1 Department of Pathology and Immunology, 660 South Euclid Avenue, Campus Box
8118, St Louis, MO 63110, USA
2 Developmental Biology Program, Washington University School of Medicine, 660
South Euclid Avenue, Campus Box 8118, St Louis, MO 63110, USA
3 Center for Vascular Biology, University of Connecticut Health Center, CT
06030, USA
4 Department of Biosciences, AstraZeneca R&D Lund, Sweden
Author for correspondence (e-mail:
kchoi{at}immunology.wustl.edu)
Accepted 17 February 2004
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SUMMARY |
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Key words: Hematopoiesis, Vasculogenesis, FLK1, SCL, BMP4, VEGF, TGFß1
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Introduction |
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Although these studies support the notion that TGFß family members and
bFGF can induce hematopoietic differentiation, they do not provide mechanisms
involved in specifying hematopoietic cell lineages. In addition, these studies
are limited to erythroid cell induction. Thus, it is still unclear how the
generation of the earliest cell population, which is committed to become
hematopoietic and endothelial cells, is regulated. Gene targeting studies
indicate that FLK1, a receptor tyrosine kinase, and SCL (TAL1 Mouse
Genome Informatics), a basic helix-loop-helix transcription factor, are
required at the initial stages of the establishment of hematopoietic and
endothelial cell development. In mice, Flk1 expression can be
detected in the presumptive mesodermal yolk sac blood island progenitors as
early as embryonic day (E) 7 (Yamaguchi et
al., 1993; Dumont et al.,
1995
). Consistent with its expression pattern,
Flk1-deficient mice display defects in blood vessels and yolk sac
blood island formation and die between E8.5 and E9.5
(Shalaby et al., 1995
).
Furthermore, Flk1/ ES cells fail to
participate in vessel formation or contribute to primitive or definitive
hematopoiesis in chimeras generated with wild-type embryos, suggesting a cell
autonomous requirement of FLK1 in hematopoietic and endothelial cell
development (Shalaby et al.,
1997
). Scl-deficient mice also exhibit defects in
hematopoietic and endothelial cell lineages and die around E10.5
(Robb et al., 1995
;
Shivdasani et al., 1995
). The
endothelial cell defects in these mice are in the remodeling of the primary
vascular plexus in the yolk sac (Visvader
et al., 1998
).
In an effort to analyze hematopoietic and endothelial cell differentiation
more systematically, we recently examined the developmental kinetics of the
expression of FLK1 and SCL by using in vitro differentiated knock-in ES cells
that express a non-functional human CD4 at the Scl locus
(Chung et al., 2002). We
demonstrated that CD4-expressing cells from in vitro differentiated
Scl+/CD4 ES cells (embryoid bodies, EBs) correlated with
that of Scl and reported that hematopoietic and endothelial cells
developed via sequentially generated FLK1 and SCL-expressing cells.
FLK1+SCL cells first emerged in differentiating
ES cells followed by FLK1+SCL+ cells, which developed
from FLK1+ cells. FLK1SCL+ cells
ultimately developed from FLK1+SCL+ cells by
downregulating FLK1. Thus, the formation of FLK1- and SCL-expressing cells
marks the onset of hematopoietic and endothelial cell differentiation. In this
study, we used an in vitro serum-free differentiation model of ES cells
(Adelman et al., 2002
) to
identify factors regulating the onset of hematopoietic and endothelial cell
lineage differentiation. We show that BMP4 was required for the generation of
the FLK1+ and SCL+ cells. We also show that VEGF, via
FLK1-mediated signals, is required for the expansion of hematopoietic and
endothelial cell progenitors. Finally, we demonstrate that the generation of
SCL+ cells by BMP4 and VEGF was inhibited by TGFß1, but
augmented by activin A. Collectively, our studies reveal a temporal,
hierarchical order of factors that function to establish hematopoietic and
endothelial cell lineages.
In this paper, CD4 refers to the non-functional human CD4 gene/protein.
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Materials and methods |
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Hematopoietic colonies were generated as described previously
(Faloon et al., 2000).
