ral
ssBioMed Cent
BMC Immunology
Open Acce
Research article
Wnt expression and canonical Wnt signaling in human bone
marrow B lymphopoiesis
Guri Døsen, Ellen Tenstad, Marit Kveine Nygren, Heidi Stubberud,
Steinar Funderud and Edith Rian*
Address: Department of Immunology, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, Medical Faculty, University
of Oslo, Norway
Email: Guri Døsen - guri.dosen@rr-research.no; Ellen Tenstad - et@hive.no; Marit Kveine Nygren - marit.nygren@rr-research.no;
Heidi Stubberud - heidi.stubberud@rr-research.no; Steinar Funderud - steinar.funderud@rr-research.no; Edith Rian* - edith.rian@rr-research.no
* Corresponding author
Abstract
Background: The early B lymphopoiesis in mammals is regulated through close interactions with
stromal cells and components of the intracellular matrix in the bone marrow (BM)
microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are
implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling
is known to be involved in embryonic development and growth regulation of tissues and cancer.
Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a
role in regulating human BM B lymphopoiesis.
Results: Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt
antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and
receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this
signaling. Furthermore, different B progenitor maturation stages showed differential expression of
Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting
canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced
stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells.
Also, Wnt3A inhibited B lymphopoiesis of CD133
+
CD10
-
hematopoietic progenitor cells and
CD10
+
B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect
was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture
showed that Wnt3A inhibits cell division of B progenitor cells.
Conclusion: These results indicate that canonical Wnt signaling is involved in human BM B
lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma
dependent manner.
Background the bone marrow (BM). Here, the B cell progeny mature
Published: 29 June 2006
BMC Immunology 2006, 7:13 doi:10.1186/1471-2172-7-13
Received: 09 November 2005
Accepted: 29 June 2006
This article is available from: http://www.biomedcentral.com/1471-2172/7/13
© 2006 Døsen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 17
(page number not for citation purposes)
In mammals, the early antigen independent phase of B
lymphopoiesis takes place in the intersinusoidal spaces in
from hematopoietic stem cells (HSC) via early lymphoid
progenitors (ELP, comprising common lymphoid progen-

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
itors and early B), pro-B, pre-B and immature B develop-
mental stages characterized by successive steps in the
rearrangement of immunoglobulin genes and consecutive
expression of cellular markers [1-3]. Using immunohisto-
chemical doublestaining we have revealed earlier that all
developmental stages of the B cell lineage in human BM
tissue are in close contact with slender CD10
+
stromal
cells or their extensions [4]. This finding correlates with
the consensus that B lymphopoiesis is tightly regulated by
signals provided by mesenchymal stromal cells and com-
ponents of the intracellular matrix in the BM microenvi-
ronment in vivo [4-6]. However, the elements of this
signaling are yet inadequately identified; stromal factors
like IL 7, Flt3 ligand [7], IL-3 [8,9] and SDF1 [10,11] are
essential, but not sufficient for BM B lymphopoiesis [2].
Clearly, there is a need for further characterization of both
the stromal phenotype as well as the autocrine and para-
crine factors that participate in the regulation of BM B
lympopoiesis.
Wnt proteins belong to a large and highly conserved fam-
ily of secreted, cystein-rich glycoprotein signaling mole-
cules, consisting of 19 members. They are likely to act
locally because of their limited solubility [12] and ten-
dency to associate with the cell surface extracellular matrix
[13]. Signaling is initiated by Wnt proteins binding to
receptors of the Frizzled family (Fzd) on the cell surface.
This binding is promiscuous and the ligand/receptor spe-
cificities are not yet properly determined. Depending on
particular Wnt/Fzd combinations, at least three signaling
cascades may be activated. Most studied is the canonical
Wnt pathway, which is activated by members of the Wnt1
class (such as Wnt1, Wnt2, Wnt3 and Wnt8) [14]. A key
regulatory molecule in this pathway is β-catenin, which in
the absence of a Wnt signal is kept low through continu-
ous phosporylation by glycogen synthase kinase-3β (GSK-
3β), resulting in a subsequent proteasome dependent
destruction of β-catenin. Binding of Wnt ligands to Fzd
receptors and coreceptors LRP5/6, leads to inactivation of
GSK3β and thereby accumulation of nonphosphorylated
β-catenin, which enter the nucleus. Here, β-catenin acts as
a coactivator of members of the lymphoid enhancer fac-
tor-1 (LEF-1)/T-cell factor (TCF) family of transcription
factors to stimulate transcription of Wnt target genes [15].
Activation of Wnt signaling can be inhibited by soluble
antagonists, including the Dickkopf (Dkk) family and the
soluble Fzd related proteins (sFRP) [16].
Recently, Wnt proteins have drawn attention as a set of
factors operating in embryonic development, growth reg-
ulation of adult tissues and cancer formation [15,17-20].
Moreover, Wnt signaling plays a central role in the com-
munication between HSC and stromal cells [21] as well as
maturation process where hematopoietic stem cells lose
their pluripotency and commit to specific lineages [24-
26]. LEF-1 and Fzd9 knockout mice show defect B lym-
phopoiesis [24,27] and Wnt signaling seems to be
involved in development of leukemia [28-30] and malig-
nant myeloma [31]. Moreover, in murine B lymphopoie-
sis this signaling pathway has a stimulatory effect on pro-
B cells from fetal liver [24]. As early B lymphopoiesis in
mice and humans to a certain extent shows distinct factor
dependency [32], and since fetal and adult lymphopoiesis
takes place in different maturation niches, the aim of the
present study was to investigate Wnt signaling in human
BM B lymphopoiesis in more detail. We have examined
which Wnt signaling pathway molecules that are
expressed in B progenitor cells and stromal cells from
human BM, and analyzed the regulated expression of sev-
eral Wnt receptors (Fzd and LRP), β-catenin and pla-
koglobin as well as the central transcription factors LEF-1
and TCF-4 during the early B lymphopoiesis. Further-
more, we have investigated the effect of recombinant
Wnt3A on progenitor B cells. We found that Wnt3A
induced β-catenin stabilization and inhibited in vitro B
lymphopoiesis in a coculture with stromal cells by sup-
pression of initial cell proliferation. Thus, canonical Wnt
signaling may be involved in human BM B lymphopoie-
sis.
Results
A distinct set of Wnt ligands, Fzd receptors and Wnt
antagonists is expressed in B progenitor cells and stromal
cells from human BM
Previous work has demonstrated expression of Wnt5A,
Wnt2B and Wnt10B in pooled human BM populations
[26]. However, the expression pattern of Wnt ligands, Fzd
receptors and Wnt antagonists in human B lineage cells
has not been explored. In the absence of available anti-
bodies to detect these large families of proteins, we per-
formed conventional RT-PCR on RNA isolated from FACS
sorted B progenitor cells (CD10
+
IgM
-
CD45
+
) pooled from
three different donors, using primers designed specifically
to detect mRNA expression of all known Wnt ligands and
Fzd receptors as well as the Wnt antagonists Dkk1, Dkk4,
sFRP1-4 and WIF1 (fig. 1 and table 1). In B progenitor
cells, Wnt 2B, 5B, 8A, 10A and 16 mRNAs were readily
detected. Interestingly, the Wnt16 PCR product had two
bands of 520 bp and 233 bp, respectively (fig. 1). The 520
bp band represents the full-length form and the 233 bp
band represents a possible splice variant lacking exon 3,
potentially giving rise to a truncated Wnt16 form. In addi-
tion, expression of several other Wnt mRNAs was detecta-
ble, however, less readily (table 1). The Fzd receptors
showed on average much higher mRNA expression levels
than the Wnts, where Fzd2, 3, 4, 5, 6 and 9 mRNAs werePage 2 of 17
(page number not for citation purposes)
in several other stem cell niches [22,23]. Several observa-
tions have established direct roles for Wnt signaling in the
easily detectable in the B progenitor population, as dem-
onstrated by strong PCR bands. Fzd1 and Fzd7 mRNA

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Page 3 of 17
(page number not for citation purposes)
mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonistsFigure 1
mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonists. RT-PCR detection of mRNAs for
Wnt ligands, Fzd receptors and Wnt antagonists in BM B progenitor cells. The + and - symbols indicate the presence and
absence of reverse transcriptase in the reaction mix, respectively. One representative of two experiments is shown. Amplicon
sizes: Wnt2B: 328 bp, Wnt5B:279 bp, Wnt8A: 400 bp, Wnt10A: 296 bp, Wnt16: 520 bp, Fzd2: 306 bp, Fzd3: 622 bp, Fzd4: 605
bp, Fzd5: 197 bp, Fzd6: 300 bp, Fzd9: 210 bp, sFRP4: 243 bp, WIF1: 200 bp, Dkk1: 235 bp, Dkk4: 241 bp. M: Size marker 1 kb
Plus DNA ladder (Invitrogen, USA). Where two different bands are detected, an arrow marks the correct band.
+-
Wnt5B
M
+-
Wnt16
M
Wnt2B
M
+-
M
+-
Fzd2
M
+-
Fzd3
M
+-
Fzd4
M
+-
Fzd9
M
+-
Fzd6
M
+-
Fzd5
M
+-
WIF1
M
Dkk1
+-
M
Dkk4
+-
+-
Wnt8A
M
+-
Wnt10A
M
M
sFRP4
+-

