Isolation of Human Progenitor Populations
Umbilical cord blood (CB) was collected from normal deliveries,
according to guidelines approved by the University of California
Los Angeles Investigational Review Board. Enrichment of CD34+
cells was performed using the magnetic-activated cell sorting
system (Miltenyi Biotec, Auburn, CA). For fluorescence-activated
cell-sorting (FACS) sorting, CD34+ enriched cells were incubated
with the following anti-human–specific monoclonal antibodies:
CD34 PerCP-Cy5.5, CD38 PE-Cy7, CD123 (interleukin-3
receptor alpha) PE, CD45RA PE-Cy5, FITC-labeled lineage-
PLOS ONE | www.plosone.org1 May 2013 | Volume 8 | Issue 5 | e63718
specific antibodies: CD2, CD3, CD4, CD8, CD7, CD10, CD11b,
CD14, CD19, CD56, and glycophorin A (Gly A); all from Becton
Dickinson, San Jose, CA). An unstained (no antibody) control was
used to define negative gates. The following, previously published
immunophenotypic definitions were used to isolate myeloid
progenitors from thawed CB CD34+ enriched cells by FACS:
CD34+CD10+lin- CLP  and CD34+CD38-lin- hematopoietic
stem/progenitor cells (HSPC) . Sorting was performed on a
FACSAria (Becton Dickinson) equipped with five lasers (355, 405,
488, 561, and 633 nm). Isolated populations were analyzed by
FACS to assess post sort purity. For all FACS sorted populations
, 95–99% purity was achieved based on re-analysis.
Cocultivation on the murine stromal line OP9 was used
to test for B lymphoid and myeloid differentiation. Freshly
sorted CD34+ cord blood cells (500–1500 cells) were seeded
onto established non-irradiated OP9 stromal cells (American
Type Culture Collection, Manassas, VA) in 96-well or 48-well
flat-bottomed plates. Cells were grown in a modified medium
(DMEM/F12, Invitrogen, Carlsbad, CA) supplemented with 5%
fetal bovine serum (Invitrogen, Carlsbad, CA) treated with
charcoal to remove LPA, 50 mM 2-mercaptoethanol (Sigma-
Aldrich), penicillin/streptomycin (Gemini Bio Products, Calaba-
sas, CA), IL-7 (5 ng/mL, R&D Systems, Minneapolis, MN),
Flt3 ligand (FL, 5 ng/mL, R&D), and thrombopoietin (TPO,
5 ng/mL, R&D). This cytokine combination is permissive for
both lymphoid (B-cells) and myeloid (monocytic, granulocytic
and megakaryocytic) lineages. Every 3 days thereafter, half the
medium was replaced with fresh medium. Lysophosphatidic acid
18:1 Oleoyl-LPA (Tocris Bioscience, MA) was reconstituted in
70% ethanol and added to the fresh culture medium at final
concentrations 0.1, 1 or 10 uM initially to determine optimal
dose response. All subsequent experiments used a concentration
of 1 mM LPA. Cells were cultured for 4 weeks followed by
harvesting, immunostaning with fluorochrome labeled antibodies
and immunophenotypic analysis of cultured cells. Sphingosine-1-
Phosphate and Ki-16425 were purchased from Tocris Biosci-
ence and reconstituted in 4% fatty acid free albumin (Sigma
Aldrich, St Louis, MO) solution in phosphate buffered saline or
70% ethanol respectively following the manufacturer’s instruc-
Immunophenotypic Analysis of Cultured Cells
FACS analysis of cultured cells was performed on an LSR II
instrument (Becton Dickinson) by direct immunofluorescence
staining with human specific monoclonal antibodies after incuba-
tion in 1.2% human intravenous immunoglobulin (IVIG; Cutter,
Berkley, CA). Lineage-specific differentiation was determined
using the following antibodies: CD45-APC Cy7, CD34-PE Cy7,
CD41a-PE Cy5, CD66b-PE, GlyA-APC or -PE, CD19-PE, APC
or Percp-Cy5.5, and CD14 PE or FITC (all from Becton
Dickinson). The following immunophenotypes were used to
identify terminally differentiated lymphoid and myeloid cells from
(CD66b+CD45+), megakaryocytes (CD41a+CD45-GLYA-) and
B-cells (CD19+CD45+) (Fig. 1A). For long term (4 week) culture
experiments, the total number of cells per well in each condition
was determined by trypan blue microscopy, and the number of
differentiated cells was calculated based on % of each lineage
phenotype by FACS multiplied to a total cell number in each well.