Briefly, cells obtained from day 5-6 EBs were replated in methyl cellulose
containing 10% plasma-derived serum (PDS, Antech; Texas), 5% protein-free
hybridoma medium (PFHM2, Gibco/BRL), L-glutamine (2 mM), transferrin (300
µg/ml; Boehringer Mannheim) and MTG (4.5x104 M),
together with the following cytokines: kit ligand (KL, 1% conditioned medium),
IL3 (1% conditioned medium), IL1 (5 ng/ml), IL6 (5 ng/ml), IL11 (5 ng/ml), Epo
(2 units/ml), MCSF (5 ng/ml), GSCF (2 ng/ml) and GMCSF (3 ng/ml).
Hematopoietic colonies were counted 5-7 days later. IL1, IL6, IL11, GCSF and
MCSF were purchased from R&D Systems. KL was obtained from medium
conditioned by CHO cells transfected with a KL expression vector (kindly
provided by Genetics Institute). EPO was purchased from Amgen (Thousand Oaks,
CA) and IL3 was obtained from medium conditioned by X63 Ag8-653 myeloma cells
transfected with a vector expressing IL3
(Karasuyama and Melchers,
1988
).
ES clones expressing SMAD6 were generated by electroporating ES cells with a linearized Flag-mouse SMAD6 expressing construct (kindly provided by Dr Miyazono at University of Tokyo, Japan). ES cells were selected with G418 (500 µg/ml). Clones were verified by a western blot analysis with an anti-Flag-tag antibody (Sigma).
FACS analysis
EB cells were dissociated with 7.5 mM EDTA/PBS (pH 7.4) for 2 minutes.
Cells were centrifuged, resuspended in staining/wash buffer (4% FCS in PBS),
passed through a 20-gauge needle four or five times to generate a single cell
suspension, and the cell number was counted. After centrifugation, cells were
resuspended at a density of 5x106 cells/ml in staining/wash
buffer. Cells were placed into each well of V-shaped 96-well plate at
5x105 cells/well. For a single color staining for FLK1,
biotinylated anti-FLK1 antibody, freshly diluted (1:1000) in staining/wash
buffer, was added and incubated for 15 minutes on ice. Subsequently, cells
were washed three times with staining/wash buffer. Streptavidin-phycoerythrin
(secondary reagent) (Pharmingen), freshly diluted in staining/wash buffer, was
added and incubated on ice for 15 minutes in the dark. Cells were washed three
times, re-suspended in staining/wash buffer, and transferred to 5 ml
polypropylene tubes for analysis. A double-color staining for human CD4 and
FLK1 was carried out by staining the cells with biotinylated mouse anti-human
CD4 monoclonal antibody (CALTAG), followed by streptavidin-allophycocyanin
(Sav-APC; Pharmingen) and phycoerythrin (PE)-conjugated anti-FLK1 monoclonal
antibody (Pharmingen). A three-color FACS analysis of FLK1, human CD4 and
TER119 was carried out by staining the cells first with biotinylated mouse
anti-human CD4 monoclonal antibody and anti-mouse TER119 antibody
(Pharmingen), followed by FITC-conjugated goat anti-rat IgG2b
(Pharmingen). Finally, Sav-APC and PE-conjugated anti-FLK1 monoclonal antibody
were added. A three-color FACS analysis of FLK1, CD4 and CD31 was carried out
by staining the cells first with biotinylated mouse anti-CD4 monoclonal
antibody and FITC-conjugated anti-mouse CD31 (Pharmingen), followed by Sav-APC
and PE-conjugated anti-FLK1 monoclonal antibody. Cells were analyzed on a FACS
Caliber (Becton-Dickinson), and FACS data were analyzed with CellQuest
software (Becton-Dickinson).
Biochemical analysis
For detecting SMAD1/5 phosphorylation, ES cells were differentiated in
serum-free conditions. Basic FGF (10 ng/ml) or BMP4 (5ng/ml) was added at the
onset of differentiation (day 0). EBs were harvested on day 1.5 or 2.75 and
lysed in 1x RIPA-B buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1%
NP-40; 0.5% deoxycholate) containing protease inhibitor cocktail (Roche), NaF,
NaOVa and phosphatase inhibitor cocktail I (Sigma). Alternatively, EBs were
generated in serum-free conditions in the absence of exogenously added
factors, collected on day 1.5, stimulated with bFGF or BMP4 for 30 to 60
minutes at 37°C, and lysed as described above. For Erk and AKT
phosphorylation detection, ES cells were differentiated in serum-free
conditions with BMP4. Three days after, EBs were harvested, washed off three
times, and then cultured for overnight in IMDM. The following day, EBs were
harvested and stimulated with VEGF for 30 minutes.
