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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2275-2283
HEMATOPOIESIS
Vascular endothelial growth factor synergistically enhances bone
morphogenetic protein-4-dependent lymphohematopoietic cell generation
from embryonic stem cells in vitro
Naoki Nakayama,
Jae Lee, and
Laura Chiu
From the Department of Cell Biology and the Flow Cytometry
Laboratory, Amgen Inc, Thousand Oaks, CA.
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Abstract |
The totipotent mouse embryonic stem (ES) cell is known to
differentiate into cells expressing the  -globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that
BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and
that vascular endothelial growth factor (VEGF) synergizes with BMP-4.
The CD45+ myelomonocytic progenitors and
Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL.
VEGF alone weakly elevated the CD34+ cell
population though no lymphohematopoietic progenitors were induced.
However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically
augmented the BMP-4-dependent generation of erythro-myeloid CFCs and
lymphoid progenitors from ES cells, which were enriched in
CD34+ CD31lo and CD34+
CD45 cell populations, respectively, in a dose-dependent
manner. Furthermore, during the 7 days of in vitro differentiation,
BMP-4 was required within the first 4 days, whereas VEGF was functional
after the action of BMP-4 (in the last 3 days). Thus, VEGF is a
synergistic enhancer for the BMP-4-dependent differentiation processes,
and it seems to be achieved by the ordered action of the 2 factors.
(Blood. 2000;95:2275-2283)
© 2000 by The American Society of Hematology.
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Introduction |
It is widely accepted that development of the
hematopoietic system during early embryogenesis is initiated by the
commitment of certain splanchnopleuric mesodermal cells (derived from
the pluripotent epiblasts) into hematopoietic and endothelial cell lineages.1 Embryologic and genetic analyses in
Xenopus, zebrafish, and mice have led to the discovery of a
number of gene products that play important roles in the embryonic
generation of primitive and definitive hematopoietic
cells.2,3 For some gene products, a temporal order of
action has also been proposed. However, definition of the specific
cellular events occurring during this process remains largely
unresolved. Therefore, taking advantage of the capacity of totipotent
embryonic stem (ES) cells, isolated from the inner cell mass of the
mouse preimplantation embryo, to differentiate in vitro into cells of
the lymphohematopoietic lineages,4-17 others and we have
begun to address this problem by characterizing the intermediate cells
in the differentiation pathway and evaluating extracellular factors
driving this pathway.
Recent progress in understanding the differentiation pathway from ES
cells to lymphohematopoietic cell lineages has been made primarily from
surface marker analysis using fluorescence-activated cell sorting
(FACS). ES cells express E-cadherin, a marker for the epiblast of the
mouse embryo, which is quickly replaced by CD44.17-19 A
fraction of differentiating ES cells also starts to express the
vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2 or flk-1)
protein. These cells represent the lateral mesodermal cell type, an
important intermediate for later hematopoietic cell
development.19 In fact, the flk-1+ cells
include hematopoietic colony-forming activity18 whereas the
vascular endothelial (VE)-cadherin+ flk-1+
subpopulation has the potential to give rise to both endothelial and
erythro-myeloid cells in vitro.19-21 These observations
have been confirmed with aorta-gonad-mesonephros-derived and yolk
sac-derived cells with similar cell surface
characteristics.22 We have also demonstrated that the
CD34+ progeny derived from ES cells, which seem to appear
later than the VE-cadherin+ flk-1+
cells,19,23 are enriched for lymphohematopoietic progenitor cells.17
In contrast, the question of how the roles of extracellular signaling
molecules that had been defined by in vivo studies would fit into the
in vitro differentiation pathway from ES cells has not been fully
addressed. It has been known that BMP-2/4 is required for hematopoietic
cell genesis during the early embryogenesis of Xenopus and
zebrafish.24-27 Furthermore, essential roles of BMP-4 on
mesoderm induction28 and the flk-1 signal transduction on
primitive and definitive hematopoietic cell development have also been
demonstrated in mice.29-32 Therefore, a physiologic
differentiation culture for ES cells to lymphohematopoietic cell
lineages should be dependent at least on BMP-4 and flk-1 ligand
(including VEGF). However, none of the available culture methods has
been 2-factor dependent, probably because they are supplemented with
fetal calf serum (FCS) or plasma, which contains sufficient
extracellular factors for the whole differentiation process to proceed.
Even the stringent in vivo requirement of the flk-1 function was not reproduced in vitro with serum-containing culture
methods32,33; however, positive effects of exogenously
added VEGF on the in vitro hematopoietic progenitor cell development
were reported.17,34 In this respect, Wiles and
Johansson35,36 have already shown that in serum-free,
chemically defined medium, BMP-4 is absolutely necessary for erythroid
cell development from ES cells.
Here we demonstrate, with a modified serum-free culture method, that
the process of lymphohematopoietic cell development from ES cells in
vitro is, in fact, dependent on BMP-4 and VEGF. Under this condition,
BMP-4 is essential for the generation of all types of erythro-myeloid
CFCs and of B and NK lymphoid progenitors. On the other hand VEGF,
which does not induce lymphohematopoietic potentials alone, shows
marked enhancement of the BMP-4-dependent lymphohematopoietic cell
genesis. Furthermore, this action of VEGF seems to follow that of
BMP-4. Possible roles of VEGF as a synergistic cofactor for BMP-4
during early embryogenesis are discussed.