Cell Migration Experiments
Migration assays were carried out in 24 well Transwell plates
from Costar with 6.5 mm diameter and 8.0 mm pore size. Freshly
sorted CMP, CLP or HSPC (1,000 cells each) were seeded into the
upper chamber in DMEM/F12 supplemented with 5% charcoal
treated serum with no growth factors in the presence or absence of
LPA (1 uM) in the lower chamber. Migration was assessed based
on the number of total cells on the bottom of the lower chamber
after 12 hours, determined separately for each cell type using
bright field microscopy. Independent experiments were carried
out using progenitor populations isolated from 3 different donors.
Cell Proliferation and Apoptosis Analyses
Freshly sorted CMP, CMP or HSPC were seeded into 48 well
plates (5,000 cells per well) on OP9 cells, with DMEM/
supplemented with 5% charcoal treated serum, with no growth
factors in the presence or absence of LPA (1 uM) and cultured for
48 hours without medium change to measure the effect of LPA on
proliferation and apoptosis. Prior to harvesting, cells were
incubated with bromodeoxyuridine (BrdU) (10 uM) for 30
minutes. Harvested cells were fixed, permeabilized, and stained
with FITC or APC conjugated antibody against BrdU. Unstained
cells were used to set negative gates. Apoptosis rates in progenitor
populations wasere assessed using FACS-based Annexin V assay
(BD Bioscience). Equal numbers (3000 of CD45 gated hemato-
poietic cells recovered from culture) of each population was
analyzed for BrdU incorporation or Annexin V binding using
Specimens of adult sponge bone (3 individual specimens) were
provided by the UCLA Translation Pathology Core Laboratory.
All specimens were from patients with no hematopoietic disorders.
Fetal bones (16–18 weeks of pregnancy) were obtained from
Novogenix (Los Angeles, CA) fixed in 4% paraformaldehyde
(Sigma-Aldrich, St. Lois, MO, in PBS). Fixed tissues were
embedded in paraffin, sectioned and subjected to histological
immunohistochemical analyses. The murine stromal cell lines
MS5 and OP9 (American Type Culture Collection (ATCC,
Rockville, MD, USA) or primary human bone marrow derived
mesenchymal stromal cells (passage 2–3) (AllCells Inc, Emeryville
CA) were seeded into chamber slides (BD Bioscience) in DMEM/
F12 (Invitrogen) medium supplemented with 20% fetal bovine
serum. The next day, cells were fixed with 4% paraformaldehyde
and subjected immunohistochemical analysis. Polyclonal antibod-
ies against autotaxin, PPAP2a and CD146 were purchased from
Abcam Inc. (Cambridge, MA) Secondary horse radish peroxidase
(HRP) conjugated IMPRESS anti-rabbit and anti-mouse antibod-
ies and 3, 39-diaminobenzidine (DAB) substrate (Vector Labs)
were used for the visualization of positively labeled regions. Images
were acquired using the Zeiss Axiovision software version 4.8 Carl
Zeiss Microscope (Carl Zeiss, Germany) equipped with Apo-
Tome.2: Modules for Axio Imager.2 and Axio Observer with 40x
(1.3 numerical aperture (NA)) and 63x (1.4 NA) oil-immersion
Quantitative Real-time PCR
Total RNA was extracted from cells (,5,000 sorted cells were
used for each population) using the RNeasy Micro Kit, and
converted to cDNA using the Omniscript RT Kit (kits were from
Qiagen Sciences, Maryland, USA). Total RNA concentration for
all samples was within 5–20 ng/ml range as determined by
Lysophosphatidic Acid Regulates Hematopoiesis
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Nanodrop analysis. No template amplification was carried out
prior to cDNA synthesis.
Next, SYBR Green RT-PCR amplification and detection was
performed using an ABI Prism 7900 HT (Applied Biosystems) as
previously described. The comparative Ct method for relative
quantification (22DDCt) was used to quantitate gene expression
according to Applied Biosystems’ recommendations [7900 HT
Real-Time fast and SDS enterprise and database user guide]. Expression of
target genes was normalized to the level of the house-keeping gene
RPL-7 and expressed relative to a calibrator (sample in each set
with lowest expression). All primer sequences were obtained from
Harvard University Primer Bank (http://pga.mgh.harvard.edu/
primerbank) Primer sequences used for QPCR are available on
LPA Stimulates Myeloid Differentiation
To dissect the role of LPA on human hematopoiesis we
began by analyzing how LPA affects differentiation of un-
fractionated CD34+ progenitors, a heterogeneous population
that includes hematopoietic
progenitors (HSPC), as well as myeloid and lymphoid-commit-
ted progenitors. CD34+ cells were co-cultured on a stromal
monolayer in medium containing 5% LPA-depleted serum ,
with or without addition of exogenous LPA and in the presence
of thrombopoietin, Flt3 Ligand and IL-7, cytokines that allow
generation of both myeloid and B lymphoid cells (Fig. 1A, 2A).