Cleared cell lysates after centrifugation were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting.
Blots were blocked in TBS containing 5% nonfat milk and 0.5% Tween 20 for 1
hour at room temperature and incubated with indicated antibodies overnight.
Antibodies used were as follows: rabbit anti-pSMAD1/5
(Rosendahl et al., 2002),
rabbit anti-SMAD1 (Upstate Biotechnique), mouse anti-phospho Erk1/2 (Santa
Cruz), rabbit anti-Erk1/2 (Santa Cruz), rabbit anti-pAKT1/2/3 (Santa Cruz) and
rabbit anti-AKT1/2/3 (Cell Signaling). One percent BSA was used for blocking
and antibody incubation instead of 5% skim milk for detecting phospho-SMAD1/5
bands. Blots were then washed and incubated with horseradish-peroxidase
conjugated anti-mouse (Sigma) or anti-rabbit (Santa Cruz) IgG antibodies for 1
hour at room temperature. Immunodetection was achieved by using an ECL-plus
detection system (Amersham).
Gene expression analysis
ES cells were differentiated in the presence of fetal calf serum (FCS) or
serum replacement (SR) media with or without BMP4. EBs were collected at
different time points as noted and RNA was purified following the Triazol
protocol (Gibco-BRL). All RNA samples were treated with DNaseI (amplification
grade from Gibco-BRL) before cDNA synthesis to eliminate any contaminating
genomic DNA. Semi-Quantitative RT-PCR was performed as described
(Choi et al., 1998;
Faloon et al., 2000
). cDNA
normalization was carried out with ß-actin gene by using Phosphorimager
Storm 840 and ImageQuant software. Specific primers used are as follows
(Choi et al., 1998
;
Faloon et al., 2000
).
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Results |
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To investigate the mechanism of how BMP4 induces FLK1+ cells, ES
cells were differentiated in serum-free conditions, with or without BMP4, and
subjected to gene expression analyses. As shown in
Fig. 2B, both T and
Gata4 genes, expressed in mesoderm and visceral endoderm
(Arceci et al., 1993),
respectively, were expressed at low levels in EB cells differentiated in
serum-free conditions. However, Fgf5, a marker for ectoderm
(Haub and Goldfarb, 1991
;
Hebert et al., 1991
), was
expressed at high levels in these cells indicating that ES cells readily gave
rise to ectoderm in serum-free conditions. When ES cells were differentiated
in the presence of BMP4, both T and Gata4 genes were
upregulated. However, Fgf5 expression was downregulated. This
suggested that BMP4 functions to induce mesoderm at the expense of ectoderm.
Moreover, the kinetics of T and Flk1 expression in BMP4 were
similar to that of serum, such that the downregulation of T coincided
with the up regulation of Flk1 gene expression. This suggested that
BMP4 could also induce FLK1+ cells from the mesoderm. Thus, we
determined if T expressing EB cells from days 2-3 were still able to
respond to BMP4 to generate FLK1+ cells. Rex1 is
downregulated at these time points. As shown in
Fig. 1C, we found similar
percentage of FLK1+ cells as long as BMP4 was added up to day 3.
The percentage of FLK1+ cells was lower when BMP4 was added on day
4 or 5 compared with earlier time points. This could be interpreted that
T-expressing mesoderm lost the ability to respond to BMP4.
Alternatively, this could be due to a decrease in the number of mesodermal
cells, which respond to BMP4. The latter view is supported by greater level of
cell death of EB cells at later time points in the absence of any added
factors (not shown). Together, this suggests that BMP4 functions at two
distinct steps. First, BMP4 can induce mesoderm. Second, BMP4 can also induce
FLK1+ cells from mesoderm.
VEGF is required for expansion of hematopoietic and endothelial progenitors
Our studies indicate that BMP4 was required for FLK1+ cell
generation. To determine if BMP4 was also necessary for SCL-expressing cell
development, we examined CD4-expressing cells from in vitro
differentiated Scl+/CD4 ES cells
(Chung et al., 2002). As shown
in Fig. 3A, very few
FLK1+ or CD4 (i.e. SCL)-expressing cells were detectable when ES
cells were differentiated in serum-free conditions. When ES cells were
differentiated in the presence of BMP4, cells expressing CD4 were detectable
at low levels in day 5-6 Ebs (5-6%, Fig.
