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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1253-1263
Maturation of Embryonic Stem Cells Into Endothelial Cells in an
In Vitro Model of Vasculogenesis
By
Masanori Hirashima,
Hiroshi Kataoka,
Satomi Nishikawa,
Norihisa Matsuyoshi, and
Shin-Ichi Nishikawa
From the Department of Molecular Genetics, Department of Geriatric
Medicine, and Department of Dermatology, Graduate School of Medicine,
Kyoto University, Kyoto, Japan.
 |
ABSTRACT |
A primitive vascular plexus is formed through coordinated regulation
of differentiation, proliferation, migration, and cell-cell adhesion of
endothelial cell (EC) progenitors. In this study, a culture system was
devised to investigate the behavior of purified EC progenitors in
vitro. Because Flk-1+ cells derived from ES cells did not
initially express other EC markers, they were sorted and used as EC
progenitors. Their in vitro differentiation into ECs, via vascular
endothelial-cadherin (VE-cadherin)+ platelet-endothelial
cell adhesion molecule-1 (PECAM-1)+ CD34
to VE-cadherin+ PECAM-1+
CD34+ stage, occurred without exogenous factors, whereas
their proliferation, particularly at low cell density, required OP9
feeder cells. On OP9 feeder layer, EC progenitors gave rise to
sheet-like clusters of Flk-1+ cells, with VE-cadherin
concentrated at the cell-cell junction. The growth was suppressed by
Flt-1-IgG1 chimeric protein and dependent on vascular endothelial
growth factor (VEGF) but not placenta growth factor (PIGF). Further
addition of VEGF resulted in cell dispersion, indicating the role of
VEGF in the migration of ECs as well as their proliferation. Cell-cell
adhesion of ECs in this culture system was mediated by VE-cadherin.
Thus, the culture system described here is useful in dissecting the
cellular events of EC progenitors that occur during vasculogenesis and
in investigating the molecular mechanisms underlying these processes.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
VASCULOGENESIS IS A process in which
angioblasts are differentiated from mesodermal cells and organized to
form a primitive vascular network. Vasculogenesis occurs in limited
embryonic sites, although angiogenesis, the formation of new blood
vessels by sprouting from preexisting ones, occurs in many situations
containing embryonic development and pathological
conditions.1-6 The most typical and earliest site of
vasculogenesis is the yolk sac. In the yolk sac, extraembryonic
mesodermal cells that have migrated through the primitive streak
differentiate to form blood islands composed of hemangioblasts,
putative common precursors of endothelial and hematopoietic
cells.7 Peripheral cells of blood islands connect to
construct a vascular network composed of capillaries, arteries, and veins.
Recent studies of mutant mice specified a set of molecules involved in
vascular development. Among these molecules, vascular endothelial
growth factor (VEGF; also known as vascular permeability factor),8-10 fetal liver kinase 1 (Flk-1),11
fms-like tyrosine kinase (Flt-1),12 and vascular
endothelial-cadherin (VE-cadherin)13,14 were proven to be
requisite for vasculogenesis.15-19 In addition, many other
molecules were proven to be essential for vascular development,20,21 but little is known about how these
molecules regulate the behavior of vascular components.
Several in vitro systems have been developed for investigating the
cellular events in vasculogenesis. The most popular systems are
embryonic stem (ES) cell-derived embryoid body formation and the
culture of mesodermal cells from embryos.3,22-26 Although these culture systems enable one to investigate vasculogenesis virtually as it occurs in embryos, the existence of many other lineage
cells generated in an uncontrollable manner hinders understanding of
the behavior of endothelial cells (ECs) in detail.
To overcome this problem, we attempted to establish a system for
analyzing the behavior of ECs specifically. In this study, we isolated
Flk-1+ EC progenitors derived from ES cells to investigate
their cellular events. The in vitro differentiation of purified
Flk-1+ cells into ECs occurs on type IV collagen-coated
dishes. In a culture system using OP9 feeder layer, the proliferation
and motility of developing ECs was regulated by dose-dependent dual
effects of VEGF and cell-cell adhesion was essentially mediated by
VE-cadherin. These results indicate that the experimental system
described here provides a new tool for dissecting the cellular events
of EC progenitors, such as differentiation, proliferation, migration, and cell-cell adhesion, whereby a primitive vascular plexus is formed.
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MATERIALS AND METHODS |
Monoclonal antibodies (MoAbs).