To determine the optimal stromal layer for these experiments,
we analysed expression of ATX, the enzyme which mediates
synthesis of LPA, in 3 stromal cell types commonly used for
hematopoietic cell support in vitro: OP9 , MS5 stroma 
and primary bone marrow derived mesenchymal stromal cells
(BM MSC). OP9 demonstrated very low levels of ATX
suggesting that this line has minimal production of endogenous
LPA in culture (Fig. 1B). In contrast to OP9, both MS5 and
primary BM MSC showed readily detectable levels of ATX
protein expression (Fig. 1B). Based on the pattern of ATX
production in the tested stromal lines we used OP9 cells for all
our co-culture experiments. We next tested the effect of
different LPA concentrations (0.1, 1 and 10 uM) on lineage
differentiation from CD34+ cord blood cells. Generation of
CD14+ monocytes was significantly enhanced in the presence of
1 and 10 mM of LPA while 0.1 uM of LPA had little or no
effect (Fig. 2A). LPA was thus used at a concentration of 1 uM
for further detailed experimentation.
The addition of LPA in culture generated significantly more
myeloid cells (CD66b+ granulocytes and CD14+ monocytes)
and CD41a+ megakaryocytes from CD34+ cells compared to
controls (otherwise identical culture conditions without LPA
added). In contrast B lymphocyte output was not altered by the
presence of LPA, and the number of CD34+ progenitors
Figure 1. In vitro system for differentiation of hematopoietic cells. A. In vitro differentiation of un-fractionated CD34+ progenitors co-
cultured on a stromal monolayer in medium containing 5% LPA-depleted serum in the presence of thrombopoietin, Flt3 Ligand and IL-7, cytokines
that allow generation of both myeloid (CD45+CD14+ monocytes, CD45+CD66b granulocytes and CD45negCD41a+ megakaryocytes) and B lymphoid
cells: CD45+CD10+ CD19neg or CD45+CD10+CD19+. Control panel represents unstained cells. B. Autotaxin protein expression in commonly used
stromal lines: MS5, OP9 and human bone marrow-derived mesenchymal stromal cells. Positive signal is shown in brown color (DAB). Magnification
20X. Images were acquired using the Zeiss Axiovision software version 4.8 Carl Zeiss Microscope (Carl Zeiss, Germany).
Lysophosphatidic Acid Regulates Hematopoiesis
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persisting after 4 weeks was not significantly changed (Fig. 2B).
LPA induced similar effects on CD34+ cells isolated from
human bone marrow to those from cord blood (not shown).
These data suggest that LPA has a selective and significant
effect on myeloid differentiation.
We next explored the specificity of observed stimulatory effects
of LPA on differentiation of CD34+ hematopoietic progenitors.
Addition of the sphingosine-1-phosphate, a lipid molecule
structurally similar to LPA, did not show any significant pro-
myelopoietic effects (Fig. 2C). LPA receptor antagonist Ki-16425
almost completely abolished stimulatory effects of LPA on CD34+
cell differentiation further confirming specificity of the LPA
mediated stimulation of myelopoiesis (Fig. 2C).
To further understand the mechanisms by which LPA induced
myeloid differentiation, we explored LPA receptor expression on
specific hematopoietic stem and progenitor populations. Real time
RT-PCR was performed on freshly isolated CD34+CD38-lin- cells
(enriched for HSPC), CD34+lin-CD45RA-IL3Ralo
myeloid progenitors (CMP), and CD34+lin-CD10+ common
lymphoid progenitors (CLP) (a population with predominantly B
cell potential  (Fig. 3A). Expression of all six LPA receptors
was higher in CMP than either HSPC or CLP (p,0.05 for LPAR
1,2,4 and 6).