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
expression was also demonstrated, but at lower levels
than the other Fzds (table 1). We also detected expression
of the Wnt antagonists Dkk1, Dkk4, sFRP4 and WIF1
mRNAs in the BM B progenitor cells (fig. 1 and table 1).
Of these, sFRP4 mRNA was most readily detectable, sug-
gesting the highest expression level. sFRP2 and sFRP3
mRNAs were variably detected (table 1), suggesting low
expression levels.
RT-PCR performed on RNA from BM stromal cells showed
expression of Wnt2B, Wnt5A, Wnt5B and Wnt8B. mRNA
expression of Wnt9B was also demonstrated in these cells,
although at a lower levels. Moreover, Fzd3, 4 and 6
mRNAs were detected in BM stromal cells, as well as
expression of the Wnt antagonists Dkk1, sFRP2 and sFRP3
mRNAs (table 1).
Regulated expression of Wnt receptors, β-catenin,
plakoglobin, LEF-1 and TCF-4 mRNAs during human BM B
lymphopoiesis
Identification of differential expression of Wnt signaling
molecules during the B lymphopoiesis may reveal at
which window in the process Wnt signaling is active.
Thus, using quantitative real-time PCR, we examined the
expression of a selection of Wnt receptors, β-catenin, pla-
koglobin and transcription factors in FACS sorted human
BM B lineage cells representing different maturation lev-
els; ELP cells (CD10
+
CD34
+
CD19
-
, also tested to be
+ + + + - -
small pre-B (CD10
+
CD34
-
CD19
+
CD20
-
IgM
-
) and imma-
ture B cells (CD10
+
CD34
-
CD19
+
CD20
+
IgM
+
). Due to lim-
ited number of cells, expression analysis in ELP cells was
restricted to seven out of ten mRNAs.
The results showed regulation of several of the important
Wnt-signaling molecules, and different expression pro-
files were recognizable (fig. 2). mRNA levels for the
plasma membrane receptors LRP5, LRP6, Fzd5 and Fzd6
dropped considerably as the cells develop from small pre-
B cells into immature B cells. Furthermore, Fzd5 mRNA
levels were strongly up-regulated as the cells commit to
the B lineage (from ELP to pro-B), with a further up-regu-
lation as the cells differentiate to pre-B cells. Fzd2 and
Fzd9 mRNA levels, on the other hand, seemed to increase
somewhat throughout the differentiation, with highest
levels in small pre-B and immature B cells. In small pre-B
cells, the mRNA levels of LRP5 and Fzd9 were about two-
fold higher than in the large cycling pre-B cells. The
expression levels of all receptors were low compared to
the expression levels of e.g. LEF-1 and β-catenin, indicat-
ing relative low mRNA expression levels. Fzd3 and Fzd4
mRNAs were not detectable with the amount of RNA tem-
plate used in these assays.
The mRNA expression of β-catenin and plakoglobin
showed little variation as the cells differentiate. β-catenin
mRNA was evenly expressed in ELP, pro-B, large pre-B and
Table 1: mRNA expression of Wnt ligands 1–19, Fzd receptors 1–10, Wnt antagonists sFRP1-4, WIF1, Dkk1 and Dkk4
BM B
progenitor cells
BM stromal
cells (BMS)
Human fetal
brain
BM B progenitor
cells
BM stromal cells
(BMS)
Human fetal brain
Wnt1 +/- - + Fzd1 +/- - -
Wnt2 - - + Fzd2 + - +
Wnt2B + + + Fzd3 + + +
Wnt3 - - + Fzd4 + + +
Wnt3A +/- - - Fzd5 + - +
Wnt4 +/- - + Fzd6 + + +
Wnt5A +/- + + Fzd7 +/- - +
Wnt5B + + + Fzd8 ND - ND
Wnt6 ND - ND Fzd9 + - +
Wnt7A - - + Fzd10 - - -
Wnt7B - - + Dkk1 + + +
Wnt8A + - + Dkk4 + - -
Wnt8B ND + ND sFRP1 - - +
Wnt9A +/- - + sFRP2 +/- + +
Wnt9B +/- + + sFRP3 +/- + +
Wnt10A + - - sFRP4 + - +
Wnt10B +/- - + WIF1 + ND +
Wnt11 +/- - +
Wnt16 + - +
Genes expressed (+), not expressed (-), variably expressed between experiments(+/-), not determined (ND). N = 2. BM B progenitor cells:
CD10
+
IgM
-
CD45
+
cells sorted by FACS and pooled from three different donors. Total RNA from human fetal brain was used as control.Page 4 of 17
(page number not for citation purposes)
CD38 ), pro-B cells (CD10 CD34 CD19 CD20 IgM ),
large pre-B cells (CD10
+
CD34
-
CD19
+
CD20
dim
IgM
-
),
immature B, with a small increase (near two-fold) in small
pre-B cells. Plakoglobin mRNA levels, in contrast,

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Page 5 of 17
(page number not for citation purposes)
Real-time PCR analysis of relative mRNA expression levels of Wnt pathway molecules in BM B progenitor sub-populationsFigure 2
Real-time PCR analysis of relative mRNA expression levels of Wnt pathway molecules in BM B progenitor sub-
populations. The sub-populations ELP, pro-B, large pre-B, small pre-B and immature B (imm.B) were isolated by FACS sort-
ing. The relative mRNA expression levels of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 were
quantified by real-time PCR analysis. Calculations of the expression levels were performed using the standard curve method
and then normalized to the expression of PGK1 mRNA. mRNA levels in pro-B cells were used as calibrators. The bars repre-
sent the mean of 3–5 experiments ± SEM.


















R
e
l
a
t
i
v
e
m
R
N
A
e
x
p
r
e
s
s
i
o
n
LRP5














































LRP6 Fzd2
Fzd5
R
e
l
a
t
i
v
e
m
R
N
A
e
x
p
r
e
s
s
i
o
n




























Fzd6
Fzd9
































R
e
l
a
t
i
v
e
m
R
N
A
e
x
p
r
e
s
s
i
o
n

-catenin Plakoglobin
R
e
l
a
t
i
v
e
m
R
N
A
e
x
p
r
e
s
s
i
o
n
Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP
Pro-B Small
Pre-B
Large
Pre-B
Imm. B Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP Pro-B Small
Pre-B
Large
Pre-B
Imm. B
Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP Pro-B Small
Pre-B
Large
Pre-B
Imm. B
















Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP
LEF-1
Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP














Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP
Pro-B Small
Pre-B
Large
Pre-B
Imm. BELP
TCF-4

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
decreased 2-fold as the cells became large pre-B cells (fig.
2).
LEF-1 and TCF-4 mRNA expression is highly regulated
during the early B lymphopoiesis, as shown previously by
microarray analysis (Hystad ME et al, manuscript in prep-
aration and [33]). Our results showed a strong up-regula-
tion of LEF-1 mRNA as the cells commit to the B lineage,
and the expression was kept continuously high until the
cells become immature B cells, where the level was
reduced to the same as in uncommitted progenitors. Here,
low LEF-1 expression was further confirmed by the
absence of LEF-1 protein in B lymphocytes from periph-
eral blood (results not shown). The relative TCF-4 mRNA
levels, on the other hand, were high in both ELP and pro-
B, and decreased (up to 5-fold) as the cells passed through
Ig rearrangement (pre-B – immature B cells) (fig. 2). It
should be noted that the LEF-1 mRNA expression was
detected 5–8 cycles earlier than the TCF-4 mRNA expres-
sion, indicating that LEF-1 mRNA is much more abundant
than TCF-4 mRNA.
Wnt3A induces β-catenin stabilization and accumulation
in BM B progenitor cells
Our data demonstrated that human BM B progenitor cells
express a set of central players in the canonical Wnt sign-
could respond to treatment with Wnt proteins, we looked
for the stabilization and subsequent accumulation of the
vital signaling molecule β-catenin in CD10
+
B progenitor
cells. When these cells were treated with Wnt3A, the
amount of β-catenin increased substantially compared to
the very low levels in untreated cells (fig. 3). Although
there were some donor variations, the results showed that
the B progenitor cells are able to receive and communicate
a signal from the Wnt pathway.
Wnt3A inhibits human in vitro B lymphopoiesis
Having identified expression of central molecules in the
canonical Wnt pathway in BM B progenitor cells, we per-
formed two variants of B lymphopoiesis assays to investi-
gate whether Wnt signaling (using recombinant Wnt3A)
had a functional effect on B lymphopoiesis in vitro. Both
assays were based on coculture with the murine stromal
cell line MS-5. In assay 1 hematopoietic progenitor cells
(HPC) were tested for their capacity to develop into B lin-
eage cells, whereas in assay 2 B progenitor cells were meas-
ured for survival and expansion. At the endpoint of the
assays, each sample was subjected to quantitative flow
cytometry and the total number of cells positive for the
pan B cell marker CD19 was measured. In assay 2, analysis
of the differentiation marker CD34 was included.
Initial analyses demonstrated that Wnt3A had an inhibi-
tory effect when BM HPC (CD133
+
CD10
-
) were grown on
stromal cells for 3 weeks at conditions that favored B lym-
phopoiesis (assay 1). The number of CD19
+
cells in the
samples treated with Wnt3A was 5 times less than the
number measured in the control samples (fig. 4A). The
inhibited B lymphopoiesis could result from Wnt3A sup-
pressing differentiation of the HSC pool found in the HPC
population [34], an indirect effect mediated by the stro-
mal cells [35], or, alternatively, Wnt3A could target more
committed lymphoid progenitor cells. To examine the lat-
ter possibility in more detail, we tested whether Wnt3A
acted on later stages of in vitro B lymphopoiesis. BM B pro-
genitor cells (CD10
+
) were grown on stromal cells in the
presence of Wnt3A or medium only for 2 weeks (assay 2).
In accordance with the results from the assays using HPC,
it was demonstrated on average near 50% reduction in the
total number of CD19
+
cells in samples treated with
Wnt3A compared with control (fig. 4B). When added
every third day, both sFRP1 and Dkk1 were able to coun-
teract the effect of Wnt3A almost completely, demonstrat-
ing a specific effect of Wnt3A on in vitro B lymphopoiesis
(fig. 4B). Similar results were obtained using Wnt3A pro-
tein from another source; Wnt3A conditioned medium
(table 2). Moreover, the effect was independent of the
source of stromal cells as the use of primary human BM
stromal cells (BMS) as supportive layer did not change the
Wnt3A induces β-catenin stabilization in BM B progenitor cellsFigure 3
Wnt3A induces β-catenin stabilization in BM B pro-
genitor cells. Western blot analysis of β-catenin levels in
BM CD10
+
B lineage progenitor cells stimulated with Wnt3A
(100 ng/ml) or vehicle (PBS with 0.1% detoxified BSA) for 3
hours. The blots were incubated with an Ab against β-cat-
enin, followed by an Ab against β-actin to ascertain equal
loading in the wells. The same results were found in cells
from 4 out of 5 different donors, indicating some degree of
donor variation in the response to Wnt3A.