MoAbs for murine E-cadherin (ECCD2), murine Flk-1 (AVAS12), and murine
VE-cadherin (VECD1) have been described previously.27-29 These MoAbs were prepared and labeled in our laboratory. Fluorescein isothiocyanate (FITC)-conjugated MoAb for murine platelet-endothelial cell adhesion molecule-1 (PECAM-1; Mec13.3) and FITC-conjugated MoAb
for murine CD34 (RAM34) were purchased from Pharmingen (San Diego, CA).
Cell culture.
CCE ES cells30 (a gift from Dr M.J. Evans, Wellcome/CRC
Institute, Cambridge, UK) were maintained as described
previously.31 MC3T3-G2/PA6 (PA6) and OP9 feeder cell lines
that were established from mouse calvaria were maintained as described
previously.32,33 NIH3T3 and Balb/c3T3 cell lines were
maintained in Dulbecco's modified Eagle's medium (GIBCO, Grand
Island, NY) supplemented with 10% fetal calf serum (FCS; GIBCO).
To induce differentiation, ES cells were cultured on type IV
collagen-coated dishes (Becton Dickinson Labware, Bedford, MA) in  minimal essential medium (MEM; GIBCO) supplemented with 10% FCS and
5 × 105 mol/L 2-mercaptoethanol (Merck,
Darmstadt, Germany) in the absence of leukemia inhibitory factor. As
described in our previous study,31 Flk-1+
VE-cadherin E-cadherin cells
corresponding to the lateral and extraembryonic mesoderm were induced.
To assess their cellular events, Flk-1+ cells were sorted
and recultured at a density of 10 cells/mm2 on each feeder
layer in the presence or absence of mFlt-1-hIgG1 (see below), human
VEGF (R&D Systems, Minneapolis, MN), human placenta growth factor
(PlGF; R&D Systems), and 20 µg/mL VECD1. Supernatant from 3-day
culture on each feeder layer in the absence of exogenous factors was
recovered and the concentration of VEGF in each culture supernatant was
measured (see below).
Flow cytometry and cell sorting.
After 4 days for ES cell differentiation, cultured cells were harvested
by incubating with cell dissociation buffer (GIBCO). The harvested
cells were incubated in mouse serum for 20 minutes on ice to block the
nonspecific Ab binding, incubated with biotinylated AVAS12 and
FITC-conjugated ECCD2 for 15 minutes on ice, and then incubated in
streptavidin-conjugated R-phycoerythrin (GIBCO) for 15 minutes on ice.
After each step, cells were washed with Hanks' balanced salt solution
(GIBCO) containing 1% bovine serum albumin (BSA; Seikagaku Kogyo,
Tokyo, Japan) and 0.01% sodium azide (Wako Chemical, Osaka, Japan).
Living Flk-1+ E-cadherin cells excluding
propidium iodide (Sigma, St Louis, MO) were sorted by FACS Vantage
(Becton Dickinson).
To test the differentiation potential of Flk-1+ cells,
sorted cells were cultured on type IV collagen-coated dishes. After 1 to 3 days of incubation, cultured cells were harvested and stained with
a mixture of MoAbs containing allophycocyanin (APC)-conjugated AVAS12,
biotinylated VECD1, FITC-conjugated Mec13.3, or FITC-conjugated RAM34
and developed by streptavidin-conjugated R-phycoerythrin as described
above. Cells were analyzed by FACS Calibur (Becton Dickinson) while
being gated to exclude small debris, dying cells, and other sources of
background interference. For analyzing Flk-1+ or
VE-cadherin+ cells, positive cells were gated.
Immunostaining.
For assessing the cellular events of Flk-1+ cells on feeder
layers, the cultured cells were fixed in situ by 4% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) in phosphate-buffered saline (PBS) for
10 minutes at 4°C for AVAS12 staining or by 5% dimethyl sulfoxide (Nacalai Tesque) in methanol for 3 minutes at 4°C for VECD1
staining. After washing with PBS, 2% skim milk in PBS was incubated as
a blocking solution for 1 hour at room temperature. The fixed dishes were incubated with AVAS12 or VECD1 MoAbs overnight at 4°C,
followed by incubating with alkaline phosphatase (ALP)-conjugated
antirat IgG (H+L) (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) for 1 hour at room temperature. After each step, the cultured cells
were washed three times with PBS containing 0.05% Tween 20 (Wako
Chemical). Cells were visualized by using 4-nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) substrate
(Roche Molecular Biochemicals, Basel, Switzerland). Endogenous ALP
activity was blocked by 2 mmol/L levamisole (Sigma) before the
incubation with MoAbs.