3A,B) and increased at day 7 (11-12%,
Fig. 3B,C). This suggested that
BMP4 could induce the generation of SCL-expressing cells. However, the
percentage of cells expressing CD4 in the presence of BMP4 was much lower
compared with that from the serum control (
6% versus
40%,
respectively, Fig. 3A). Thus,
we searched for additional factor(s) that could cooperate with BMP4 to
generate CD4-expressing cells. As shown in
Fig. 3A, the addition of BMP2,
bFGF, activin A or TGFß1 did not affect the generation of CD4-expressing
cells. However, the level of cells expressing CD4 dramatically increased when
both BMP4 and VEGF were added to serum-free differentiation conditions.
Importantly, the CD4+ levels approximated the level of serum
differentiation (
49%). The percentage of CD4+ cells increased
up to 10 ng/ml of VEGF in the presence of BMP4. VEGF at higher concentrations
(50 ng/ml) did not further increase CD4+ cells (data not
shown).
|
In an effort to determine if VEGF is constitutively required for the
generation of SCL-expressing cells, we determined the window of time in which
VEGF can function to generate SCL-expressing cells. Our experimental strategy
was to differentiate ES cells in serum-free conditions in the presence of BMP4
and VEGF up to day 3 or day 4. Factors were then washed out, and
CD4-expressing cells were analyzed on day 5.5
(Fig. 4A, upper panel).
Alternatively, ES cells were differentiated with BMP4 alone, VEGF was added on
day 3 or day 4, and CD4-expressing cells were analyzed on day 5.5
(Fig. 4A, lower panel). This
scheme is based on our previous studies that FLK1-expressing cells emerged
between days 1.5 and 2 of EB differentiation, expanded up to day 4, and then
decreased (Chung et al., 2002).
When BMP4 and VEGF were removed on day 3
(Fig. 4A, upper middle panel),
there was a remarkable decrease in CD4+ cells. However, the removal
of BMP4 and VEGF on day 4 (Fig.
4A, upper right panel) did not affect the percentage of
CD4+ cells generated compared with the control
(Fig. 4A, upper left panel).
Conversely, when VEGF was added on day 3, CD4+ cells developed as
expected (Fig. 4A, lower middle
panel), while the addition of VEGF on day 4 did not augment CD4+
cell numbers significantly (Fig.
4A, lower right panel). We next determined if VEGF induced
SCL-expressing cells between days 3 and 4 were sufficient to generate mature
hematopoietic and endothelial cells. Similar to previous experiments, BMP4 and
VEGF were added at the time of differentiation and removed on day 3 or day 4,
and EB cells were subjected to FACS analyses for TER119 (erythroid) or CD31
(endothelial) expression on day 6. As shown in
Fig. 4B, both TER119 and
CD31-expressing cells decreased greatly when BMP4 and VEGF were removed on day
3. However, the percentage of both TER119+ and CD31+
cells was largely unchanged when BMP4 and VEGF were removed on day 4.
Collectively, our findings suggested that VEGF is only required for a short
time to induce SCL, TER119 and CD31-expressing hematopoietic and endothelial
cells.
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|
The cell analyses suggested that VEGF is required for the expansion of SCL-expressing cells. As the MAPK pathway and AKT activation via PI3 kinase have been implicated in cell proliferation and cell survival, we examined ERK1/2 and AKT phosphorylation by VEGF. To achieve this, ES cells were differentiated in serum-free conditions with BMP4. Three days later, BMP4 was washed out overnight and EBs were treated with VEGF for 30 minutes. Cells were then collected and analyzed for ERK1/2 phosphorylation. The AKT phosphorylation was also measured to determine if the PI3-kinase pathway was activated. As shown in Fig. 5C, ERK1/2 phosphorylation was induced when EBs were stimulated with VEGF, while AKT phosphorylation was not affected. Collectively, our studies indicate that the generation of FLK1+ and SCL+ cells by BMP4 and VEGF involves the activation of the SMAD1/5 and MAP kinase pathways, respectively.