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Materials and methods |
Cells and reagents
The A3-1 ES cell line and OP9 stroma cell line were obtained as
described before,17 and the E14 ES cell line was kindly provided by C. Saris at Amgen (Thousand Oaks, CA). Iscove's modified Dulbecco's medium,  -minimum essential medium, and
phosphate-buffered saline without Mg2+ and
Ca++ (PBSA) were purchased from Gibco (Gaithersburg, MD).
All the tissue culture flasks and plates were from Falcon (Franklin
Lakes, NJ).
Recombinant human erythropoietin (EPO), human leukemia inhibitory
factor (LIF), human granulocyte colony-stimulating factor (G-CSF),
human interleukin (IL)-2, mouse IL-3, human IL-6, and rat stem cell
factor (SCF) were prepared at Amgen. Recombinant human BMP-4, mouse
IL-7, mouse VEGF, and mouse granulocyte macrophage colony-stimulating
factor (GM-CSF) were purchased from R&D Systems (Minneapolis, MN). The
monoclonal antibody for mouse CD34 (clone RAM34) conjugated with
fluorescein isothiocyanate (FITC) was purchased from Pharmingen (San
Diego, CA). Phycoerythrin (PE)-conjugated monoclonal antibodies for
mouse B220 (clone RA3-6B2), CD19 (clone 1D3), CD31 (clone MEC13.3),
CD45 (clone 30F11), Sca-1 (clone E13-161.7), and Ter119 were also
purchased from Pharmingen. For FACS, monoclonal antibody for mouse
CD16/CD32 (clone 2.4G2) from Pharmingen was added for blocking
nonspecific staining.
Maintenance of embryonic stem cells and induction of differentiation
by the embryoid body formation method
Both A3-1 and E14 ES cells were maintained and differentiated in a
serum-containing medium as described before17 except that
the differentiation medium contained 0.9% methylcellulose (Stem Cell
Technology, Vancouver, Canada) and the dish used for the embryoid body
(EB) formation was made of polymethylpentene (Nalge-Nunc, Milwaukee,
WI). The serum-free differentiation was performed by replacing FCS with
Knockout-SR (Gibco) from the preculture stage, and standard
bacterial-grade polystyrene dishes were used for the EB formation.
Knockout-SR is a bovine serum albumin (BSA) solution with lipids,
vitamins, trace elements, and low concentrations of transferrin and
insulin. The initial cell concentration was 500 to 700 cells/mL for the
serum-containing culture and 2500 to 4500 cells/ml for the serum-free
culture. The differentiation culture included 100 ng/mL SCF unless
otherwise stated.
Harvesting and staining EB cells for FACS
Embryoid bodies were collected, washed twice with PBSA, and
resuspended in 0.25% (wt/vol) collagenase mix in 15% FCS containing PBSA. The collagenase mix is a 1:1 mixture of Collagenase D (Boehringer Mannheim, Mannheim, Germany) and Collagenase XI (Sigma, St Louis, MO).
The EBs were incubated at 37°C for 60 minutes and dissociated into
a single-cell suspension by passing through a 20-gauge needle. Remaining small aggregates were removed by filtration through a 40-µm
mesh (Falcon). The EB cells were spun and resuspended in 0.5% BSA
(Path-O-Cyte 4; Miles, Kankakee, IL) in PBSA at
5 × 106 cells/mL. Cells were stained with 2 to 20 µg/mL antibodies. Stained samples were analyzed on a FACScan (Becton
Dickinson, San Jose, CA) or were sorted for CD34, CD31, and CD45
markers using a Vantage cell sorter (Becton Dickinson).
For factor-exchange experiments, EBs were collected on day 4 and washed
once with PBSA, and half of them were subjected to the same collagenase
treatment for FACS analysis. The rest were replated in the serum-free
methylcellulose medium in the presence of a different set of factors
and were analyzed in the same way on day 7.
Colonogenic cell assay
Total EB cells obtained through the collagenase-treatment were mixed
with the 1% methylcellulose (Stem Cell Technology) containing CFC
medium17 and distributed onto 2 to 4 35-mm bacterial-grade dishes at 5 × 104 cells/plate. For erythroid
progenitors, 100 ng/mL SCF, 10 ng/mL IL-3, and 3 U/mL EPO were added to
the culture, and colony forming unit (CFU)-erythrocyte
(E), burst forming unit (BFU)-E, and CFU-Emix (CFU-EM(macrophage) + CFU-n(neutrophil)E + CFU-mastE + CFU-nEM + CFU-mastEM) were counted on day 8. For myeloid colonies, 100 ng/mL SCF,
10 ng/mL IL-3, 10 ng/mL GM-CSF, 50 ng/mL G-CSF, and 25 ng/mL
IL-6 were added, and CFU-M, CFU-n/mast (CFU-n + CFU-mast), and
CFU-nonEmix (CFU-nM + CFU-mastM) were counted on day 9. For FACS
purified EB cells, 0.5 to 1 × 104 cells were plated
per 35-mm dish, cultured in the presence of 100 ng/mL SCF, 10 ng/mL
IL-3, 10 ng/mL GM-CSF, 50 ng/mL G-CSF, 25 ng/mL IL-6, and 3 U/mL EPO
for 9 days, and all types of colonies were counted at once.