Staining of DiI-acetylated low-density lipoprotein (DiI-Ac-LDL).
Cultured cells were incubated in MEM supplemented with 10%
lipoprotein-deficient serum and 10 µg/mL DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA) for 4 hours. Cells were then washed with
MEM and observed by fluorescent microscopy (Axiovert 135M; Zeiss,
Jena, Germany) using a rhodamine filter.
Preparation of a chimeric protein of murine Flt-1 extracellular
domain and Fc portion of human IgG1 (mFlt-1-hIgG1).
Plasmid vector containing murine Flt-1 cDNA was kindly provided by Dr
W. Risau (Max-Planck-Institute for Physiological and Clinical Research,
Bad Nauheim, Germany). cDNA encoding amino acids from 607 to 755 were
amplified by polymerase chain reaction (PCR) from this vector with
sense and antisense primers that had Xho I-Bal I and
BamHI restriction sites at their 5' ends, respectively. Amplified DNA fragments were subcloned into the expression vector pCDM8-hIgG1,34 and then cDNA encoding amino acids from 1 to 607 that had Xho I and Bal I restriction sites at the
5' and 3' ends, respectively, was subcloned into this
vector. Production and purification of this chimeric protein were
performed as described previously.35
Enzyme-linked immunosorbent assay (ELISA).
The specific binding of mFlt-1-hIgG1 to human VEGF and human PlGF was
examined in microtiter plate immunosorbent assay by comparing its
reactivity to human macrophage colony-stimulating factor (M-CSF; a gift
from Dr T. Sudo, Toray Industries, Inc, Kamakura, Japan). Triplicate
wells were coated with 250 ng/mL of each ligand overnight at 4°C
and 1% BSA in PBS was incubated as a blocking solution for 1 hour at
room temperature. mFlt-1-hIgG1 diluted to 5 µg/mL in blocking
solution was applied, followed by incubating with peroxidase-conjugated
antihuman IgG Fc (Organon Teknika Corp, West Chester, PA) for 1 hour at
room temperature. After each step, wells were washed five times with
PBS containing 0.05% Tween 20. Binding of mFlt-1-hIgG1 to each ligand
was detected by using substrate of
3',3',5',5'-tetramethylbenzidine (a gift from
Dr M. Hirashima, The Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan) and quantified by ImmunoMini NJ-2300 (Intermed, Tokyo, Japan).
The concentration of VEGF produced by feeder cells was examined. The
concentration of VEGF in recovered culture supernatant was measured by
using Quantikine M Mouse VEGF Immunoassay (R&D Systems).
| |
RESULTS |
Sequential expression of EC markers during the differentiation of
Flk-1+ cells generated from ES cells.
Our previous study demonstrated that the differentiation of ES cells
into endothelial and hematopoietic cell lineages occurs through two
successive intermediate stages, Flk-1+
VE-cadherin and Flk-1+
VE-cadherin+ stages. Sorting and reculture of
Flk-1+ VE-cadherin+ cells showed that ECs are
generated through this differentiation pathway.31
To further characterize the differentiation of Flk-1+
VE-cadherin cells into Flk-1+
VE-cadherin+ cells, we sorted Flk-1+ cells
generated from 4-day culture of ES cells on type IV collagen-coated dishes, recultured them under the same conditions, and analyzed the
expression pattern of EC markers (Flk-1, VE-cadherin,
PECAM-1/CD31,36,37 and CD3438,39) by flow
cytometry. Because E-cadherin+ cells that may include
totipotent ES cells were excluded from Flk-1+ cells, the
contribution of contaminating immature cells in this culture should be
negligible (Fig 1A). Sorted
Flk-1+ cells were negative in the expression of
VE-cadherin, PECAM-1, and CD34 (Fig 1B and C; sorted population). As
demonstrated previously,31 a considerable fraction of
Flk-1+ cells rapidly lost the expression of Flk-1. However,
concomitantly, some Flk-1+ cells maintained the expression
of Flk-1 and started to express VE-cadherin and PECAM-1 almost
simultaneously, indicating that the differentiation into EC lineage
progresses under this culture condition. Interestingly, CD34 was
expressed about 1 day after the appearance of VE-cadherin and PECAM-1
in the Flk-1+ population (Fig 1B and C; days 1 through 3).