LPA Stimulates the Migration, Proliferation and Survival
of Myeloid but not Lymphoid Progenitors
Functional studies were next performed to define the effects
ofLPA stimulationon lineage-specific
migratory potential of CMP but had no detectable effect on
either HSPC or lymphoid progenitors (Fig. 3B). Cell prolifer-
ation after 48 hours of LPA stimulation (measured by BrdU
uptake) was significantly increased in CMP but not HSPC or
CLP (Fig. 3C). In addition, LPA reduced apoptosis of CMP but
had no effect on either HSPC or CLP (Fig. 3D). Of note, all
migration assays were performed in stroma-free conditions and
therefore represented direct effects of LPA on hematopoietic
ATX and PPAP2A are Differentially Expressed in Human
Published data suggest that the hematopoietic niche is spatially
organized between the endosteal region and the sinusoidal
perivascular zones [16,17,18]. As myeloid and lymphoid commit-
ment from HSPC is regulated in large part by differential signals
emanating from the microenvironment, it is plausible that these
lineages develop in spatially distinct compartments within the bone
marrow. Indeed, some studies have indicated that myeloid cells
accumulate in perisinusoidal regions before they enter systemic
circulation [16,18], and others that pre-B cells migrate and
accumulate in close proximity to osteoblasts , The mechanisms
controlling this separation of lymphoid and myeloid cells in the
bone marrow niche are not known.
In view of our findings that LPA induces migration and growth
specifically of myeloid progenitors, we hypothesized that LPA
might play a role in the compartmentalization of the bone marrow
niche. As LPA levels cannot be assayed in situ directly, we studied
the expression and the spatial localization within adult bone
marrow of ATX and PPAP2A, the enzymes responsible for
synthesis and degradation of LPA respectively.
In adult bone marrow, PPAP2A was highly expressed by
osteoblasts throughout the endosteal zone (Fig. 4A–h). PPAP2A
was largely absent in the perivascular region (Fig. 4A–g) with the
exception of vessels near the endosteal zone where perivascular
cells form ‘‘stromal bridges’’ with osteoblasts (Fig. 4A–h). Little or
no ATX expression was found in the osteoblastic region of adult
human bone marrow (Fig. 4A–j). However, ATX was highly
expressed by CD146+ perivascular stromal cells of blood vessels
To investigate whether the spatial distribution of LPA-
generating and -metabolising enzymes was age specific and/or
related to bone marrow involution and fat deposition associated
with aging, ATX and PPAP2A expression was analysed in human
fetal (16–18 week old) bone marrow (Fig. 4B). Consistent with the
pattern observed in adult bone marrow, high levels of PPAP2A
and no/little ATX expression were seen in the endosteal region
(Fig. 4B–h,j), whereas ATX expression was clearly present in
Figure 2. LPA stimulates generation of myelopoietic lineages from cord blood CD34+ + progenitor cells. A. Dose response of CD34+ cord
blood FACS sorted cells to increasing concentration (0.1, 1, 10 uM of LPA). Detection of CD14+ monocytes was used as readout of activity. Mean 6
standard deviation (SD), Mean 3 *p,0.05 compared to control cells. B. LPA stimulated generation of myeloid (monocytes, granulocytes and
megakaryocytes), but not lymphoid (B-cell) differentiation from CD34+ cells. Freshly sorted CB CD34+ cells were cultured on OP9 stroma for 4 weeks
in medium supplemented with 5% LPA-depleted (charcoal treated) serum and growth factor combinations permissive for both myeloid and
lymphoid differentiation in the absence (CON=control) or presence of LPA (1 uM). The total number of cells per well in each condition was
determined by counting in hematocytometer, and the number of cells of each immunophenotype (shown on the y-axis) was calculated based on %
of each lineage phenotype by FACS multiplied to total cell number in each well. Shown is Mean 6 standard deviation (SD), N=4 independent
experiments, *p,0.05. C. Stimulatory effects of LPA on myelopoiesis can be ablated using LPA receptor antagonist Ki16425. Myelopoietic
differentiation of CD34+ cord blood cells was assessed by the generation of CD14+CD45+ monocytes at 7 days of culture. Concentration of tested
compounds: LPA and S1P –1 uM, Ki16425–5 uM. Cord blood samples from 4 donors were analyzed independently and results shown as Mean 6 SD.
** P,0.01, * P,0.05. CON=Control.
Lysophosphatidic Acid Regulates Hematopoiesis
PLOS ONE | www.plosone.org4May 2013 | Volume 8 | Issue 5 | e63718
perivascular regions of blood vessels (Fig. 4B–i). Thus spatial
distribution of ATX and PPAP2A during fetal hematopoiesis is
similar to that seen in adult.