-catenin

-actin
W
n
t
3
A
C
o
n
t
r
o
lPage 6 of 17
(page number not for citation purposes)
aling pathway, potentially allowing a Wnt signal to be
conveyed. To further examine whether B progenitor cells
outcome of the experiment (table 2).

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Page 7 of 17
(page number not for citation purposes)
Wnt3A inhibits in vitro B lymphopoiesisFigure 4
Wnt3A inhibits in vitro B lymphopoiesis. BM CD133
+
CD10
-
HPC (A: assay 1) or CD10
+
B progenitor cells (B: assay 2)
were cocultured with a confluent layer of the murine stromal cell line MS-5 for 3 or 2 weeks, respectively, while treated with
Wnt3A (100 ng/ml), Wnt3A + sFRP1 (2 µg/ml), Wnt3A + Dkk1 (500 ng/ml) or medium only. The number of resulting CD19
+
B lineage cells in each sample was determined by quantitative flow cytometry. The percentage of CD34
+
cells among the
CD19
+
cells were measured before and after culturing, with and without treatment with Wnt3A (C). The bars represent the
mean of N experiments performed in duplicate, ± SEM. A) N = 6. B) Cells treated with control medium or Wnt3A: N = 11,
Wnt3A + sFRP1: N = 3, Wnt3A + Dkk1: N = 2. C) day 0: N = 7, day 7: N = 3, Day 14: N = 8. *p ≤ 0.01, Wilcoxon Signed
Ranks Test.
R
e
l
a
t
i
v
e
n
u
m
b
e
r
o
f
C
D
1
9
c
e
l
l
s
p
e
r
w
e
l
l
R
e
l
a
t
i
v
e
n
u
m
b
e
r
o
f
C
D
1
9
c
e
l
l
s
p
e
r
w
e
l
l
















*














*
B) CD10 B progenitor cells

A) CD133 CD10 hematopoietic progenitor cells


0714
















C) Percentage of CD34 cells among the CD19 cells

%
C
D
3
4
c
e
l
l
s
a
m
o
n
g
t
h
e
C
D
1
9
+
+
c
e
l
l
s
Days of culturing
Control Wnt3A
+ sFRP1
Wnt3A Wnt3A
+ Dkk1
Control Wnt3A

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
To check whether Wnt3A affected distinct early B subpop-
ulations differently, the cells in assay 2 were additionally
analyzed for expression of the CD34 differentiation
marker to distinguish between pro-B and pre-B cells. The
relative frequency of CD34
+
cells (pro-B) decreased from
38 % before culturing (day 0), to approximately 30 % and
15 % after one and two weeks of culturing, respectively.
This decrease was independent of treatment with or with-
out Wnt3A (fig. 4C). Furthermore, separation of the pre-B
population into large cycling and small resting pre-B cells
by surface expression of CD20 [33] revealed inhibitory
effect of Wnt3A on all subpopulations (results not
shown). Thus, we conclude that Wnt3A does not affect the
relative proportions of different BM B subpopulations,
but has a general inhibitory effect on pro-B, pre-B and
immature B cells in a stroma coculture.
Wnt3A inhibits BM B progenitor cell division in vitro
The inhibitory effect of Wnt3A on in vitro B lymphopoiesis
could be explained by increased apoptosis, an inhibitory
effect on proliferation, or both. However, measurements
of apoptosis in cells cultured without stromal cells for 1,
2 or 3 days showed no effect of Wnt3A (results not
shown), suggesting an effect on proliferation only. To ver-
ify this, we used high-resolution cell division tracking to
study the initial effects of Wnt3A on B progenitor cells
grown on a stromal layer. Sorted CFSE labeled CD10
+
B
progenitor cells were cocultured with MS-5 for 3 days in
the presence of Wnt3A or medium only, and examined for
the number of cell divisions by flow cytometry as well as
the surface markers CD34 and CD19. The data clearly
demonstrated that Wnt3A inhibited the initial divisions
of B progenitor cells taking place in the coculture (fig. 5A).
When gating for pro-B cells (CD34
+
CD19
+
) and pre-B
cells (CD34
-
CD19
+
) separately, we found that Wnt3A
inhibited proliferation of both these populations in a
dose-dependent manner (fig. 5B). This effect was blocked
Discussion
Several studies have identified the canonical Wnt pathway
as a regulator of the homeostasis of human and murine
HSC and hematopoietic progenitor cells [26,34,36]. Fur-
thermore, knockout studies (LEF-1 and Fzd9) in mice
have indicated a central role for Wnt signaling in B lym-
phopoiesis [24,27]. The Wnt pathway also seems to be
involved in development of leukemia [28-30]. In the
present work, we wanted to study in more detail the
implications of canonical Wnt signaling in human BM B
lymphopoiesis. Here, we describe that a set of Wnt lig-
ands, Fzd receptors and Wnt antagonists is expressed in
BM B progenitor cells, allowing a Wnt signal to be con-
veyed and modulated in these cells. We demonstrate reg-
ulated expression of several Wnt receptors, β-catenin and
plakoglobin as well as the transcription factors LEF-1 and
TCF-4 mRNAs during early differentiation steps in the B
cell lineage, supporting the hypothesis that Wnt signaling
is active in BM B lymphopoiesis. Furthermore, we show
that canonical Wnt signaling, as measured by the accumu-
lation of β-catenin levels, is induced in human BM B pro-
genitor cells. Finally, we demonstrate that Wnt3A inhibits
human stromal dependent B lymphopoiesis and that this
effect is a consequence of decreased cell proliferation.
We show that CD10
+
human B progenitor cells express a
set of Wnt ligand mRNAs (2B, 5B, 8A, 10A and 16), of
which Wnt16 is of particular interest, since this gene is
activated by the E2A-Pbx1 translocation in some cases of
acute lymphocytic leukaemia (ALL) [28]. However, sev-
eral pre-B leukemia cell lines studied [28] do not express
Wnt16, suggesting a distinct role for this factor in early B
lymphopoiesis that is turned off during leukemiagenesis,
except in cases where Wnt16 is aberrantly activated by the
E2A-Pbx1 fusion protein. Further, we demonstrate that
primary BM stromal cells express mRNA of several Wnt
ligands, including Wnt2B, Wnt5A, Wnt5B, Wnt8B and
Table 2: Number of CD19 cells after two weeks of culturing BM CD10
+
cells on stromal cells
Exp. No. (with MS5) Control-CM Wnt3A-CM Inhibition Index*
1 2182 ± 427 184 ± 91 0.08
2 9440 ± 1953 2652 ± 721 0.28
3 7292 ± 1928 2524 ± 475 0.35
Exp. No. (with BMS) Medium rmWnt3A Inhibition Index*
1 1746 ± 300 920 ± 64 0.53
BM CD10
+
cells were cultured on a layer of the murine stromal cell line MS-5 in the presence of Wnt3A-conditioned medium (Wnt3A-CM) or
control-conditioned medium (control-CM), or on a layer of human bone marrow stromal cells (BMS) in the presence of rmWnt3A or control
medium. The numbers in the table represent the mean of duplicate wells ± SD. *Number of CD19
+
cells in wells containing Wnt3A divided by
number of cells in wells containing control-medium.Page 8 of 17
(page number not for citation purposes)
by the Wnt antagonist sFRP1 (fig. 5B). Wnt9B. This is partly in accordance with previous studies
[24,26]. Taken together, these results show that both