During prolonged culture, the expression of Flk-1 was downregulated in
the VE-cadherin+ population that coexpresses PECAM-1 (Fig
1D). The differentiation pathway of ECs on type IV collagen-coated
dishes is summarized in Fig 2.
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| Fig 1.
Flow cytometric analysis of the differentiation of ES
cells into EC lineage. (A) CCE ES cells were cultured on type IV
collagen-coated dishes in the absence of leukemia inhibitory factor.
After 4 days for ES cell differentiation, Flk-1+
E-cadherin cells were sorted and recultured under the
same conditions. (B) The expression of Flk-1 and VE-cadherin on sorted
Flk-1+ cells during 3 days in culture. (C) The expression
of VE-cadherin and PECAM-1 or CD34 on the Flk-1+
population during 3 days in culture. (D) The expression of Flk-1 and
PECAM-1 or CD34 on the VE-cadherin+ population during 3 days in culture.
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| Fig 2.
Schematic representation of the differentiation pathway
from ES cells into EC lineage on type IV collagen-coated dishes. The
sorted population in the bold box was used as EC progenitors in this
study.
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Culture of Flk-1+ cells supported by feeder layers.
After characterizing the in vitro differentiation of sorted
Flk-1+ cells into ECs, we attempted to establish a culture
system for dissecting the cellular events of these developing ECs.
However, they could not grow at low cell density on type IV
collagen-coated dishes even in the presence of VEGF (data not shown).
Thus, we considered the support of feeder layers to overcome this problem.
Four different types of established feeder cell lines (NIH3T3,
Balb/c3T3, PA6,32 and OP933) were compared for
their abilities to support the growth of Flk-1+ cells. The
former two represent embryonic fibroblasts, whereas the latter two are
derived from neonatal calvaria. Flk-1+ cells were cultured
at low cell density on a monolayer of each feeder cell line in the
absence of exogenous factors. After 3 days of culture, cells were
stained by MoAb for Flk-1 to detect the growth of Flk-1+
cells. All feeder layers except NIH3T3 could support the growth of
sorted Flk-1+ cells, but PA6 supported it to a lesser
extent than Balb/c3T3 and OP9
(Fig
3A through D). Interestingly, Flk-1+ cells grew to form the
cord-like structure on Balb/c3T3 feeder layer, whereas they grew to
form the sheet-like structure on OP9 feeder layer. We
supposed that the formation of Flk-1+ cell sheets on OP9
feeder layer was due to insufficiency of molecules that enhance cell
motility and, therefore, are suitable for analyzing various molecules
modulating this basal level. Thus, we selected OP9 feeder layer for
subsequent studies.
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| Fig 3.
Growth of Flk-1+ cell clusters supported by
feeder layers. Sorted Flk-1+ cells were cultured at low
cell density on a monolayer of each feeder cell line in the absence of
exogenous factors. After 3 days of culture, immunostaining with MoAb
for Flk-1 was performed as described in Materials and Methods. (A)
NIH3T3 could not support their growth. (B) PA6 supported it poorly. (C
and D) Notably, Flk-1+ cells grew to form the cord-like
structure on Balb/c3T3 feeder layer (C), whereas they grew to form the
sheet-like structure on OP9 feeder layer (D). Scale bar = 500 µm.
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| Fig 4.
A sheet-like cluster of ECs generated from
Flk-1+ cells cultured on OP9 feeder layer. Sorted
Flk-1+ cells were cultured at low cell density on OP9
feeder layer in the absence of exogenous factors. After 3 days of
culture, immunostaining with MoAb for Flk-1 (A) or VE-cadherin (B) was
performed as described in Materials and Methods. As compared with the
diffuse distribution of Flk-1 over the cell surface, VE-cadherin was
concentrated at cell-cell junctions. (C) Uptake of DiI-Ac-LDL was
observed by fluorescent microscopy using a rhodamine filter. Virtually
all cells in this cluster showed uptake of DiI-Ac-LDL. Scale bar = 100 µm.
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| Fig 5.
Blocking effect of mFlt-1-hIgG1 on the growth of
Flk-1+ cells on OP9 feeder layer. (A) The preparation of
mFlt-1-hIgG1 and assessment of its binding to M-CSF, VEGF, or PlGF were
performed as described in Materials and Methods. The bars show the mean
(±SD) of triplicate samples. (B, C, and D) Sorted
Flk-1+ cells were cultured on OP9 feeder layer in the
presence of 100 ng/mL mFlt-1-hIgG1 (B), 100 ng/mL mFlt-1-hIgG1 and 3 ng/mL VEGF (C), or 100 ng/mL mFlt-1-hIgG1 and 100 ng/mL PlGF (D). After
3 days of culture, immunostaining with MoAb for Flk-1 was performed as
described in Materials and Methods. Scale bar = 500 µm.