Although numerous studies have investigated the hematopoietic
stem cell niche, relatively few have been specifically focused on the
localisation of more mature hematopoietic cells within the bone
marrow. Injection of radiolabelled pre-B cells to mice with severe
combined immunodeficiency lacking lymphoid cells demonstrated
that most of the injected cells migrated into the bone marrow and
homed near osteoblasts  selectively occupying microenviron-
ments near the surrounding bone. In contrast, myeloid cells have
been described as concentrated in close proximity to sinusoids
. Moreover megakaryocytes can invaginate into the luminal
space of sinusoids where they give rise to terminally differentiated
platelets migrating directly into the circulating blood . The
exact mechanisms regulating hematopoietic cell compartmental-
isation in the bone marrow niche are not clear. It is plausible to
predict that regulatory gradients are needed to separate different
hematopoietic lineages in the bone marrow and also provide
migration signals for differentiated blood cells prepared to enter
systemic blood circulation. The spatial expression of ATX and
PPAP2A suggests that the highest levels of LPA in this system will
appear in the close proximity to the small blood vessels where LPA
molecules diffuse directly from blood plasma. High levels of LPA
would also be predicted in perivascular regions near larger
microvessels (20–100 mkm in diameter) where ATX is expressed.
In close proximity to osteoblasts, especially those located remotely
from microvessels, levels of LPA would be expected to be minimal
due to the high PPAP2A activity and minimal expression of ATX.
S1P, another member of the lysophospholipid family, has
previously been demonstrated to play a role in both HSPC and
lymphoid compartmentalization ; high levels of S1P in
peripheral blood compared with the bone marrow tissue create
a gradient that promotes migration of B-cell progenitors from the
bone marrow to secondary lymphoid organs . Disruption of
this gradient abrogates HSPC mobilization following AMD3100
treatment thus proposing a critical role for bioactive lipid gradients
Figure 3. Myeloid progenitors are functional targets of LPA. A. LPA receptor mRNA expression in hematopoietic stem-progenitor cells
(HSPC), common lymphoid (CLP) and myeloid progenitors (CMP) by qPCR. N=4 independent experiments; *p,0.05. B. Migration in TranswellH
experiments in the presence or absence of 18:1 Oleoyl-LPA, 12 hours after seeding of HSPC, CMP or CLP. C. Proliferation of each cell type shown
measured by 48 hour BrdU uptake. Y axis shows % of BrdU positive cells) D. Apoptosis of each cell type measured by the Annexin V assay (Y axis-
shows the % of Annexin V positive cells). Mean 6 SD; N=3 independent experiments; *p,0.05.
Lysophosphatidic Acid Regulates Hematopoiesis
PLOS ONE | www.plosone.org5 May 2013 | Volume 8 | Issue 5 | e63718
in hematopoietic cell migration . The data presented here,
identify LPA as a regulator of migration, growth and survival of
myeloid progenitors. We propose that LPA provides a novel
mechanism through which anatomical partitioning of the bone
marrow microenvironment creates spatial regulation of myeloid
differentiation during hematopoiesis.
We are grateful for technical support from the BSCRC flow cytometry core
and UCLA Translational Core Laboratory.
Conceived and designed the experiments: DE BL. Performed the
experiments: DE BL SC RC YZ AS WR MC. Analyzed the data: DE
BD GMC. Wrote the paper: DE BL GMC.
Figure 4. Spatial distribution of autotaxin and PPAP2A in adult bone marrow. A. Adult Bone marrow. Low power (10x) unstained sections
showing two different focal plans (a, b). (A–c,e,g,i) shows high power views (43x) of boxed area in (a). (A–d,f,h,j) show high power views of boxed area
in (b). (Aa–d) controls stained with secondary antibody only, (A–e, f) CD146 detection of perivascular cells, (A–g, h) PPAP2A expression in endosteal
region (arrows), but not perivascular cells, (A–i, j) ATX expression limited to perivascular cells (arrow heads). V=vascular space. B. Fetal Bone marrow.
Consistent with the expression pattern of PPAP2A in adult BM, PPAP2A immunoreactivity in fetal BM was predominantly located in bone surfaces
lining osteoblasts (arrows), ATX expression limited to perivascular cells (arrow heads) (Fig. 2B–g, h). Low power views (10x) are shown in a, and b.
High power views (63x) of areas boxed in panels at left are shown in Bc, e, g, i. High power views (63x) of areas boxed in panels at right are shown in
Bd, f, h and j. Positive signals in A and B are shown in brown color (DAB). Images were acquired using the Zeiss Axiovision software version 4.8 Carl
Zeiss Microscope (Carl Zeiss, Germany) equipped with ApoTome.2: Modules for Axio Imager.2 and Axio Observer with 40x (1.3 numerical aperture
(NA)) and 63x (1.4 NA) oil-immersion objectives.
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Lysophosphatidic Acid Regulates Hematopoiesis
PLOS ONE | www.plosone.org7 May 2013 | Volume 8 | Issue 5 | e63718