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Page 9 of 17
(page number not for citation purposes)
Wnt3A inhibits the initial phase of stromal supported cell division of BM B progenitorsFigure 5
Wnt3A inhibits the initial phase of stromal supported cell division of BM B progenitors. Highly purified BM
CD10
+
CFSE
mean
cells were grown on a confluent layer of MS-5 and treated with Wnt3A (25–400 ng/ml) or medium only. After
three days, the cells were analyzed on a FACScan flow cytometer for the number of cell divisions of CD19
+
cells. A) Tracking
histograms of cell divisions of CFSE-labeled BM B progenitor cells in the presence or absence of Wnt3A (100 ng/ml) One rep-
resentative experiment of six is shown. B) Dose dependent inhibition of cell division of CD34
+
pro-B cells and CD34
-
pre-B
cells by Wnt3A (closed circles). The inhibitory effect of Wnt3A was blocked by Wnt antagonist sFRP1 (2 µg/ml) (open circle).
Data are shown as percentage of cells that had gone through one or more cell divisions, as determined by cell division tracking
with CFSE. One representative experiment of two is shown, except for Wnt3A (100 ng/ml) and Wnt3A + FRP1 (2 µg/ml)
where one representative experiment of six is shown (*p < 0.05, Wilcoxon Signed Ranks Test, n = 6).
%
c
e
l
l
s
w
i
t
h
1
o
r
m
o
r
e
c
e
l
l
d
i
v
i
s
i
o
n
s
0 100 200 300 400 500
5
10
15
20
25
30
35
40
*
*
Wnt3A (ng/ml)
Wnt3A (ng/ml)
B)
Pre-B cells (CD34CD19 )
-+
A)
Relative fluorescence (CFSE)
30
20
10
0
1 100010010
18.5%
N
u
m
b
e
r
o
f
c
e
l
l
s
30
20
10
0
32%
1 100010010
Control Wnt3A
0 100 200 300 400 500
15
20
25
30
35
40
45
50
*
*
Pro-B cells (CD34 CD19 )
++

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
hematopoietic cells and the supporting stromal cells may
produce Wnt ligands. Different Wnt ligands may have dis-
tinct effects during early B lymphopoiesis, which is a topic
for future investigations.
So far, only scarce knowledge is available about both lig-
and specificity and tissue-restricted expression of the Fzd
receptors. In our studies we found expression of a wide
range of Fzd receptor mRNAs, including Fzd2, 3, 4, 5, 6
and 9, in BM B progenitor cells. Compared to the Wnt
mRNAs, these are more readily detectable, indicating
higher expression levels, which suggests that Wnt-signal-
ing is important for B progenitor cells. Real-time PCR
assays demonstrated differential expression of several Fzd
receptor mRNAs, including Fzd5 and Fzd6, which are
strongly down-regulated as the cells become immature B
cells. Notably, LRP5 and LRP6 mRNAs showed a similar
down-regulation. Furthermore, both Fzd5 and Fzd9 are
up-regulated as the cells commit to the B cell lineage and
go through differentiation. Interestingly, Fzd9
-/-
mice
show a depletion of developing B cells in the BM, particu-
larly in the cycling pre-B population [27]. In contrast to
this, our results show that the large cycling pre-B cells
express lower levels of LRP5, LRP6, Fzd6, Fzd9, β-catenin
and plakoglobin than the small resting pre-B cells.
Although one should be cautious in trying to predict func-
tional consequences from mRNA expression data, this
trend suggests that Wnt signaling is not likely to be
involved in a positive regulation of cycling of the large
pre-B cells after Ig heavy chain rearrangement. And even
though the absolute expression levels of the receptor
mRNAs are low, these data suggest that during a narrow
window of the development comprising pro- and pre-B
cells, B progenitor cells might be target for Wnt signaling
through these receptors.
To be able to convey a Wnt-signal, the cells have to express
either of the two important molecules, β-catenin or pla-
koglobin. Our results show that levels of β-catenin mRNA
change little during the differentiation. Although it has
been demonstrated that levels of cytoplasmic β-catenin
protein may vary throughout the development of thymo-
cytes [37], these variations may not necessarily be
reflected by the mRNA levels. In fact, as β-catenin is
needed both for signaling purposes as well as for adhesion
purposes, the mRNA levels may have to be kept relatively
stable. Plakoglobin mRNA, on the other hand, decreases
after the pro-B differentiation level. This corresponds to
the observations made in developing murine thymocytes
[37], where plakoglobin is down-regulated at the level of
immature single positive thymocytes, suggesting that pla-
koglobin may play a central, but hitherto unexplored role
in conveying a Wnt signal during lymphopoiesis. In fact,
prompted the authors to suggest that plakoglobin may
stand-in for β-catenin in this respect.
The LEF-1/TCFs are directly activated by canonical Wnt
signaling, and LEF-1 knockout mice show defects in pro-B
cell proliferation and survival [24]. However, it cannot yet
be ruled out that this effect might be a result of abolish-
ment of the repressive functions or other non-Wnt related
activities of LEF-1 [15]. Here, we have verified microarray
data showing regulation of LEF-1 and TCF-4 during B lym-
phopoiesis (Hystad ME et al, manuscript in preparation
and [33]). Interestingly, it has been reported that LEF-1 is
a target gene for the B lymphopoiesis key transcription
factor Pax-5 [39]. Moreover, LEF-1 interacts with Pax-5
and c-Myb to activate the Rag-2 promoter [40], but the
accurate role of LEF-1 in B lymphopoiesis is still elusive.
In contrast to LEF-1, we found TCF-4 mRNA levels to be
high in ELP and pro-B cells, and lower in the more mature
pre-B and immature B populations. Although expressed at
lower levels, one could speculate that TCF-4 steps in for
LEF-1 in the earliest lymphoid progenitors before LEF-1 is
properly switched on, potentially in conveying a Wnt sig-
nal or, alternatively, in acting as a transcription repressor
of B lineage genes before commitment. These are topics
for further studies.
Wnt antagonists play important roles in preventing or fine
tuning the Wnt signal [16]. Our data show expression of
the Wnt antagonists Dkk1, Dkk4, sFRP4 and WIF1
mRNAs in B progenitor cells. Dkk1, sFRP2 and sFRP3
were expressed in bone marrow stromal cells. Of these fac-
tors, Dkk1 in particular is known to be involved in a feed-
back loop to adjust or shut down canonical Wnt signaling
[41]. It is likely that these factors are important in adjust-
ing the incoming Wnt signals in the bone marrow micro-
environment, where several cell types are able to express a
wide range of ligands and Wnt receptors.
The inhibitory effect of Wnt3A on the generation and cell
division of B progenitor cells in vitro, both with regard to
pro- and pre-B cells, is in contrast to several reports on the
functional effects of canonical Wnt signaling in mice.
Both in murine HSC [34], developing thymocytes [25]
and a wide range of cancer cells [31,42], elevated levels of
β-catenin lead to increased cell proliferation. Further-
more, in fetal murine pro-B cell [24], Wnt3A conditioned
medium leads to increased BrdU incorporation. Our
divergent results may be due to different species, microen-
vironments and/or cell context. For instance, murine and
human B lymphopoiesis require to a certain extent differ-
ing factor dependency [32]. However, by culturing murine
BM B progenitor cells, we have not been able to demon-
strate increased cell proliferation in the presence ofPage 10 of 17
(page number not for citation purposes)
the lack of effect of knocking down β-catenin in early
hematopoiesis, including B and T lymphopoiesis [38],
Wnt3A (results not shown). Thus, we suspect the Wnt
response to be different in fetal and adult B progenitor