ES cells over expressing SMAD6 display defects in FLK1+ cell generation
The finding that the SMAD1/5 pathway was activated at the time of
FLK1+ cell formation suggested that it plays a functional role in
this process. To determine whether the SMAD1/5 activation by BMP4 signaling is
crucial for FLK1+ cell development, we generated ES clones over
expressing SMAD6, which inhibits either the recruitment of SMAD1 to the
receptor or the heterodimer formation between phosphorylated SMAD1 and SMAD4
(Imamura et al., 1997;
Hata et al., 1998
). If
activation of the SMAD1/5 pathway is crucial for BMP4-mediated
FLK1+ cell generation, the overexpression of SMAD6 should block the
formation of FLK1+ cells. As shown in
Fig. 6, FLK1+ cells
developed as expected from control clones (Flag 6 and Flag 8). However, SMAD6
overexpressing clones (Flag SMAD6-6 and Flag SMAD7, and data not shown) were
not able to respond to BMP4 and failed to generate FLK1+ cells in
serum-free conditions. Importantly, SMAD6 overexpressing clones generated much
lower FLK1+ cells even in the full complement of serum compared
with controls (Fig. 6).
Therefore, activation of the SMAD1/5 pathway is crucial for FLK1+
cell formation.
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To compare the generation of SCL-expressing cells to hematopoietic and endothelial cell differentiation, we examined day 6.5 EB cells differentiated with BMP4+VEGF for TER119 and CD31 as a measure of hematopoietic (erythroid) and endothelial cell differentiation, respectively (Fig. 9B). As seen before in Fig. 4, TER119-expressing cells increased significantly in the presence of BMP4+VEGF compared with BMP4 alone. CD31+ cells also increased in the presence of BMP4+VEGF compared with BMP4 alone. Importantly, when TGFß1 was added to BMP4 + VEGF, the generation of TER119+ cells decreased considerably. CD31+ cells also decreased in response to TGFß1, although the decrease in CD31+ cells by TGFß1 was not as evident as in the TER119+ cells.
To verify the FACS data, EB cells generated in the presence of BMP4, BMP4+VEGF, BMP4+VEGF+TGFß1 or BMP4+VEGF+activin A were subjected to hematopoietic replating (Fig. 9C). As shown, erythroid, macrophage and erythroid/macrophage bipotential colonies all developed in BMP4 alone. Importantly, the number of these colonies increased when VEGF was added to the BMP4 culture. Again, TGFß1 inhibited the generation of hematopoietic progenitors. We again saw a slight increase of hematopoietic differentiation by activin A. There did not seem to be a qualitative difference in the generation of primitive and definitive erythroid progenitors by BMP4 or BMP4+VEGF, as both types of colonies were present in EBs generated in BMP4 or BMP4+VEGF (data not shown). Collectively, our data demonstrate that efficient hematopoietic and endothelial cell generation requires an ordered function of BMP4, VEGF, TGFß1 and activin A. BMP4 is required initially, followed by VEGF. TGFß1 and activin A then modulate the function of VEGF by inhibiting or enhancing the generation of SCL-expressing cells.
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Discussion |
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Our kinetic studies show that T was induced in EBs differentiated
in BMP4. Furthermore, we also observed that FLK1+ cells still
developed when BMP4 was added to EB cells expressing T but not
Rex1. This suggests that BMP4 functions at two distinct steps. First,
BMP4 can induce mesoderm. Second, BMP4 can also induce FLK1+ cells
from mesoderm. It is not clear whether these FLK1-expressing cells still
represent the mesoderm. Fehling et al.
(Fehling et al., 2003)
recently showed that T-expressing, but FLK1, cells
progressed to give rise to T- and FLK1-expressing cells. Therefore, it is
possible that FLK1 expression can specify a subset of mesoderm that ultimately
generates the hematopoietic and vascular systems.
Our studies have demonstrated that ES cells differentiated in serum-free
conditions in the presence of BMP4 alone could still give rise to
SCL-expressing cells and hematopoietic progenitors. In addition, we observed
SCL gene induction in Flk1/ EBs
differentiated in BMP4 (data not shown). Previous studies also support the
idea that BMP4-mediated signals are required for hematopoietic specific gene
expression. For example, Johansson and Wiles
(Johansson and Wiles, 1995)
reported that BMP4 could induce T and ß-H1globin from ES cells
differentiated in chemically defined medium. Furthermore, Adelman et al.