Stroma coculture method for developing lymphokine-activated killer
cells and pre-B cells from embryoid body cells
The lymphoid potential in total and in a subfraction of EBs was
quantified based on the "switch" culture method described before.17 First, the OP9 stroma cell line was maintained
and prepared for coculture. Then total EB cells or FACS-purified EB cells were seeded at 5 × 103 to
4 × 104 cells/well (6-well plate) on a confluent
layer of OP9 and cultured in the low-serum medium17 with
100 ng/mL IL-2 and 5 ng/mL IL-7. The medium was changed one-third to
one-half volume every 3 days. Generation of pre-B cells and
lymphokine-activated killer (LAK) cells was quantified first by
visually counting hematopoietic cell foci on day 10 or 11, which were
reinspected under the microscope to further distinguish them between
the dense pre-B-type foci, and hallow-containing LAK-type foci on day
14 or 15. Nonadherent cells and loosely attached cells were then
mechanically harvested; this was followed by FACS phenotyping using
anti-Sca-1-FITC, anti-B220-PE, and anti-CD19-PE monoclonal antibodies
to confirm the generation of B220+ CD19+ pre-B
cells and B220+ CD19
(Sca-1hi/lo) LAK cells as described
before.17
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Results |
Spontaneous differentiation of embryonic stem cells to CD34
CD31 cells in the serum-free medium
Because EB cells expressing the hematopoietic stem cell markers CD34
and ER-MP12 (CD31)37 were detected during the ES cell differentiation in the serum-containing medium,17 EBs
developed in the serum-free medium were first analyzed with these
markers. As shown in Figure 1A, the
undifferentiated ES cells were CD34
CD31+. On induction of differentiation in the serum-free
medium, virtually all became CD34 CD31
(approximately 97% and 97.6% of total A3-1 and E14 EB cells,
respectively; Figure 1B). In contrast, in the presence of 15% FCS,
CD34 CD31+ and CD34+
CD31+ (R5 + R6 in Figure 1B) cells became readily
detectable. Furthermore, 2 subpopulations within the CD34+
CD31+ cell fraction, CD34+ CD31hi
and CD34+ CD31lo (R5 and R6, respectively, in
Figure 1B), became apparent. On the other hand, neither the
erythroid-specific Ter119-expressing cells (R3 in Figure 1B) nor the
leukocyte-specific CD45 expressing cells (R7 in Figure 1B) were
detected in the serum-free medium, whereas both were consistently
observed in the serum-containing medium. It was noted that the
CD45+ cells were also CD34+.
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| Fig 1.
Effect of BMP-4 on the differentiating ES cells.
Undifferentiated E14 ES cells (day 0) were stained with anti-CD31-PE
and anti-CD34-FITC monoclonal antibodies (A). The ES cells were also
induced to differentiate for 7 days in the serum-free medium with or
without 2 ng/mL BMP-4 or in the serum-containing medium, and they were
analyzed with anti-CD45-PE, Ter119-PE, or CD31-PE, and anti-CD34-FITC
monoclonal antibodies (B). The scatter patterns as well as the isotype
control staining patterns (rIgG-FITC/rIgG-PE) for the undifferentiated
ES cells and EB cells are shown in the top two panels of A, and in the
two panels of the left most column of B, respectively. The region R7
represents the CD45+ cell population, and R3 represents the
Ter119+ cell population. R5 corresponds to
CD34+ CD31hi cells, and R6 corresponds to
CD34+ CD31lo cells. (A) R5: 0.16%; R6: 0.03%.
(B, left to right) R7: 0.0%, 1.2%, 3.1%; R3: 0.41%, 3.2%, 14.1%;
R5: 0.30%, 3.4%, 2.0%; R6: 0.37%, 2.4%, 6.5%. R4 was used as a
gate for sorting CD34+ CD45 cells in
Table 2.
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Generation of CD34+, Ter119+, and
CD45+ progeny is dependent on BMP-4 in the serum-free
medium
Next we examined the effect of BMP-4 on the CD34+ EB
cell generation in our serum-free culture. As also shown in Figure 1B, when 2 ng/mL BMP-4 was added to the culture, not only CD34
CD31+ cells and CD34+ CD31+
cells but also Ter119+ cells and CD45+ cells
became readily detectable. Furthermore, the dose-dependency analysis
showed that both CD34 CD31+ (data not
shown) and CD34+ CD31+ (CD34+
CD31hi+CD34+ CD31lo in Figure
2) cells appeared with 0.1 to 0.15 ng/mL
BMP-4, and the levels were saturated at 1.5 to 2 ng/mL. The
CD34+ CD31hi and the CD34+
CD31lo subpopulations also became apparent with BMP-4
(Figure 1B). Thus the overall distribution of CD34 and CD31 expression
among EB cells developed in the presence of BMP-4 was essentially the
same as that generated with FCS. Furthermore, both Ter119+
cells and CD45+ cells began to be detected with 0.5 ng/mL
BMP-4, and the numbers were maximal with 2 ng/mL (Figure 2).
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| Fig 2.
Effects of BMP-4 on Ter119+ and
CD45+ EB cell generation are concentration dependent.