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VE-cadherin+ or PECAM-1+ cell sheets on OP9
feeder layer were detected in number and size similar to
Flk-1+ cell sheets (Fig 4A
and B, data not shown), indicating that VE-cadherin and PECAM-1 were
coexpressed on Flk-1+ cells. In addition, the expression of
CD34 was also detected on cells in similar clusters (data not shown).
These results are consistent with results by flow cytometry. As
compared with the diffuse distribution of Flk-1 over the cell surface,
VE-cadherin was concentrated at cell-cell junctions. This suggests that
OP9 feeder layer supports the proliferation, differentiation, and cluster formation of Flk-1+ VE-cadherin+ cells
with adherens junction. In addition, virtually all cells in these
sheets showed uptake of DiI-Ac-LDL (Fig 4C), indicating that they
represent ECs.40
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| Fig 6.
VEGF-induced enhancement of the motility of
Flk-1+ cells on OP9 feeder layer. Sorted
Flk-1+ cells were cultured on OP9 feeder layer in the
presence of VEGF or PlGF. After 3 days of culture, immunostaining with
MoAb for Flk-1 was performed as described in Materials and Methods.
Flk-1+ cells in the periphery of the sheets started to
disperse at 3 ng/mL VEGF (A; arrows). At 50 ng/mL VEGF,
Flk-1+ cells formed spiky clusters composed of spindle
form cells (B; arrows). (C) In contrast to VEGF, 100 ng/mL PlGF had no
effect on the motility of Flk-1+ cells. Scale bar = 100 µm.
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Requirement of VEGF for in vitro proliferation of
Flk-1+ cells.
We investigated the molecular mechanisms supporting the proliferation
of sorted Flk-1+ cells on OP9 feeder layer. Because VEGF
has been implicated as the major growth factor of ECs during
vasculogenesis15,16 and the expression of VEGF was detected
by reverse transcription-PCR (RT-PCR) of OP9 (data not shown), we
investigated whether OP9-derived VEGF plays an important role in the
proliferation of Flk-1+ cells. Taking the previous in vivo
study of Aiello et al41 into consideration, we constructed
a chimeric protein of murine Flt-1 extracellular domain and Fc portion
of human IgG1 (mFlt-1-hIgG1) and used it to block the activity of VEGF
family molecules. As shown in Fig 5A, prepared mFlt-1-hIgG1 could bind
to both VEGF and PlGF.
The number of Flk-1+ cell clusters on OP9 feeder layer was
decreased and the size of each cluster was reduced by the addition of
mFlt-1-hIgG1 (Fig 5B). Indeed, the effective amount of VEGF measured by
ELISA was reduced up to 20% by 100 ng/mL mFlt-1-hIgG1 (data not
shown). This blocking effect of 100 ng/mL mFlt-1-hIgG1 was compensated
by the addition of 3 ng/mL VEGF but not 100 ng/mL PlGF,42,43 another ligand for Flt-1 (Fig 5C and D).
Moreover, VEGF overrode the blocking effect of mFlt-1-hIgG1 in a
dose-dependent manner (Table 1). Thus,
OP9-derived VEGF should be involved in the proliferation of
Flk-1+ cells. However, interestingly, small
Flk-1+ cell clusters were formed even in the presence of a
higher dose of mFlt-1-hIgG1 that is sufficient to bind all VEGF in this
culture (data not shown), suggesting that other molecules are involved in their growth.
Enhancement of cell motility by VEGF.
Because previous reports on VEGF+/ mice
suggested that the dose of VEGF is important for establishment of a
primitive vascular plexus,15,16 1 to 100 ng/mL VEGF was
added to investigate the effect of additional VEGF on
Flk-1+ cells cultured on OP9. A problem in this experiment
was that two distinct receptors, Flk-1 and Flt-1, share VEGF as the
ligand,44-46 and the expression of Flt-1 was also detected
by RT-PCR of purified Flk-1+ cells (data not shown). Thus,
to distinguish the involvement of Flk-1 from that of Flt-1, the effect
of PlGF, which stimulates Flt-1 specifically,47 was also evaluated.