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
cells, potentially affected by the cellular microenviron-
ment and/or context. Indeed, the fetal pro-B cells are
exposed to the microenvironment of the liver and this is
very different from that of the BM. For instance several
regulators of the Wnt pathway are more highly expressed
in fetal liver stroma than in BM stroma [43], which sug-
gest that Wnt signaling might be regulated in a different
manner and have a different role in the fetal liver than in
the BM. Another important aspect that has to be taken
into consideration, is that different Wnt ligands, although
able to activate canonical Wnt signaling, indeed show dis-
tinct activities [44]. In addition there may also be species
and location differences. However, as mentioned above,
Cobas et al have demonstrated a lack of an essential role
for β-catenin in BM hematopoiesis, including prolifera-
tion of B lymphocytes [38]. Thus, in contrast to findings
in the fetal liver, our results may very well represent a
physiological situation in the adult organism, where Wnt
signaling via β-catenin is not essential for B lymphocytes,
but may be used to fine tune the delicate balance between
proliferation, differentiation and apoptosis taking place
during early BM B lymphopoiesis.
In support of our data on an inhibitory effect of Wnt3A on
cell division, it has been reported that canonical Wnt sig-
naling hampers fibroblast cell proliferation through cell
cycle blocks, potentially mediated via p53 [45]. Moreover,
Wnt signaling inhibits proliferation and regulates cell-
cycle arrest at distinct stages of development in Dro-
sophila wing development [46]. Thus, it is likely that the
cellular context, in some cases represented by the ability of
a central regulatory molecule like p53 to respond, will
affect how the cells react to vital stimuli like Wnt. It has
been speculated that aberrant p53 is necessary to convey
the strong tumor promoting effect of abnormal Wnt sign-
aling seen in colon cancer [47,48]. It is also interesting
that Wnt5A has been found to inhibit B cell proliferation
and can function as a tumor suppressor in hematopoietic
tissue, albeit via the non-canonical Wnt/Ca
2+
pathway
[49].
We show expression of Wnt2B, 5B, 8A, 10A and 16 in BM
CD10
+
cells and of Wnt2B, Wnt5A, Wnt5B, Wnt8B and
Wnt9B mRNAs in human primary BMS cells. Further we
demonstrate that Wnt3A acts directly on B progenitor cells
by increasing the levels of β-catenin, suggesting that the
microenvironment may use Wnt signaling to regulate the
fate of developing B lymphocytes. Yet, we cannot exclude
that the functional effect of Wnt3A on in vitro B lym-
phopoiesis is indirect and mediated via the stromal cells,
as observed for in vitro hematopoiesis [35]. The BM micro-
environment is composed of a heterogeneous population
of cells including fibroblasts, adipocytes, endothelial cells
in adipogenesis may be relevant here, as it has been dem-
onstrated that Wnt10B [51,52] inhibits adipogenesis, and
there seems to be a positive correlation between adipo-
genesis and hematopoiesis [52]. This emphasizes the
complexity of the interactions in the B lymphopoiesis
maturation niche and opens for the possibility that B pro-
genitor cells may manipulate the stromal support via
these Wnt factors. However, it is not uncommon in devel-
opmental niches that morphogenic signals have the
potential to act on several cells in the microenvironment.
Therefore, it has been suggested that Wnt signaling might
influence the HSCs both directly and indirectly by main-
tenance of the cellular elements of the stem cell niche
[21]. In line with this theory, several studies have demon-
strated expression of multiple Wnt mRNAs in thymocytes
and the thymic microenvironment. It is likely that partic-
ular Wnts serve distinct roles, thus, cell specific effects may
be achieved by "playing the Wnt repertoire" as well as
through combinations with other signaling events.
Conclusion
In this study, we have demonstrated mRNA expression of
several Wnt ligands, Fzd receptors and Wnt antagonists in
human BM B progenitor cells and regulated expression of
Fzd receptors and co-receptors, β-catenin, plakoglobin,
LEF-1 and TCF-4 mRNA in these cells during differentia-
tion. Furthermore, we find that Wnt3A induced an accu-
mulation of β-catenin in the BM B progenitor cells and
inhibition of in vitro B lymphopoiesis. These results sug-
gest the Wnt/β-catenin pathway as a negative regulator of
human stromal dependent B lymphopoiesis. This is in
contrast to observations on Wnt effects in fetal murine
pro-B cells, and may represent a distinction between the
fetal liver and adult BM microenvironments.
Methods
Reagents and antibodies for FACS and western blot
analysis
Recombinant murine (rm) Wnt3A, recombinant human
(rh) secreted frizzled related protein 1 (sFRP1), rh Dick-
kopf 1 (Dkk1), rh interleukin (IL)-7, rh IL-3 and rh Flt3
ligand (FL) were purchased from R&D Systems (Great
Britain). The following monoclonal antibodies (mAbs)
were used for flow cytometry: anti-CD34 PE, anti-CD10
APC, anti-CD10 PE-Cy7 and anti-CD20 APC from Becton
Dickinson, Biosciences Pharmingen (San Jose, CA, USA),
anti-CD19 PE-Cy5 and anti-CD34 PE-Cy5 from Immu-
notech (Marseille, France) and anti-CD19 PE, anti-CD45
PE and anti-IgM Fitc from Dako Cytomation (Denmark).
Irrelevant isotype matched Abs were used as controls. The
following Abs were used in western blot analysis: anti-β-
catenin (Mouse IgG1, BD Transduction Laboratories),
anti-β-actin (Goat polyclonal IgG, Santa Cruz Biotechnol-Page 11 of 17
(page number not for citation purposes)
and osteoblasts, all derived from a common mesenchy-
mal precursor [50]. In particular, the role of Wnt signaling
ogy), rabbit anti-mouse IgG1-HRP and rabbit anti-goat
IgG-HRP (Dako cytomation, Denmark).

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Primary cells and cell lines
BM aspirates were from the iliac crest of normal adult vol-
unteers (approved by the Regional Ethical Committee).
Mononuclear cells (MNC) were separated by Ficoll-
Hypaque density gradient centrifugation (Lymphoprep,
Nycomed, Norway). CD10
+
B progenitor cells (ELP, pro-B
and pre-B cells) were isolated from BM MNC using Dyna-
beads
®
M-450 Epoxy (Dynal, Oslo, Norway) directly
coated with anti-CD10 mAb (clone RFAL-3, Sigma-
Aldrich, UK) followed by detachment using CD4/CD8
DETACHaBEAD (Dynal, Norway) according to the pro-
ducer 's protocol. The CD10
+
cells were of 90–95% purity,
they were CD45
+
and contained 4–7% IgM
+
cells (imma-
ture B cells). CD34
+
and CD19
+
cells were isolated in a
similar manner from MNC using Dynabeads
®
M-450 con-
jugated with anti-CD34 or anti-CD19 mAb, respectively,
and CD34 or CD19 DETACHaBEAD (Dynal, Norway),
respectively. CD133
+
CD10
-
cells (HPC) were isolated
from the CD10
-
fraction of BM MNC (see above) using the
MACS system (Magnetic cell sorting of human cells) and
a CD133 Cell Isolation Kit (Miltenyi Biotec, Germany).
Briefly, the mononuclear cells were magnetically labeled
with CD133 MicroBeads and separated on a column,
which was placed in the magnetic field of a MACS Separa-
tor. The magnetically labeled CD133
+
cells were retained
in the column while the unlabeled CD133
-
cells passed
through. After removal of the column from the magnetic
field, the magnetically retained CD133
+
cells were eluted
as the positively selected cell fraction. The CD133
+
cells
were typically of 97–98% purity. In monoculture, the cells
were kept in X-VIVO 15™ (BioWhittaker, Walkersville,
USA) with 0.1% detoxified BSA.
The murine stromal cell line MS-5 [53] was cultured in α-
MEM with 10% FCS and 100 µg/ml of penicillin and
streptomycin (PAA Laboratories, Pasching, Austria) and
was passaged twice a week. Cultures of human BM stro-
mal (BMS) cells were established as previously described
[54]. Briefly, total BM MNC cells depleted of CD34
+
cells
were seeded into 75-cm
2
tissue culture flasks in RPMI-
1640 with 10% FCS, penicillin and streptomycin. Non-
adherent cells were washed off after 2 hours at 37°C, and
the adherent cells were cultured in EX-CELL 610 (JRH Bio-
sciences, USA) with 10% FCS, penicillin and streptomy-
cin. The BMS cells were passaged twice before they were
used for experiments.
The human ALL cell lines Reh (no ACC 22, DSMZ) and
Nalm-6 (no ACC 128, DSMZ) (Hurwitz et al, 1979) were
kept in X-VIVO 15™ supplemented with 100 µg/ml of
penicillin and streptomycin.
FACS analysis and cell sorting
(argon-ion laser tuned at 488 nm; Becton Dickinson).
Quantitative analysis of CD19
+
cells in cocultures was per-
formed using Flow Cytometry Absolute Count Standard,
from Bangs Laboratories Inc., (Fishers, IN 46038 USA).
Data acquisition and analysis were performed using CEL-
LQuest software (Becton Dickinson).
Highly purified (98–99%) B progenitor cells for RT-PCR
analysis of Wnt, Fzd and Wnt antagonist mRNA expres-
sion were obtained by sorting of BM CD10
+
CD45
+
IgM
-
B
progenitor cells using a FACSDiVa flow cytometer (Becton
Dickinson, USA) after staining of BM CD34
+
and CD19
+
isolated cells with anti-CD45 PE, anti-CD10 APC and
anti-IgM FITC Abs. Highly purified BM cell populations
for real-time PCR were obtained by staining BM CD34
+
and CD19
+
cells with anti-CD10 PE-Cy7, anti-CD34 PE,
anti-CD19 PE-Cy5, anti-CD22 APC and anti-IgM Fitc Abs
and the following subpopulations were sorted using a
FACSDiVa flow cytometer: ELP (CD10
+
CD34
+
CD19
-
IgM
-
), pro-B (CD10
+
CD34
+
CD19
+
CD20
-
IgM
-
), large pre-B
(CD10
+
CD34
-
CD19
+
CD20
dim
IgM
-
), small pre-B
(CD10
+
CD34
-
CD19
+
CD20
-
IgM
-
) and immature B
(CD10
+
CD34
-
CD19
+
CD20
high
IgM
+
). Separation of large
and small pre-B cells was based on both CD20 expression
and size (forward scatter, FSC).
PCR analysis
Total RNA from freshly isolated and sorted BM
CD45
+
CD10
+
IgM
-
cells was isolated using Absolutely
RNA™ RT-PCR Mini-prep kit (Stratagene Europe, Amster-
dam, Netherland) according to the manufacturer's
instructions. RNA from human fetal brain was purchased
from BioChain Institute, Inc., USA. cDNA was synthesized
from 1 µg total RNA primed with random hexamers in a
50 µl reaction using TaqMan Reverse Transcription Rea-
gents (Applied Biosystems, Foster City, CA, USA). Control
reactions lacking reverse transcriptase were always
included. RT-PCR of 20 ng of total RNA was performed
with a titanium polymerase (BD Biosciences, USA) in a 25
µl reaction for 37 cycles at 95°C for 30 seconds, 60°C for
30 seconds, and 68°C for 30 seconds. The primer
sequences used to identify Wnt, Fzd and Wnt antagonist
gene expression are listed in Table 3. The primer
sequences was partly designed specifically for this work
and partly copied from previous expression analyses [55].
For all mRNAs expressed, the amplified products have
been sequenced and confirmed to represent the correct
target gene.
Real-time PCR
Total RNAs from 5–20 000 freshly isolated and sorted BM
B progenitor cells (ELP, pro-B, large pre-B, small pre-B and
immature B cells) were purified using Absolutely RNA™Page 12 of 17
(page number not for citation purposes)
Cells were stained with anti-CD19 PE Ab for 30 min at
4°C before analysis on FACSCalibur flow cytometer
RT-PCR Micro-prep kit (Stratagene Europe, Amsterdam,
Netherland) according to the manufacturer's instructions.