Both A3-1 and E14 ES cells were differentiated in various
concentrations of BMP-4, and analyzed as in Figure 1. Generation of
Ter119+ (R3), CD45+ (R7), CD34+
CD31hi (R5), and CD34+ CD31lo (R6)
cells were quantified as % total EB cells. Numbers derived from 2 to 5 independent experiments were averaged and plotted
according to the BMP-4 concentration with the corresponding SD
(vertical line). Open symbols are results from A3-1 ES cells, and
closed symbols are from E14 ES cells. Squares indicate results from
serum-free medium, and circles indicate results from 15%
FCS-containing medium.
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The absolute percentage of some of the cell populations analyzed (eg,
CD34+ CD31hi) varied significantly among
experiments, resulting in the large standard deviation (SD) values.
However, the dose-dependent response to BMP-4 in each experimental set
was essentially the same as shown, and it reproduced with both A3-1 and
E14 ES cell lines. We therefore concluded that in serum-free ES cell
differentiation, BMP-4 was a strong inducer of progeny expressing
various hematopoietic markers, and all the BMP-4 effects tested were
maximal at 1.5 to 2 ng/mL.
VEGF alone weakly up-regulates the generation of
CD34+ CD31+ progeny but does not induce
Ter119+ and CD45+ EB cells
We demonstrated previously that VEGF up-regulates the
CD34+ cell population in EBs, where lymphohematopoietic
progenitors were highly enriched, in the serum-containing
medium.17 Therefore, we determined whether this would also
be the case in the serum-free medium. Addition of 0.2 to 20 ng/mL VEGF
alone weakly elevated the CD34+ CD31+ EB cell
population (R5 and R6 in Figure 3;
CD34+ CD31hi + CD34+
CD31lo in Figure 4) in the
serum-free medium. However, contrary to the BMP-4 effect, the VEGF
effect was so weak that no clear CD34+ CD31hi
and CD34+ CD31lo subpopulations were observed,
and no concomitant increase in the CD34
CD31+ cell population was detected (Figure 3).
Furthermore, neither the Ter119+ nor the CD45+
cells were generated (Figures 3 and 4). Thus, although VEGF was an
up-regulator for the CD34+ CD31+ cells, it
seemed to achieve this through a pathway different from BMP-4.
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| Fig 3.
Synergistic effect of VEGF on the BMP-dependent
generation of CD34+ CD31+,
Ter119+, and CD45+ EB cell population.
E14 ES cells were induced to differentiate for 7 days in the serum-free
medium without additional growth factor, with 20 ng/mL VEGF, with low
concentration of BMP-4 (0.15 ng/mL), and with 0.15 ng/mL BMP-4 + 20
ng/mL VEGF. EB cells were harvested and stained with anti-CD45-PE,
Ter119-PE or CD31-PE, and anti-CD34-FITC antibodies. Scatter patterns
and isotype control staining patterns for EB cells are essentially the
same as shown in Figure 1B. The designation of the regions is the same
as in Figure 1. (left to right) R7: 0.0%, 0.05%, 0.22%, 3.5%; R3:
0.09%, 0.43%, 0.44%, 4.9%; R5: 0.25%, 0.71%, 1.0%, 3.5%; R6:
0.24%, 0.70%, 0.71%, 5.7%.
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| Fig 4.
Effects of VEGF on Ter119+ and
CD45+ EB cell generation are concentration dependent.
Both A3-1 and E14 ES cells were differentiated in various
concentrations of BMP-4 and VEGF analyzed as in Figures 1 and 3.
Generation of Ter119+ (R3), CD45+ (R7),
CD34+ CD31hi (R5), and CD34+
CD31lo (R6) cells were quantified as % total EB cells.
Except for the results with 0.15 ng/mL BMP4, which were obtained once
with each ES cell line, numbers were derived from an average of 2 to 5 independent experiments and were plotted according to the VEGF
concentration with the corresponding SD (vertical line). Open symbols
are results from A3-1 ES cells, and closed symbols are from E14 ES
cells. Squares indicate results from 0 ng/mL BMP-4, triangles indicate
results of 0.15 ng/mL BMP-4, circles indicate results from 1.5 ng/mL
BMP-4, and the diamond indicates results of 15 ng/mL BMP-4.
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Synergistic enhancement of BMP-4-dependent generation of
CD34+ CD31lo, Ter119+, and
CD45+ cells by VEGF
However, when 10 ng/mL VEGF was added with BMP-4 at 0.15 ng/mL, the
concentration insufficient for inducing Ter119+ or
CD45+ cells by itself, both cell populations became readily
detectable (induced by 10- to 19-fold and 11- to 24-fold, respectively;
Figure 3). The CD34+ CD31+ cell population (R5 + R6, Figure 3) was also elevated 3- to 5-fold. Furthermore,
dose-dependency analyses revealed that these VEGF-dependent enhancements were functions of both VEGF and BMP-4 concentrations (Figure 4). At a given BMP-4 concentration, CD34+
CD31+ and CD45+ EB cell generation was
dependent on the VEGF concentration from 2 to 20 ng/mL. The
Ter119+ EB cell formation was also VEGF concentration
dependent and was maximal at 2 ng/mL. However, these VEGF effects
became less pronounced when the BMP-4 concentration was increased. In
fact, compared with the results using 0.15 ng/mL BMP-4, the enhancement
was reduced to 3- to 4-fold for Ter119+ cells and
CD45+ cells and to 2- to 3-fold for CD34+
CD31+ cells (CD34+ CD31hi + CD34+ CD31lo, Figure 4) when using 1.5 ng/mL
BMP-4, even though the VEGF concentration was brought up to 20 ng/mL.