VEGF at 1 to 10 ng/mL induced a slight increase in number and size of
Flk-1+ cell clusters, indicating that VEGF derived from OP9
is insufficient to induce the maximum growth. On the other hand, at a
higher dose, VEGF displayed neither an enhancing nor a suppressive
effect on the proliferation of Flk-1+ cells (Table 1).
Interestingly, VEGF induced a marked change of cluster shape.
Flk-1+ cells in the periphery of the sheets started to
disperse at 3 ng/mL VEGF (Fig 6A). At 50 ng/mL VEGF, Flk-1+
cells formed spiky clusters composed of spindle form cells, instead of
smooth round clusters (Fig 6B). In contrast to VEGF, 100 ng/mL PlGF had
no effect on the proliferation or motility of Flk-1+ cells
(Fig 6C), indicating that Flt-1 stimulation was not involved in cell
dispersion induced by VEGF in this system. These results indicate that
VEGF has dual roles in regulating the cellular events of ECs in
vasculogenesis; supporting the proliferation of ECs at low dose and
enhancing cell motility with shift of cell morphology at high dose.
Role of VE-cadherin in cell adhesion of Flk-1+ cell
clusters.
It was reported that the formation of an organized vascular network is
incomplete in embryoid bodies generated from
VE-cadherin/ ES cells, whereas the
differentiation of ECs is not impaired.19 To evaluate the
role of VE-cadherin in our in vitro culture system of vasculogenesis,
Flk-1+ cells were cultured on OP9 feeder layer in the
presence of blocking MoAb against VE-cadherin (VECD1).29
When VECD1 was added from the beginning of culture, the differentiation
and proliferation of Flk-1+ cells were not affected.
However, the formation of sheet-like clusters was inhibited in the
presence of VECD1 (Fig 7A). These results
are consistent with a previous report on
VE-cadherin/ ES cells.
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| Fig 7.
VE-cadherin-mediated cell-cell adhesion of
Flk-1+ cell clusters on OP9 feeder layer. (A) Sorted
Flk-1+ cells were cultured on OP9 feeder layer in the
presence of blocking MoAb against VE-cadherin (VECD1). After 3 days of
culture, immunostaining with MoAb for Flk-1 was performed as described
in Materials and Methods. Cell-cell contact was disrupted by this
treatment, whereas the proliferation of Flk-1+ cells was
not affected. (B, C, and D) Sorted Flk-1+ cells were
cultured on OP9 feeder layer. After 3 days, the cultures were left
untreated (B) or added either with 50 ng/mL VEGF (C) or 50 ng/mL VEGF
and VECD1 (D) and were further incubated for another 24 hours. Each
Flk-1+ cell preserved cell-cell contact in culture with
VEGF (C), whereas cell-cell contact of Flk-1+ cells was
completely disrupted in the presence of VEGF and VECD1 (D). Scale bar
= 100 µm.
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To further determine the role of VE-cadherin in cell-cell adhesion in a
more dynamic situation in which motility of ECs is induced by high dose
of VEGF, 50 ng/mL VEGF and VECD1 were added simultaneously after 3 days
of culture, when Flk-1+ cells formed tightly packed sheets
on OP9 feeder layer. Treated or untreated culture was analyzed after 24 hours. Compared with the culture with OP9 alone (Fig 7B), 50 ng/mL VEGF
enhanced cell motility of Flk-1+ cells in the periphery of
sheets. Notably, although the area with cell-cell contact was markedly
reduced, with shift of cell morphology from oval to spindle form, each
Flk-1+ cell preserved cell-cell contact, particularly in
the central region of sheets (Fig 7C). On the other hand, further
addition of VECD1 in this culture completely disrupted cell-cell
contact of all cells in the Flk-1+ cell clusters, although
it had no effect on their survival or proliferation (Fig 7D).
These results suggest that VE-cadherin is the essential molecule
maintaining the cell-cell connection of ECs during vasculogenesis.
| |
DISCUSSION |
ECs are thought to be the major component of a primitive vascular
plexus, although the interaction between ECs and neighboring cells such
as pericytes is required for remodeling of a primitive vasculature into
a more complex form.48 During vasculogenesis, ECs are
supposed to be generated from mesodermal cells and proliferate, migrate, and eventually form the endothelial lining. Thus, we attempted
to establish an experimental system for dissecting the cellular events
of EC progenitors required for vasculogenesis.