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
Table 3: Primer sequences used for mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonists
Primer Sequence Amplicon (bp)
Wnt1 F-5' TAG CCT CCT CCA CGA ACC TG-3' 239
F-5' CAG CCT CGG TTG ACG ATC TTG-3'
Wnt2 F-5' TGG TGG TAC ATG AGA GCT ACA GGT G-3' 297
R-5' CCC TGG TGA TGG CAA ATA CAA C-3'
Wnt2B F-5' TCA TGC TCA GAA GTA GCC GAG A -3' 328
R-5' TGG CAC TTA CAC TCC AGC TTC A -3'
Wnt3 F-5' CTG GCT ACC CAA TTT GGT GGT-3' 225
R-5' CAT CTA TGG TGG TGC AGT TCC A-3'
Wnt3A F-5' AAG CAG GCT CTG GGC AGC TA-3' 234
R-5' GAC GGT GGT GCA GTT CCA-3'
Wnt4 F-5' GAG GAG ACG TGC GAG AAA CTC AA-3' 346
R-5' ATC CTG ACC ACT GGA AGC CCT GT-3'
Wnt5A F-5' ATC CTG ACC ACT GGA AGC CCT GT-3' 358
R-5' GGC TCA TGG CGT TCA CCA C-3'
Wnt5B F-5' CAG CTT CTG ACA GAC GCC AAC T-3' 279
R-5' GCC TAT CTG CAT GAC TCT CCC A-3'
Wnt6 F-5' GCT CCA GCC ACA GCA AGG-3' 378
R-5' CAG CCT GCC CGC CTC GTT-3'
Wnt7A F-5' CCT GGG CCA CCT CTT TCT CAG-3' 573
R-5' TCC AGC TTC ATG TTC TCC TCC AG-3'
Wnt7B F-5' TTT CTC TGC TTT GGC GTC CT-3' 391
R-5' TGG TTG TAG TAG CCC TGC TTC TC-3'
Wnt8A F-5' TCC CAA GGC CTA TCT GAC CTA C-3' 400
R-5' CCG GCC CTG TTG TTG TGA-3'
Wnt8B F-5' GCC CAG AGT GGT ATT GAA GAA TG-3' 266
R-5' TTG TCA CTG CAG CCT CCC-3'
Wnt9A F-5' AAG TAC AGC AGC AAG TTC GTC AAG G-3' 538
R-5' GCA CTC CAC ATA GCA GCA CCA AC-3'
Wnt9B F-5' AGT TTC AGT TCC GGC ATG AGC-3' 340
R-5' TTC ACA GCC TTG ATG CCC A-3'
Wnt10A F-5' ACA CAG TGT GCC TAA CAT TGC C-3' 296
R-5' AGG CCT TCA GTT TGC CCA G -3'
Wnt10B F-5' CCT CGC GGG TCT CCT GTT C-3' 563
R-5' GGT TAC AGC CAC CCC ATT CC-3'
Wnt11 F-5' ACA ACC TCA GCT ACG GGC TCC T-3' 394
R-5' CCC ACC TTC TCA TTC TTC ATG C-3'
Wnt16 F-5' CTG TGC AAG AGG AAA CCG TAC CTG-3' 520
R-5' CAG CAC AGG AGC CGG AAA CT-3'
Fzd1 F-5' CTC TAC TTC TTC AGC ATG GCC A-3' 230
R-5' TCC ACG TTG TTA AGC CCC A-3'
Fzd2 F-5' CCA TCC TAT CTC AGC TAC AAG TTT CT-3' 306
R-5' GCA GCC CTC CTT CTT GGT-3'
Fzd3 F-5' TCC CCT CTG CCT GTA TGT GGT AGT-3' 622
R-5' GCT GCT CAC TTT GCT GTG GA-3'
Fzd4 F-5' CTC GGC TAC AAC GTG ACC AAG AT-3' 605
R-5' AAT ATG ATG GGG CGC TCA GGG TA-3'
Fzd5 F-5' GTG CCC ATT CTG AAG GAG TCA C-3' 197
R-5' TCC ATG TCG ATG AGG AAG GTG-3'
Fzd6 F-5' ACT CTT GCC ACT GTG CCT TTG-3' 300
R-5' GTC GAG CTT TTG CTT TTG CCT-3'
Fzd7 F-5' CAA GAC CGA GAA GCT GGA GAA G-3' 248
R-5' TGC CGA CGA TCA TGG TCA T-3'
Fzd8 F-5' GGA CTA CAA CCG CAC CGA CCT-3' 407
R-5' ACC ACA GGC CGA TCC AGA AGA C-3'
Fzd9 F-5' TCA AGG TCA GGC AAG TGA GCA-3' 210
R-5' AGC TTC CAG AGG AAC GCA ACA-3'
Fzd10 F-5' CAG GTG TGC AGC CGT AGG TTA A-3' 212
R-5' AAG CAC CAC ATC TTA GCT CCG G-3'Page 13 of 17
(page number not for citation purposes)
WIF1 F-5' ACG GAC CTC ACT GTG AGA AAG C-3' 200

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
cDNAs were synthesized from total RNA primed with ran-
dom hexamers using TaqMan Reverse Transcription Rea-
gents (Applied Biosystems, Foster City, CA, USA). LEF-1
and TCF-4 (gene name TCF-7L2) mRNA expression was
analyzed by real-time quantitative RT-PCR using Taqman
technology according to the manufacturers procedure
(Applied Biosystems). Predeveloped assay reagents
including primers and probes for LRP5
(Hs00182031_m1), LRP6 (Hs00233935_m1), Fzd2
(Hs00361432_s1), Fzd5 (Hs00361869_g1), Fzd6
(Hs00171574_m1), Fzd9 (Hs00268954_s1), β-catenin
(CTNNB1, Hs00170025_m1), plakoglobin (JUP,
Hs00158408_m1), LEF-1 (Hs00212390_m1) and TCF-4
(Hs00181036_m1) mRNAs as well as the endogenous
control phosphoglycerate kinase 1 (PGK1)
(Hs99999906_m1) were supplied by Applied Biosystems
and the PCR reactions were performed according to the
manufacturer's instructions using Taqman Universal PCR
Master Mix. Each measurement was performed in dupli-
cate and the expression level for each gene was calculated
using the standard curve method for relative quantitation
of gene expression as described by the manufacturer (ABI
Prism 7700 Sequence Detection System, User Bulletin 2,
PE Applied Biosystems, Foster City, CA). Total RNA from
the ALL cell lines Reh and Nalm6 as well as total RNA
from human fetal brain were used for standard curves.
Expression values for PGK1 mRNA, initially determined
to be a suitable endogenous control for BM populations,
were used for normalization of the expression levels. The
expression level of the different genes in pro-B cells was
used as a calibrator, and the expression of the other pop-
ulations were calculated relative to the expression in pro-
B cells.
Western blot analysis
The cells were treated with Wnt3A or vehicle only (PBS
with 0.1% detoxified BSA) for 3 hours and total cell
lysates were analyzed by Western blot using 10% SDS
containing 0.1% Tween-20 (PBS-T) and 5% dry milk,
incubated overnight with anti-β-catenin Ab or 1 hour with
anti-β-actin Ab and then washed 2 × 15 min in PBS-T. The
filters were then incubated with the secondary Ab rabbit
anti-mouse IgG1-HRP Ab or rabbit anti-goat IgG-HRP Ab,
respectively, for 60 minutes at room temperature and
washed 2 × 15 min in PBS-T before the proteins were vis-
ualized using ECL
+
Western Blotting Detection Reagents
from Amersham Biosciences (Piscataway, NJ, USA).
Hematopoietic cell-stromal cell coculture
Assay 1: HPC (CD133
+
CD10
-
) were cultured in 24 well
tissue plates (2000 cells/well) pre-seeded with MS-5 (2.5
× 10
4
cells/well). Assay 2: B progenitor cells (CD10
+
) were
cultured in 96 well tissue plates (8000 cells/well) pre-
seeded with MS-5 (1 × 10
4
cells/well). Both sets of cocul-
tures were in α-MEM containing 1% FCS, 100 µg/ml of
penicillin and streptomycin, and supplemented with
cytokines (for HPC: SCF, 25 ug/ml and G-CSF, 2,5 ug/ml,
for B progenitor cells: IL-7, 50 ng/ml, IL-3, 20 ng/ml and
FL, 50 ng/ml). In one additional experiment, the wells
were pre-seeded with BMS (1 × 10
4
cells/well) in EX-CELL
610 with 1% FCS, 100 µg/ml of penicillin and streptomy-
cin and cytokines (IL-7, 50 ng/ml, IL-3, 20 ng/ml and FL,
50 ng/ml). Where indicated, Wnt3A (10–100 ng/ml),
Dkk1 (500 ng/ml) or sFRP1 (2 µg/ml) were added to the
cultures. 50% of the medium was replaced weekly. After 3
(HPC) or 2 (B progenitor cells) weeks of culturing, single
wells were harvested by trypsination and the B progenitor
cells were immunophenotyped using the pan B cell
marker CD19 as well as the CD34 differentiation marker
and subjected to quantitative analyses (see above). Wnt3A
conditioned medium and control medium were collected
from L-Wnt3A cells and control nontransfected L-cells,
respectively (purchased from American Type Culture Col-
lection (ATCC), Manassas, USA), according to the manu-
facurer's procedure.
R-5' GCT GAT TTC ACA CTG CTC TCC C-3'
sFRP1 F-5' GGT CAT GCA GTT CTT CGG CT-3' 206
R-5' TCC TCA GTG CAA ACT CGC TG-3'
sFRP2 F-5' ACC GAG GAA GCT CCA AAG GTA T -3' 259
R-5' TCA TCT CCT CAC AGG TGC ACT G -3'
sFRP3 F-5' CTC ATC AAG TAC CGC CAC TCG TG-3' 210
R-5' CCG GAA ATA GGT CTT CTG TGT AGC TC-3'
sFRP4 F-5' GCA CAT GCC CTG GAA CAT CAC-3' 243
R-5' ATC TTC ATG AGG GGC TCG CAG T-3'
Dkk1 F-5' ACC ATT GAC AAC TAC CAG CCG T -3' 235
R-5' TGG TTT CCT CAA TTT CTC CTC G -3'
Dkk4 F-5' CGT TCT GTG CTA CAT GTC GTG G-3' 241
R-5' TCT TGT CCC TTC CTG CCT TGT-3'
Table 3: Primer sequences used for mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonists (Continued)polyacrylamide gels from Pierce (Rockford, USA) asPage 14 of 17
(page number not for citation purposes)
described earlier [56]. The filters were pretreated with PBS