It was noted that the VEGF effects on the CD34+
CD31hi and CD34+ CD31lo
subpopulations were not necessarily equal. Especially in the case of
E14 ES cells, the CD34+ CD31lo EB cells were
more sensitive to VEGF than the CD34+ CD31hi EB
cells. The former were elevated 2- to 3-fold even at 2 ng/mL, whereas
the latter were not significantly up-regulated until 10 ng/mL VEGF
(Figure 4). However, when the VEGF concentration was brought to
20 ng/mL or higher, both subpopulations were significantly enhanced.
Although the absolute percentage of some of the cell populations
analyzed (eg, Ter119+) varied significantly, the
dose-dependent responses to VEGF shown in Figure 4 were representatives
of the individual results obtained and essentially were reproduced with
2 ES cell lines. Therefore, in conclusion, VEGF served as a synergistic
stimulator for BMP-4 to develop progeny expressing hematopoietic
markers from ES cells, and it seemed to be especially effective when
the BMP-4 concentration was low (0.1-0.15 ng/mL).
Generation of erythro-myeloid CFCs from embryonic stem cells in the
serum-free medium requires BMP-4, and VEGF is a synergistic stimulator
Next we addressed whether the synergistic increase in the number of
erythro-myeloid progenitor cells would be observed with BMP-4 and VEGF.
Therefore, BMP-4 at 1.5 ng/mL or 15 ng/mL with or without 2 ng/mL or 20 ng/mL VEGF was added to the serum-free differentiation medium, and the
occurrence of CFC produced was compared. As shown in Figure
5, BMP-4 was absolutely necessary for any
CFC types to be generated in EBs, and the addition of VEGF
synergistically elevated the numbers of all type of CFCs in a
dose-dependent manner (2- to 4-fold with 20 ng/mL). In the presence of
1.5 ng/mL BMP-4, VEGF induced a synergistic enhancement of the CFC
numbers from 2 ng/mL. Increasing the VEGF concentration to 20 ng/mL
preferentially elevated the numbers of myeloid CFCs (CFU-n/mast and
CFU-M), whereas erythroid CFCs (total E: CFU-E + BFU-E) did not
respond as clearly. These observations correlated well with the
previous FACS phenotyping results for CD45+ and
CD34+ CD31lo cell populations and for the
Ter119+ cell population, respectively (Figure 4). The
number of multipotential progenitor cells (CFU-mix) varied
significantly among experiments. However, in every experiment, 2 to 20 ng/mL VEGF did show enhancement in the presence of 1.5 ng/mL BMP-4
(data not shown). In addition, consistent with the FACS phenotyping
results, the response to VEGF was significantly weaker at a high
concentration of BMP-4 (15 ng/mL) than at the lower concentration (1.5 ng/mL). Erythroid CFCs and CFU-mix in particular appeared to become
insensitive to VEGF at 15 ng/mL BMP-4 (Figure 5).
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| Fig 5.
Effects of BMP-4 and VEGF on the development of
erythro-myeloid CFCs in EBs. A3-1 and E14 ES cell lines were
differentiated for 7 days in the serum-free medium in the presence of
1.5 ng/mL BMP-4 or 15 ng/mL BMP-4 with or without 2 ng/mL VEGF or 20 ng/mL VEGF. EB cells were collected and subjected to erythro-myeloid
CFC assays. Total-E (CFUE + BFU-E), CFU-M, CFU-n/mast, and CFU-mix
were counted separately, and individual colony numbers were averaged
over 3 to 4 independent experiments. The colony numbers are
plotted/5 × 104 total EB cells according to the
VEGF concentration with the corresponding SD (vertical line). Open
symbols are results from A3-1 ES cells, and closed symbols are from E14
ES cells. Squares indicate results obtained with 0 ng/mL BMP-4, circles
indicate results obtained with 1.5 ng/mL BMP-4, and the diamond
indicates results obtained with 15 ng/mL BMP-4.
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These results indicated that BMP and VEGF synergistically induced the
development of all types of erythro-myeloid CFCs during the
differentiation of ES cells in the serum-free medium and that a high
concentration of BMP-4 (15 ng/mL) may be detrimental for CFCs to
respond to VEGF.
Generation of lymphoid progenitor cells from embryonic stem cells in
the serum-free medium also requires BMP-4 and is enhanced by VEGF
Embryoid bodies were formed under the same conditions, and total EB
cells were cocultured with OP9 stroma cells for 10 to 14 days in the
presence of IL-7 and IL-2 as described.17 As summarized in
Table 1, regardless of the presence of
VEGF, cells derived from EBs formed in the absence of BMP-4 generated
neither pre-B nor LAK cells. Conversely, the addition of 1.5 ng/mL
BMP-4 during EB formation reproducibly induced 1 to 2 foci of pre-B cells, LAK cells, or both. The addition of VEGF at 2 or 20 ng/mL with
1.5 ng/mL BMP-4 resulted in a significant increase in the pre-B/LAK
focus numbers (4 to 9 foci). Furthermore, at the end of the OP9
coculture, the yield of nonadherent cells and the fraction of cells
expressing the lymphocyte markers, such as Sca-1, B220, and CD19, were
found to be highest when EBs were formed with VEGF + 1.5 ng/mL BMP-4
(data not shown).