(Adelman et al., 2002
) reported
that BMP4 could induce expression of Eklf and Gata1
erythroid-specific genes. Whether BMP4 can induce hematopoietic cells directly
from mesoderm is not clear. All of these studies, including our own, used an
ES/EB system, in which hematopoietic differentiation occurs via the mesoderm
followed by generation of FLK1 expressing cells. Thus, future studies are
required to examine if BMP4 can directly induce Scl within
FLK1+ cells.
Gene targeting studies largely support the notion that BMP4-mediated
signals are important for hematopoietic and vascular development. For example,
Bmp4-deficient mice die between E7.5 and E9.5 with defects in
mesoderm formation and patterning. Those that survive up to E9.5 show severe
defects in blood island formation (Winnier
et al., 1995). Additionally, mice lacking the type I BMP receptor
(Alk3) fail to complete gastrulation and die by E9.5
(Mishina et al., 1995
). Mice
deficient in Smad1 or Smad5, downstream signaling molecules
of TGFß family members, display varying degrees of defects in
hematopoietic and vascular development, perhaps owing to overlapping function
between SMAD1, SMAD5 and SMAD8 (Tremblay
et al., 2001
). For example, Smad1-deficient mice display
defects in chorioallantoic fusion and die between E9.5 and E10.5
(Tremblay et al., 2001
;
Lechleider et al., 2001
).
Although overall hematopoietic and vascular development appears to be normal,
some Smad1-deficient embryos display defects in yolk sac angiogenesis
(Lechleider et al., 2001
).
Smad5-deficient mice also show early embryonic lethality. The
primitive plexus can be found in mutant embryos, but they fail to form
organized vessels. There seems to be more primitive blood cells in E8.5 mutant
yolk sacs, although E9.5 mutant yolk sacs contained almost none
(Chang et al., 1999
).
Furthermore, Smad5-deficient yolk sacs contained higher frequency of
high-proliferative potential colony forming cells (HPP-CFCs) and
Smad5-deficient ES cells gave rise to an increased number of
hematopoietic progenitors including blast colonies in vitro
(Liu et al., 2003
).
Collectively, these studies demonstrate the importance of BMP4-mediated
signals in early stages of mouse development including hematopoietic and
endothelial cells.
VEGF, TGFß1 and activin A collectively regulate hematopoietic differentiation
We showed herein that SCL-expressing cells developed when ES cells were
differentiated in BMP4 alone. However, efficient expansion and differentiation
of SCL-expressing cells required VEGF. Our studies indicate that VEGF function
was required within a narrow window of time, such that the presence of VEGF
between days 3 and 4 of EB differentiation readily generated SCL-expressing
cells. The presence of VEGF between days 3 and 4 was sufficient for subsequent
hematopoietic and endothelial cell differentiation
(Fig. 4). Our studies are
consistent with Endoh et al. (Endoh et
al., 2002) who demonstrated that Scl gene reactivation
from day 2 to day 4 after initiation of differentiation in Scl-null
ES cells could rescue both primitive and definitive hematopoiesis.
Our studies indicate that VEGF signaling through FLK1 was responsible for
augmenting SCL-expressing cells. First,
Flk1/ ES cells failed to respond to VEGF and
gave rise to a similar percentage of TER119+ or CD31+
cells in the presence of BMP4 versus BMP4 and VEGF. Second,
Flt1/ ES cells responded to VEGF and
generated higher levels of TER119+ or CD31+ cells as
well as hematopoietic progenitors. Finally, our studies demonstrated that
VEGF121, which does not use neuropilin receptors, efficiently
induces CD4-expressing cells. Collectively, our studies establish a
hierarchical role of BMP4 and VEGF. BMP4 is required for the generation of
FLK1- and SCL-expressing cells. VEGF function is to enhance the hematopoietic
differentiation, as judged by the expansion of SCL expressing and
hematopoietic progenitors. Our interpretation is consistent with studies by
Nakayama et al. (Nakayama et al.,
2000) that the sequential treatment of BMP4 followed by VEGF
enhanced hematopoietic differentiation of ES cells and studies by Cerdan et
al. (Cerdan et al., 2004
),
which showed that VEGF augmented erythroid development from human ES
cells.