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
High-resolution cell division tracking
BM CD34
+
and CD19
+
cells were labeled with 5- and 6-
carboxyfluorescein diacetate succinimidyl ester (CFSE;
Molecular Probes, Eugene, OR, USA) as described earlier
[57]. To allow unbound dye to diffuse from cells, labeled
cells were seeded on a confluent layer of MS-5 and incu-
bated for 18–24 hours at 37°C in α-MEM with 1% FCS.
Subsequently, the cells were stained with CD10 APC mAb
and CD10
+
CFSE
mean
cells were sorted on a BD FACSDiVa
flow cytometer (Becton Dickinson). Sorted cells (1.5–2 ×
10
4
/well) were cultured in 48 well tissue plates pre-seeded
with MS-5 (2 × 10
4
cells/well) supplemented with IL-7 (50
ng/ml) and FL (50 ng/ml) and treated with Wnt3A (25–
400 ng/ml), Wnt3A + sFRP1 (2 µg/ml) or medium only.
IL-3 was left out of these cultures, because earlier experi-
ments showed that IL-7 and FL were sufficient to support
survival and proliferation of the B progenitor cells (data
not shown). After three days the cells were harvested by
trypsination and analyzed on a FACSCalibur flow cytom-
eter for the number of cell divisions as well as expression
of the cell surface markers CD34 and CD19.
Statistical analysis
The statistical significance of differences between groups
was determined using the paired two-tailed Wilcoxon's
nonparametric test, by applying SPSS 11.5 software.
Abbreviations
Wnt, Wingless-type MMTV integration site family
BM, bone marrow
Fzd, Frizzled
HSC, hematopoietic stem cell
HPC, hematopoietic progenitor cell
CLP, common lymphoid progenitor
ELP, early lymphoid progenitor
IL-7, interleukin 7
IL-3, interleukin 3
FL, Flt3 ligand, Fms-related tyrosine kinase 3 ligand
SDF-1, stromal cell-derived factor 1
Dkk, Dickkopf
sFRP, soluble Fzd related protein
LRP, low density lipoprotein receptor-related protein
LEF-1, lymphoid enhancer-binding factor 1
TCF-4, transcription factor 4
WIF1, Wnt inhibitory factor 1
CFSE, carboxyfluorescein diacetate succinimidyl ester
ALL, acute lymphocytic leukaemia
CLL, chronic lymphocytic leukaemia
BSAP, B-cell lineage specific activator protein
Pax-5, paired box gene 5
Rag-2, Recombination-Activating Gene-2
Rm, recombinant murine
Rh, recombinant human
mAb, monoclonal antibody
MNC, mononuclear cells
BMS, bone marrow stroma
BSA, bovine serum albumin
PGK1, phosphoglycerate kinase 1
Authors' contributions
GD designed and conducted experiments, oversaw
research, and wrote paper. ET designed and conducted
experiments, oversaw research, and wrote paper. MKN
designed and conducted experiments, oversaw research.
HS designed and conducted experiments. SF designed
experiments, oversaw research, and wrote paper. ER
designed experiments, oversaw research, and wrote paper.
Acknowledgements
We are grateful to MDs Dag Josefsen and Gunnar Kvalheim, The Norwe-
gian Radium Hospital, for harvesting BM aspirates. We thank Mali Strand
Ellefsen, Hans Christian Dalsbotten Aass and Trond Stokke at the Flow
cytometry core facility for assistance in sorting the B progenitor cells. The
project was partly financed with grants from the Norwegian Cancer Society
and the Norwegian Research Council.
References
1. LeBien TW: Fates of human B-cell precursors. Blood 2000, 96:
9-23.
2. Bertrand FE, Eckfeldt CE, Fink JR, Lysholm AS, Pribyl JA, Shah N, LeB-Page 15 of 17
(page number not for citation purposes)
GSK3β, glycogen synthase kinase-3β
ien TW: Microenvironmental influences on human B-cell
development. Immunol Rev 2000, 175:175-186.

BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
3. Blom B, Spits H: Development of human lymphoid cells. Annu
Rev Immunol 2006, 24:287-320.
4. Torlakovic E, Tenstad E, Funderud S, Rian E: CD10+ stromal cells
form B-lymphocyte maturation niches in the human bone
marrow. J Pathol 2005, 205:311-317.
5. Tokoyoda K, Egawa T, Sugiyama T, Choi BI, Nagasawa T: Cellular
niches controlling B lymphocyte behavior within bone mar-
row during development. Immunity 2004, 20:707-718.
6. Hirose J, Kouro T, Igarashi H, Yokota T, Sakaguchi N, Kincade PW:
A developing picture of lymphopoiesis in bone marrow.
Immunol Rev 2002, 189:28-40.
7. Sitnicka E, Brakebusch C, Martensson IL, Svensson M, Agace WW,
Sigvardsson M, Buza-Vidas N, Bryder D, Cilio CM, Ahlenius H, Mar-
askovsky E, Peschon JJ, Jacobsen SE: Complementary signaling
through flt3 and interleukin-7 receptor alpha is indispensable
for fetal and adult B cell genesis. J Exp Med 2003, 198:
1495-1506.
8. Muench MO, Humeau L, Paek B, Ohkubo T, Lanier LL, Albanese CT,
Barcena A: Differential effects of interleukin-3, interleukin-7,
interleukin 15, and granulocyte-macrophage colony-stimu-
lating factor in the generation of natural killer and B cells
from primitive human fetal liver progenitors. Exp Hematol
2000, 28:961-973.
9. Namikawa R, Muench MO, de Vries JE, Roncarolo MG: The FLK2/
FLT3 ligand synergizes with interleukin-7 in promoting stro-
mal-cell-independent expansion and differentiation of
human fetal pro-B cells in vitro. Blood 1996, 87:1881-1890.
10. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T,
Bronson RT, Springer TA: Impaired B-lymphopoiesis, myelopoi-
esis, and derailed cerebellar neuron migration in C. Proc Natl
Acad Sci USA 1998, 95:9448-9453.
11. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kita-
mura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lym-
phopoiesis and bone-marrow myelopoiesis in mice lacking
the CXC chemokine PBSF/SDF-1. Nature 1996, 382:635-638.
12. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya
T, Yates JR III, Nusse R: Wnt proteins are lipid-modified and can
act as stem cell growth factors. Nature 2003, 423:448-452.
13. Papkoff J, Schryver B: Secreted int-1 protein is associated with
the cell surface. Mol Cell Biol 1990, 10:2723-2730.
14. Golan T, Yaniv A, Bafico A, Liu G, Gazit A: The human Frizzled 6
(HFz6) acts as a negative regulator of the canonical Wnt.
beta-catenin signaling cascade. J Biol Chem 2004, 279:
14879-14888.
15. Staal FJ, Clevers HC: WNT signalling and haematopoiesis: a
WNT-WNT situation. Nat Rev Immunol 2005, 5:21-30.
16. Kawano Y, Kypta R: Secreted antagonists of the Wnt signalling
pathway. J Cell Sci 2003, 116:2627-2634.
17. Wodarz A, Nusse R: Mechanisms of Wnt signaling in develop-
ment. Annu Rev Cell Dev Biol 1998, 14:59-88.
18. Moon RT, Brown JD, Torres M: WNTs modulate cell fate and
behavior during vertebrate development. Trends Genet 1997,
13:157-162.
19. Polakis P: Wnt signaling and cancer. Genes Dev 2000, 14:
1837-1851.
20. Reya T, Clevers H: Wnt signalling in stem cells and cancer.
Nature 2005, 434:843-850.
21. Rattis FM, Voermans C, Reya T: Wnt signaling in the stem cell
niche. Curr Opin Hematol 2004, 11:88-94.
22. Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X,
Cheng L: Defining the Role of Wnt/{beta}-Catenin Signaling in
the Survival, Proliferation and Self-Renewal of Human
Embryonic Stem Cells. Stem Cells 2005.
23. Ille F, Sommer L: Wnt signaling: multiple functions in neural
development. Cell Mol Life Sci 2005, 62:1100-1108.
24. Reya T, O'Riordan M, Okamura R, Devaney E, Willert K, Nusse R,
Grosschedl R: Wnt signaling regulates B lymphocyte prolifer-
ation through a LEF-1 dependent mechanism. Immunity 2000,
13:15-24.
25. Staal FJ, Meeldijk J, Moerer P, Jay P, van de Weerdt BC, Vainio S,
Nolan GP, Clevers H: Wnt signaling is required for thymocyte
development and activates Tcf-1 mediated transcription.
Eur J Immunol 2001, 31:285-293.
26. Van Den Berg DJ, Sharma AK, Bruno E, Hoffman R: Role of mem-
27. Ranheim EA, Kwan HC, Reya T, Wang YK, Weissman IL, Francke U:
Frizzled 9 knock-out mice have abnormal B-cell develop-
ment. Blood 2005, 105:2487-2494.
28. McWhirter JR, Neuteboom ST, Wancewicz EV, Monia BP, Downing
JR, Murre C: Oncogenic homeodomain transcription factor
E2A-Pbx1 activates a novel WNT gene in pre-B acute lym-
phoblastoid leukemia. Proc Natl Acad Sci USA 1999, 96:
11464-11469.
29. Lu D, Zhao Y, Tawatao R, Cottam HB, Sen M, Leoni LM, Kipps TJ,
Corr M, Carson DA: Activation of the Wnt signaling pathway
in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2004,
101:3118-3123.
30. Simon M, Grandage VL, Linch DC, Khwaja A: Constitutive activa-
tion of the Wnt/beta-catenin signalling pathway in acute
myeloid leukaemia. Oncogene 2005, 24:2410-2420.
31. Derksen PW, Tjin E, Meijer HP, Klok MD, MacGillavry HD, van Oers
MH, Lokhorst HM, Bloem AC, Clevers H, Nusse R, van der NR,
Spaargaren M, Pals ST: Illegitimate WNT signaling promotes
proliferation of multiple myeloma cells. Proc Natl Acad Sci USA
2004, 101:6122-6127.
32. Sitnicka E, Buza-Vidas N, Larsson S, Nygren JM, Liuba K, Jacobsen SE:
Human CD34+ hematopoietic stem cells capable of multilin-
eage engrafting NOD/SCID mice express flt3: distinct flt3
and c-kit expression and response patterns on mouse and
candidate human hematopoietic stem cells. Blood 2003, 102:
881-886.
33. van Zelm MC, van der BM, de Ridder D, Barendregt BH, de Haas EF,
Reinders MJ, Lankester AC, Revesz T, Staal FJ, van Dongen JJ: Ig gene
rearrangement steps are initiated in early human precursor
B cell subsets and correlate with specific transcription factor
expression. J Immunol 2005, 175:5912-5922.
34. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, Hintz
L, Nusse R, Weissman IL: A role for Wnt signalling in self-
renewal of haematopoietic stem cells. Nature 2003, 423:
409-414.
35. Yamane T, Kunisada T, Tsukamoto H, Yamazaki H, Niwa H, Takada
S, Hayashi SI: Wnt signaling regulates hemopoiesis through
stromal cells. J Immunol 2001, 167:765-772.
36. Austin TW, Solar GP, Ziegler FC, Liem L, Matthews W: A role for
the Wnt gene family in hematopoiesis: expansion of multilin-
eage progenitor cells. Blood 1997, 89:3624-3635.
37. Weerkamp F, Baert MR, Naber BA, Koster EE, de Haas EF, Atkuri KR,
van Dongen JJ, Herzenberg LA, Staal FJ: Wnt signaling in the thy-
mus is regulated by differential expression of intracellular
signaling molecules. Proc Natl Acad Sci USA 2006, 103:3322-3326.
38. Cobas M, Wilson A, Ernst B, Mancini SJ, MacDonald HR, Kemler R,
Radtke F: Beta-catenin is dispensable for hematopoiesis and
lymphopoiesis. J Exp Med 2004, 199:221-229.
39. Nutt SL, Morrison AM, Dorfler P, Rolink A, Busslinger M: Identifica-
tion of BSAP (Pax-5) target genes in early B-cell develop-
ment by loss- and gain-of-function experiments. EMBO J
1998, 17:2319-2333.
40. Jin ZX, Kishi H, Wei XC, Matsuda T, Saito S, Muraguchi A: Lym-
phoid enhancer-binding factor-1 binds and activates the
recombination-activating gene-2 promoter together with c-
Myb and Pax-5 in immature B cells. J Immunol 2002, 169:
3783-3792.
41. Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki Y, Sug-
ano S, Akiyama T: DKK1, a negative regulator of Wnt signaling,
is a target of the beta-catenin/TCF pathway. Oncogene 2004,
23:8520-8526.
42. Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL,
Gotlib J, Li K, Manz MG, Keating A, Sawyers CL, Weissman IL: Gran-
ulocyte-macrophage progenitors as candidate leukemic
stem cells in blast-crisis CML. N Engl J Med 2004, 351:657-667.
43. Martin MA, Bhatia M: Analysis of the human fetal liver hemat-
opoietic microenvironment. Stem Cells Dev 2005, 14:493-504.
44. Castelo-Branco G, Wagner J, Rodriguez FJ, Kele J, Sousa K, Rawal N,
Pasolli HA, Fuchs E, Kitajewski J, Arenas E: Differential regulation
of midbrain dopaminergic neuron development by Wnt-1,
Wnt-3a, and Wnt-5a. Proc Natl Acad Sci USA 2003, 100:
12747-12752.
45. Damalas A, Kahan S, Shtutman M, Ben Ze'ev A, Oren M: Deregu-
lated beta-catenin induces a p53- and ARF-dependentPage 16 of 17
(page number not for citation purposes)
bers of the Wnt gene family in human hematopoiesis. Blood
1998, 92:3189-3202.
growth arrest and cooperates with Ras in transformation.
EMBO J 2001, 20:4912-4922.

Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
BMC Immunology 2006, 7:13 http://www.biomedcentral.com/1471-2172/7/13
46. Johnston LA, Edgar BA: Wingless and Notch regulate cell-cycle
arrest in the developing Drosophila wing. Nature 1998, 394:
82-84.
47. Levina E, Oren M, Ben Ze'ev A: Downregulation of beta-catenin
by p53 involves changes in the rate of beta-catenin phospho-
rylation and Axin dynamics. Oncogene 2004, 23:4444-4453.
48. Thorstensen L, Lind GE, Lovig T, Diep CB, Meling GI, Rognum TO,
Lothe RA: Genetic and epigenetic changes of components
affecting the WNT pathway in colorectal carcinomas strati-
fied by microsatellite instability. Neoplasia 2005, 7:99-108.
49. Liang H, Chen Q, Coles AH, Anderson SJ, Pihan G, Bradley A, Ger-
stein R, Jurecic R, Jones SN: Wnt5a inhibits B cell proliferation
and functions as a tumor suppressor in hematopoietic tissue
. Cancer Cell 2003, 4:349-360.
50. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD,
Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage
potential of adult human mesenchymal stem cells. Science
1999, 284:143-147.
51. Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lucas PC, Opp
MR, MacDougald OA: Wnt10b inhibits development of white
and brown adipose tissues. J Biol Chem 2004, 279:35503-35509.
52. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL,
MacDougald OA: Inhibition of adipogenesis by Wnt signaling.
Science 2000, 289:950-953.
53. Itoh K, Tezuka H, Sakoda H, Konno M, Nagata K, Uchiyama T, Uchino
H, Mori KJ: Reproducible establishment of hemopoietic sup-
portive stromal cell lines from murine bone marrow. Exp
Hematol 1989, 17:145-153.
54. Pribyl JAR, Shah N, Dittel BN, LeBien TW: Methods for purifica-
tion and growth of human B cell precursors in bone marrow
stromal cell-dependent cultures. In The Immunology Methods
Manual 2nd edition. Edited by: Lefkovits I. San Diego, CA: Academic;
1997:903-923.
55. Etheridge SL, Spencer GJ, Heath DJ, Genever PG: Expression pro-
filing and functional analysis of wnt signaling mechanisms in
mesenchymal stem cells. Stem Cells 2004, 22:849-860.
56. Myklebust JH, Smeland EB, Josefsen D, Sioud M: Protein kinase C-
alpha isoform is involved in erythropoietin-induced eryth-
roid differentiation of CD34(+) progenitor cells from human
bone marrow. Blood 2000, 95:510-518.
57. Bryder D, Jacobsen SE: Interleukin-3 supports expansion of
long-term multilineage repopulating activity after multiple
stem cell divisions in vitro. Blood 2000, 96:1748-1755.yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Page 17 of 17
(page number not for citation purposes)