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Table 1.
Generation of the B and NK potentials was also dependent
on BMP-4 + VEGF in the serum-free EB-forming culture
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Consistent with results from the FACS phenotyping and CFC analyses, the
lower concentration (1.5 ng/mL) of BMP-4 gave reproducibly higher
numbers of lymphoid foci (4 to 9 foci) than the high concentration (1 to 2 foci at 15 ng/mL) in response to VEGF. This synergistic effect of
VEGF was already evident at 2 ng/mL, and no additional enhancement was
observed at 20 ng/mL. Thus the development of lymphoid progenitors from
ES cells in vitro was also dependent on BMP-4 and enhanced by VEGF.
VEGF may also enhance the BMP-4-dependent generation of
nonhematopoietic CD34+ cells in the serum-free medium
We also addressed whether the synergistic enhancement of
BMP-4-dependent CD34+ EB cell generation by VEGF was
achieved specifically through the increase in the CD34+
lymphohematopoietic progenitor cell numbers. Therefore,
CD45+ and CD34+ CD45 or
CD34+ CD31lo and CD34+
CD31hi fractions were isolated from EBs treated with BMP-4
with or without VEGF, and the frequencies of CFCs and B/NK progenitor
numbers were compared.
No significant increases in the concentration of erythro-myeloid CFCs
were observed in all the CD34+ EB cell subsets (1.2-fold on
average; Table 2). However,
the calculated frequency of the CD34+ CFCs per
106 total EB cells increased by 3.9-fold, consistent with
the results using the whole EB cells (Figure 5). Similar results were
obtained with the B/NK potentials: a 0.8-fold increase in the
subfractions of CD34+ cells and a 3-fold increase in the
total EB cell population (Table 2). Thus no indication of preferential
increase of lymphohematopoietic progenitor cells by VEGF was detected
within the CD34+ cell fraction. This supports the idea that
VEGF may enhance the generation not only of the lymphohematopoietic
CD34+ cells but also of the other types of
CD34+ cells by BMP-4.
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Table 2.
Distribution of erythro-myeloid (EM) CFCs and pre-B/LAK
cell potentials in the subfractions of CD34+ EB cells:
CD45+ and CD34+ CD45 cell
fractions and CD34+ CD31hi and
CD34+ CD31lo cell fractions.
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It is worth noting that marked differences in the distribution of the
lymphohematopoietic cell potentials within the CD34+ cell
fraction were observed (Table 2). The CD34+
CD31lo fraction was enriched for BFU-E, CFU-M, CFU-n/mast,
and CFU-mix, and it represented 70% to 94% of the total
CD34+ CFCs, whereas the CD45+ fraction was
enriched for CFU-M and represented only 37% to 40% of the total
CD34+ CFCs. In fact, the CD45+ EB cells were
CD34+ CD31lo, and CD34+
CD45 cells were primarily CD34+
CD31hi (data not shown). On the other hand, the B/NK
progenitors were highly enriched in the CD34+
CD45 fraction, which is consistent with the in vivo
observation reported previously,22 and the
CD34+ CD31hi fraction showed more B/NK
potentials than the CD34+ CD31lo fraction.
Therefore, CD34+ erythro-myeloid CFCs were primarily
CD34+ CD31lo cells, and the CD34+
lymphoid progenitors were likely to be in the CD34+
CD31hi CD45 fraction.
Ordered function of BMP-4 and VEGF
It has been expected from genetic analyses in zebrafish and mice
that the mesoderm-inducing function of BMP-4 should be followed by
flk-1 for successful hematopoietic cell
development.25,28,30,38 This ordered function of BMP-4 and
the VEGF receptor can explain the synergy between BMP-4 and VEGF for
the in vitro production of lymphohematopoietic progenitor cells.
Therefore, we addressed this question by changing factors on day 4. Whether with VEGF (Figures 6e, 6g) or
without VEGF (Figures 6c, 6d) for the last 3 days, continuous exposure
to BMP-4 for longer than 4 days (Figures 6d, 6g) did not show any
effects on the levels of Ter119+ and CD45+ cell
types in the day 7 EBs compared with those treated only for the first 4 days (Figures 6c, 6e). On the contrary, 3-day treatment of BMP-4 after
ES cells had been differentiated for 4 days in the absence of BMP-4
with VEGF (Figure 6f) or without VEGF (Figure 6b) induced neither
Ter119+ nor CD45+ cell formation. Therefore,
BMP-4 was necessary and sufficient to work during the initial 4 days of
differentiation regardless of VEGF.
View larger version (27K):
[in this window]
[in a new window]
| Fig 6.
4-day treatment with BMP-4 followed by 3-day treatment
with VEGF induced Ter119+ and CD45+ EB
cells. E14 ES cells were induced to differentiate for 4 days with
or without 1.5 ng/mL BMP-4, 10 ng/mL VEGF, or both in the serum-free
medium followed by another 3 days in the presence or absence of these
factors. Generations of Ter119+ erythroid cell and
CD45+ macrophage progenitors were analyzed by FACScan on
day 7. Scatter pattern, gate setting, and staining pattern of the
isotype controls were as shown in Figure 1. Note that this series of
experiments was performed without SCF. (a) Without factor for 7 days.