As described in previous papers,45,49,50 mesodermal cells
containing EC progenitors can be defined by the expression of Flk-1. We
confirmed here that purified Flk-1+ cells, which did not
initially express other EC markers, differentiated in vitro into ECs,
although Flk-1 cells of undefined lineages were also
generated. All the EC markers examined here were expressed in a
sequential manner: at first VE-cadherin and PECAM-1 and subsequently
CD34. Although a previous report51 demonstrated that the
expression of PECAM-1 was detected earlier than that of VE-cadherin in
the differentiation of ES-derived ECs, we confirmed simultaneous
expressions of both molecules at least in the Flk-1+
population. However, we also detected the expression of PECAM-1 earlier
than that of VE-cadherin in the Flk-1 populations
(data not shown). In several days, the expression of Flk-1 was
downregulated, whereas that of VE-cadherin was maintained, which is
consistent with previous reports on the expression of Flk-1 in
embryos.45,49 In contrast, another previous
report11 showed that the expression of Flk-1 was also
detected in some adult tissues, such as brain, kidney, heart, spleen,
and muscle. In fact, the expression of Flk-1 on OP9 feeder layer was
maintained at least for 7 days (data not shown). Thus, we found that
the differentiation pathway of ECs on type IV collagen-coated dishes was very similar to that during embryogenesis. While a number of
culture systems using either ES cell-derived embryoid bodies, epiblasts, or whole yolk sac tissues have been devised to investigate the process in vasculogenesis, our culture system described here is
unique and advantageous in that purified Flk-1+ mesodermal
cells are used as the starting population.
By the culture of these purified Flk-1+ cells at low cell
density, we attempted to establish a culture system for dissecting the
cellular events of developing ECs. However, they could not grow at low
cell density on type IV collagen-coated dishes, even in the presence of
VEGF. Because feeder cell lines have been frequently used for
supporting the growth of various cell lineages, we searched for feeder
cell lines that can maintain the differentiation and proliferation of
Flk-1+ cells. As we expected, three of four feeder cell
lines tested in this study were effective in supporting the growth and
differentiation of Flk-1+ cells, whereas the distribution
pattern of Flk-1+ cells on each feeder layer was different.
The variations in size and shape of Flk-1+ cell clusters on
different feeder layers may reflect the differences of molecules
affecting the cellular events of Flk-1+ cells. The
variations did not seem to be correlated with the concentration of VEGF
that was measured in supernatant recovered from 3-day culture of sorted
Flk-1+ cells on each feeder layer; NIH3T3 (409.5 pg/mL),
PA6 (82.3 pg/mL), Balb/c3T3 (137.4 pg/mL), and OP9 (38.5 pg/mL). At
present, it remains obscure what kinds of molecules regulate the
morphological diversity. It is difficult to exclude the possibility
that the morphological diversity was derived from a small number of
contaminating cells or different subpopulations within sorted
Flk-1+ cells. We observed very similar results when we
performed second-round cell sorting of Flk-1+
E-cadherin cells to greater than 99% purity (data
not shown). Furthermore, the number of Flk-1+ cell clusters
on Balb/c3T3 feeder layer was similar to that on OP9 feeder layer (data
not shown). Thus, we suppose that the morphological difference between
Flk-1+ cell clusters on each feeder layer resulted from the
difference of microenvironment.
The growth of Flk-1+ cell clusters on OP9 feeder layer was
significantly inhibited by the addition of mFlt-1-hIgG1, but not completely even by a larger amount of mFlt-1-hIgG1, suggesting that
VEGF plays an important role in the proliferation of Flk-1+
cells but that other molecules produced by OP9 also play a role in
their proliferation. This is consistent with a previous report demonstrating the presence of ECs even in
VEGF/ embryos.15 Indeed,
the presence of VEGF was insufficient for the proliferation of
Flk-1+ cells in the absence of feeder cells. The growth and
survival of ECs during vasculogenesis are thus regulated by multiple
molecules. It is important to specify these molecules expressed on
feeder cell lines.
In contrast to the proliferation, the differentiation of EC lineage, as
assessed by the expression of VE-cadherin and PECAM-1, was not
inhibited by the addition of mFlt-1-hIgG1 into the culture both before
and after sorting of Flk-1+ cells (data not shown),
indicating that the differentiation of Flk-1+ cells is
independent of VEGF. This is consistent with our results that the
differentiation of Flk-1+ cells occurs in the absence of
exogenous growth factors (Fig 1).
The number and size of Flk-1+ cell clusters on OP9 feeder
layer increased to some extent by the addition of 1 to 10 ng/mL VEGF, indicating that VEGF regulates the proliferation of Flk-1+
cells in a dose-dependent manner. After reaching a plateau of proliferation, moreover, further increase of VEGF resulted in an
enhancement of cell motility, with a marked change of cell morphology
from oval to spindle form. This result may explain the cellular events
of ECs in Flk-1LacZ/LacZ mice reported
previously.17 In this embryo, the number of LacZ-positive cells decreases and these cells form a cell-mass in the posterior region. This appears to result from impaired proliferation and migration. In contrast to VEGF, PlGF, a specific ligand for Flt-1, had
no effect on the proliferation or motility of Flk-1+ cells
in our culture system. These results indicate that Flt-1 is not
involved positively in the regulation of the proliferation and motility
of ECs during vasculogenesis. In fact, although Flt-1 is demonstrated
to be a higher affinity receptor of VEGF than Flk-1,46
previous reports showed that ECs overgrew and formed aberrant but not
organized vascular systems in Flt-1/
mice as well as avian embryos with overexpression of VEGF or those
injected with VEGF.18,52,53 Moreover, it was recently demonstrated that Flt-1 lacking the tyrosine kinase domain is sufficient for normal vascular development in mice, suggesting that
Flt-1 plays a role in vasculogenesis as a negative regulator by
preventing VEGF from binding Flk-1.54 These results
together with our present findings suggest that VEGF signals
through Flk-1 in a strictly concentration-dependent manner during
vasculogenesis of early embryos. In summary, our results indicate that
VEGF has dual roles in regulating the cellular events of ECs in
vasculogenesis, supporting the proliferation of ECs at low dose and
enhancing cell motility at high dose. These dose-dependent dual effects of VEGF may account for previous reports that
VEGF+/- mice are embryonic lethal.15,16
During the differentiation of Flk-1+ cells, VE-cadherin
started to express from an early stage and was concentrated at
cell-cell junctions in the sheet-like structure. Because previous
reports described that VE-cadherin is essential for establishment of
organized vascular plexus as well as maintenance of the integrity of
ECs,19,29 VE-cadherin should contribute to the formation of
Flk-1+ cell sheets in our culture system. This was
confirmed by an experiment demonstrating that VECD1 inhibited the
formation of Flk-1+ sheet-like structure on OP9 feeder
layer. Thus, VE-cadherin is the major molecular component regulating
cell-cell adhesion of ECs. Interestingly, dispersion of
Flk-1+ cells on OP9 feeder layer could be also induced by
the addition of a high dose of VEGF. In this case, Flk-1+
cells maintained cell-cell junctions, although the area of cell-cell contact was markedly reduced. This contrasts with the cell dispersion induced by VECD1 in which all of the cells loosened cell-cell contact.
Such diversity in the shape of Flk-1+ cell clusters formed
in our culture system suggests that alteration of VEGF concentration
and VE-cadherin expression profoundly affect the proliferation,
motility, and cell-cell adhesion of ECs, thereby playing
a major role in morphogenesis of a primitive vascular plexus.
As compared with previous systems using ES cell-derived embryoid bodies
or the culture of mesodermal cells from embryos, our culture system is
still insufficient to induce the formation of tubular networks in
vascular development. However, such complex structures should be
achieved by regulating the differentiation, proliferation, migration,
and cell-cell adhesion of each individual EC progenitor. In this sense,
our culture system is unique in that (1) it is the first system to use
purified EC progenitors; (2) it is possible to dissect the cellular
events of ECs in vasculogenesis, such as differentiation,
proliferation, migration, and cell-cell adhesion; and (3) it is devoid
of complex embryoid structures.
| |
ACKNOWLEDGMENT |
The authors thank Dr M.J. Evans for providing CCE ES cell line, Dr H. Kodama for both MC3T3-G2/PA6 and OP9 feeder cell lines, Dr A
Nagafuchi for ECCD2 MoAb, and Drs G. Breier and W. Risau for
murine Flt-1 cDNA. We also thank Dr M. Ogawa for help in flow cytometry.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted October 13, 1998.
Supported by grants from the Ministry of Education, Science, Sports and
Culture of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Masanori Hirashima, MD,
Department of Molecular Genetics, Graduate School of Medicine, Kyoto
University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan;
e-mail: mnhirash{at}virus.kyoto-u.ac.jp.
| |
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Abstract
Introduction