Gene targeting studies also support the notion that VEGF is crucial for
proper hematopoietic and endothelial cell differentiation. For example, mice
heterozygous for Vegf (Vegf+/) are
embryonic lethal due to defects in vascular development
(Ferrara et al., 1996;
Carmeliet et al., 1996
). The
production of hematopoietic cells is significantly reduced in these mice.
Conversely, mice with slightly higher levels of VEGF expression (two- to
threefold) result in early embryonic lethality because of severe abnormalities
in heart development (Miquerol et al.,
2000
). As for its mode of action, recent studies by Damert et al.
(Damert et al., 2002
)
demonstrated that VEGF production in the yolk sac visceral endoderm was
crucial for proper hematopoietic and endothelial cell development. In this
study, the authors generated chimeras between Vegf wild-type
tetraploid embryos and diploid Vegflo/lo embryos and
showed that defects in blood island formation and vascular development of
Vegflo/lo animals were rescued. Moreover, the hematopoietic cell
population in the embryo proper of these chimeras increased as the
contribution of Vegf wild-type tetraploid cells to the yolk sac
visceral endoderm augmented. Importantly, chimeras generated between
Vegflo/lo tetraploid embryos with
Vegf+/+ ES cells showed defects in yolk sac vascular
development. These studies indicate that tight regulation of VEGF expression
is crucial for correct vascular and hematopoietic differentiation of the
developing embryo.
Our studies suggest that coordinated VEGF, TGFß1 and activin A
function was important for efficient generation of hematopoietic progenitors.
We observed that TGFß1 inhibited BMP4+VEGF effect on hematopoietic and
endothelial cell differentiation (Fig.
9). Activin A could slightly augment BMP4+VEGF effect.
Consistently, mice with targeted mutations of TGFß1 and TGFß
receptor II display abnormal yolk sac hematopoietic and endothelial cell
development (Dickson et al.,
1995; Oshima et al.,
1996
). The initial vasculogenesis occurs in these mice, but
subsequent angiogenesis and capillary formation are defective. As for the
hematopoiesis, Larsson et al. (Larsson et
al., 2001
) have shown that the number of erythroid progenitors was
largely increased in TGFß receptor I-deficient yolk sac compared with
wild-type yolk sac, while CFU-GM and CFU-Mix appeared to be similar.
Role of the map kinase and SMAD pathways in hematopoietic and endothelial cell differentiation
We demonstrated that the SMAD1/5 and map kinase pathways were activated by
BMP4 and VEGF, respectively, and that the activation of these pathways was
crucial for the generation of FLK1+ and SCL+ cells (Figs
5,6,7).
ES cells overexpressing SMAD6 showed a decrease in FLK1+ cells in
response to BMP4. The MKK1-specific inhibitor U0126 was able to block the
generation of SCL+ cells. Furthermore, we did not observe ERK1/2
phosphorylation in Flk1/ EBs when stimulated
with VEGF (not shown). Consistent with our studies that the activation of map
kinase pathways is crucial for hematopoietic and endothelial cell development,
the yolk sac of Mkk1-deficient mice show diminished levels of blood
cells and distended blood vessels (Giroux
et al., 1999). Additionally, Mkk1-deficient embryos show
defects in placental angiogenesis. Moreover, recent studies
(Corson et al., 2003
)
demonstrate that there was a transient activation of ERK in nascent blood
vessels. Collectively, these studies indicate that the activation of both the
SMAD and MAP kinase pathways is crucial for blood and blood vessel
formation.
In conclusion, we have positioned factors implicated in hematopoietic differentiation at each developmental stage of hematopoietic and endothelial cell formation (Fig. 10). Specifically, BMP4 is required sequentially from ES cells to mesoderm, from mesoderm to FLK1+ cells, and from FLK1+ to SCL+ cells. VEGF then acts through FLK1 to expand SCL+ cells. The activation of the SMAD and map kinase pathways by BMP4 and VEGF, respectively, is crucial in this process. TGFß1 and activin A function to further modulate the expansion of hematopoietic and endothelial cells by BMP4 and VEGF. Future in vivo studies are required to verify the observations made in the ES/EB system.
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ACKNOWLEDGMENTS |
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Footnotes |
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