(b) Without factor for 4 days followed by BMP-4 for 3 days. (c) With
BMP-4 for 4 days, then without factor for 3 days. (d) With BMP-4 for 7 days. (e) With BMP-4 for 4 days followed by VEGF for 3 days. (f) With
VEGF for 4 days followed by BMP-4 for 3 days. (g) With BMP-4 for 4 days
followed by BMP-4 + VEGF for 3 days. (h) With BMP-4 + VEGF for 7 days. Data are displayed as averaged percentage total EB cell
population obtained from 4 (for CD45) to 5 (for Ter119) independent
experiments with the corresponding SD. Exception: (c) experiments were
performed 3 times each.
|
|
When VEGF was added to (Figure 6g) or in place of (Figure 6e) BMP-4 on
day 4, significant enhancement of Ter119+ and
CD45+ cell generation was observed on day 7 (2.9% to 3.3%
for Ter119+; 1.1% to 1.2% for CD45+) compared
with the levels achieved without VEGF (1.1% to 1.2% for
Ter119+; 0.5% to 0.8% for CD45+) (Figures 6c,
6d). This indicated that VEGF could still be effective after the BMP-4
treatment, supporting the idea of the ordered function of the 2 factors: BMP-4 followed by VEGF. However, when VEGF was added from the
beginning with BMP-4 (Figure 6h), it gave higher levels of
Ter119+ cells and CD45+ cells (5.0% and 1.3%,
respectively). Therefore, it is still possible that VEGF functions at
the same time with BMP-4 to produce the synergistic increase in
progenitor cell number.
| |
Discussion |
We have demonstrated that VEGF synergistically enhanced the effect
of BMP-4 on the in vitro development of lymphohematopoietic cells from
ES cells and that the synergy was likely to be achieved by the ordered
action of the 2 factors during the differentiation process. We have
also provided evidence that BMP-4 was the essential factor not only for
developing erythroid cells, as reported,35,36 but also for
generating myeloid-CFCs and lymphoid cell potentials from ES cells.
Differences between the serum-containing culture and the serum-free
culture in the ability to support the differentiation of ES cells into
a variety of progeny-cell types were striking. Without FCS, ES cells
were differentiated, but apparently only into CD34
CD31 cell types (Figure 1). No sign of
lymphohematopoietic cell development was detected (Figure 5; Table 1).
One interpretation for these observations is that ES cells may be
differentiated through a default pathway without FCS, which does not
lead to hematopoietic cell development. The fact that ES cells are
spontaneously differentiated into cell populations expressing pax-6, a
neuroectoderm marker, in a chemically defined medium suggests that the
presumptive default pathway may lead to neuroectoderm cell
formation.36 On the other hand, when BMP-4 was added, it
appeared to convert the serum-free culture to a serum-containing-like
culture; the distribution of CD34- and CD31-expressing cells became
similar, Ter119+ cells and CD45+ cells were
detected (Figures 1, 3), and all types of CFCs and B/NK lymphoid
progenitors were generated (Figure 5; Table 1). Therefore, as
demonstrated for the erythroid cell development from ES
cells,35,36 BMP-4 seemed to induce all the necessary signals for ES cells to differentiate into lymphohematopoietic cell
lineages. However, there were some differences between the effects of
BMP-4 and FCS. The maximal levels of the erythro-myeloid CFC-containing
cell fractions such as Ter119+ cells, CD45+
cells, and CD34+ CD31lo cellswere lower with
BMP-4 than with 15% FCS, whereas those of CD34+
CD31hi cells were higher with BMP-4 than with 15% FCS
(Figure 2). Thus FCS contained activity that seemed to preferentially
support the generation of erythro-myeloid progenitor cells from ES cells.
VEGF clearly induced the number of Ter119+ erythroid cells
and CD45+ and CD34+ CD31lo
erythro-myeloid progenitors when added with BMP-4 (Figures 3, 4). In
fact, EBs produced by BMP-4 and VEGF were red, whereas those developed
only with BMP-4 were white (data not shown). Especially when the
concentration of BMP-4 was insufficient for inducing these cells by
itself (0.1-0.15 ng/mL), the culture seemed to become totally dependent
on BMP-4 and VEGF (Figures 3 and 4; the results shown in Figure 6
provide more definitive evidence because SCF was omitted). However,
even at the optimal concentration of BMP-4 (1.5-2 ng/mL), VEGF still
increased the frequency of erythro-myeloid CFCs and lymphoid potentials
(Figure 5; Table 1). As far as we know, this is the first demonstration
that the in vitro differentiation of ES cells toward
lymphohematopoietic cell lineages is dependent on a flk-1 ligand. Given
that the flk-1 signaling is essential for the hematopoietic cell
development in vivo,30 the in vivo concentration of BMP-4
might be in the range of 0.1 to 0.15 ng/mL. Interestingly, at a higher
concentration of BMP-4 (15 ng/mL), the VEGF effect was less pronounced
(Figures 4, 5), and many cells (mainly macrophage-like cells) formed
either no colonies or microcolonies during the CFC analysis (data not
shown). Therefore, a high concentration of BMP-4 might also stimulate
differentiation of hematopoietic progenitor cells or, alternatively,
desensitize their ability to respond to various growth factor/cytokine signals.
VEGF as a synergistic enhancer of BMP-4-dependent lymphohematopoietic
cell generation from ES cells appears to contradict the finding by
Hidaka et al33 in that VEGF is inhibitory for generating
CFCs in 6- to 12-day-old EBs. However, because their EB culture method
includes hematopoietic cytokines and FCS and because the VEGF effect on
the CFC generation is stimulating (ie, positive) in day 4 EBs, it is
likely that the inhibitory effect of VEGF is on the long-term
growth/maturation of CFCs during EB culture. All our results were
derived from day 7 EBs. Therefore, day 7 EB in the serum-free culture
might correspond to day 4 EB in the serum-containing culture. In this
regard, it is worth mentioning that during the 14-day culture of
various subsets of CD34+ cells on OP9 cells, VEGF was
inhibitory for pre-B/LAK cell formation (data not shown).
The synergistic effect of VEGF on the BMP-4-dependent differentiation
processes does not seem to be restricted to the hematopoietic cell
lineages because the CD34+ cell subpopulations, all of
which were elevated with VEGF, contained concentrations of CFCs and
lymphoid progenitors virtually identical to those obtained without VEGF
(Table 2). However, this result also implies that there may be no
preferential enhancement of nonhematopoietic CD34+ cell
types (presumably including endothelial cells). This observation seems
to contradict the finding with chicken embryonic flk-1+
cells in that VEGF preferentially shifts their differentiation toward
the endothelial cell lineage, thereby inhibiting their differentiation
into hematopoietic progenitor cells.39 However, the
difference may account for the intrinsic differences in the characteristics of chicken cells and mouse cells.
Although the ES cell differentiation into lymphohematopoietic
progenitor cells was dependent on VEGF in the serum-free medium, VEGF
was dispensable when BMP-4 was supplied at the optimal concentration (Figures 1, 2, 5; Table 1). However, with flk-1/
mutant ES cells, it has been shown that an active role of the VEGF receptor, flk-1, is necessary for the in vitro generation of
-globin-expressing erythroid cells in the presence of BMP-4 alone.32 The former suggests that the VEGF receptor
signaling is not required, and the latter indicates that it is
required. One possible explanation for the apparent differences is
that, at the optimal concentration, BMP-4 may induce the expression of
VEGF or other VEGF family members, which helps the in vitro lymphohematopoietic cell generation through flk-1, presumably at low
levels, so that further activation of flk-1 by exogenous VEGF is still
able to show the synergistic effect. Nevertheless, this notion is an
interesting parallel with the in vivo observations that VEGF is not so
strictly required as flk-1 for the primitive erythroid cell
development.29-31,40
How, then, is the synergistic effect achieved? Genetic analyses in
zebrafish and mice suggest a model that implicates BMP-4 as an
earlier-acting factor that is followed by the stimulation of flk-1
action for successful hematopoietic cell
development.25,28,30,38 Based on this model, BMP-4 may
effectively induce the differentiation of ES cells into uncommitted
mesodermal precursors but only poorly support the further
differentiation of such cells into lymphohematopoietic progenitor
cells. In contrast, VEGF may be a later-acting factor that facilitates
the latter stage of development. Alternatively, the synergy may result
from the presence of a critical mesoderm intermediate whose
proliferation/differentiation is dependent on both factors. In search
of the critical timing required for each factor to work during the 7 days of in vitro differentiation, the BMP action was found to be
required within the first 4 days, whereas VEGF was able to exert the
synergistic effect after the BMP-4 treatment (Figure 6). Thus although
the latter possibility has not been entirely ruled out, this result
would support the former model that fits with the in vivo observations.
The idea of VEGF as the "second-stage factor" is supported by the
recent report that flk-1/ ES cells are able
to generate levels of blast-CFCs in vitro similar to those for
wild-type ES cells.32 The blast-CFC represents a
VEGF-dependent common progenitor cell for primitive and definitive erythropoiesis34 and has endothelial
potential21 that leads to the hypothesis that VEGF may be a
growth/differentiation factor for the hypothetical
hemangioblast.41 Therefore, although the experiments were
performed in serum-containing media, the results suggest that the
flk-1-dependent signal may not be required for the generation of the
"hemangioblast." Furthermore, flk-1 does not seem to be necessary
for generating the erythro-myeloid CFC activity in early mouse embryos
(E7.5), whereas it is required for maintaining the activity through the
later stages of embryogenesis (E8.5).30,32 Thus it is
tempting to speculate that VEGF-sensitive cells, which are induced by
BMP-4 from ES cells during the first 4 days of in vitro
differentiation, might be the hemangioblast. This is a question
currently being addressed.
| |
Acknowledgments |
We thank L. Souza for G-CSF, T. Boone for IL-3 and IL-6, K. Langley for
SCF, W. Kenney for IL-2, and B. Samal for LIF. We also thank C. Saris
for providing E14 cells and E. Medlock, C. Saris, and I. Ponting for
sharing information and interesting discussions and particularly for
critically reading this manuscript. We give warm thanks to W. Boyle for
support and encouragement.
| |
Footnotes |
Submitted November 10, 1999; accepted December 7, 1999.
Reprints: Naoki Nakayama, Department of Cell Biology,
Amgen Inc, MS14-1-D, One Amgen Center Drive, Thousand Oaks, CA 91320;
e-mail: naoki.nakayama{at}amgen.com.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction