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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1549-1556
In Vitro Hematopoietic and Endothelial Cell Development From Cells
Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region
By
Isao Hamaguchi,
Xu-Ling Huang,
Nobuyuki Takakura,
Jun-ichi Tada,
Yuji Yamaguchi,
Hiroaki Kodama, and
Toshio Suda
From the Department of Cell Differentiation, Institute of Molecular
Embryology and Genetics, Kumamoto University School of Medicine,
Kumamoto; and Research Center Kyoto, Bayer Yakuhin Ltd, Kyoto, Japan.
 |
ABSTRACT |
Recent studies have shown that long-term repopulating hematopoietic
stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM)
region. Our immunohistochemistry study showed that TEK+
cells existed in the AGM region. Approximately 5% of AGM cells were
TEK+, and most of these were CD34+ and
c-Kit+. We then established a coculture system of AGM
cells using a stromal cell line, OP9, which is deficient in macrophage
colony-stimulating factor (M-CSF). With this system, we showed that AGM
cells at 10.5 days postcoitum (dpc) differentiated and proliferated
into both hematopoietic and endothelial cells. Proliferating
hematopoietic cells contained a significant number of colony-forming
cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated
hematopoietic cells and platelet endothelial cell adhesion molecule
(PECAM)-1+ endothelial cells on the OP9 stromal layer,
while TEK cells did not. When a ligand for TEK,
angiopoietin-1, was added to the single-cell culture of AGM,
endothelial cell growth was detected in the wells where hematopoietic
colonies grew. Although the incidence was still low (1/135), we showed
that single TEK+ cells generated hematopoietic cells and
endothelial cells simultaneously, using a single-cell deposition
system. This in vitro coculture system shows that the
TEK+ fraction of primary AGM cells is a candidate for
hemangioblasts, which can differentiate into both hematopoietic cells
and endothelial cells.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
DURING MOUSE EMBRYOGENESIS, hematopoiesis
beginning in the yolk sac at 7.5 days postcoitum (dpc) shifts to the
fetal liver and later to the spleen and bone marrow.1-3
Hematopoiesis before the formation of the fetal liver is known as
primitive hematopoiesis and is distinguished from the adult-type
definitive hematopoiesis by specific expression of embryonic-type
globin in nucleated erythrocytes. Although primitive hematopoiesis and committed hematopoietic progenitors can be detected in the yolk sac as
early as 7 to 8.5 dpc,2,4 neither the colony-forming units
in spleen (CFU-S) nor long-term repopulating hematopoietic stem cells
(LTR-HSCs) are present in the yolk sac before
circulation.5-7 Recently, in the mouse embryo, a preliver
intraembryonic site of potent definitive hematopoietic activity has
been identified.7 This mesodermally derived region of the
mouse embryo containing the dorsal aorta, genital ridge/gonads, and
pro/mesonephros (aorta-gonad-mesonephros [AGM]) has been shown to
harbor adult-type multipotent hematopoietic progenitors and
LTR-HSCs.7-9
The formation of the hematopoietic organ closely relates to
angiogenesis, indicating the existence of common progenitors, hemangioblasts.10 Among endothelial cell receptor tyrosine
kinases (RTKs), the vascular endothelial growth factor (VEGF)
receptors, Flt-111,12 and Flk-1/KDR,13,14
including Flt-4,15,16 are well characterized. These RTKs
are critical to the development of the vascular and hematopoietic
system. We and other groups have characterized the second subfamilies,
TEK and TIE,17,18 both of which have similar domain
structures, and similar expression patterns. TEK and TIE have complex
extracellular domains consisting of two immunoglobulin-like loops
separated by three EGF-like repeats that are followed by three
fibronectin type III-like repeats. Their intracellular portions
contain a split kinase domain.18 In embryo at 7.5 dpc, TEK
is expressed in both the extraembryonic mesoderm and embryonic mesoderm
in regions thought to give rise eventually to the embryonic
vasculature, which follows the TIE expression in the same region at 8.0 dpc.17,19,20 Mice lacking TEK die from defects in
angiogenesis and vascular remodeling, as well as vessel integrity
between 9.5 and 10.5 dpc.21 These periods are important for
the definitive hematopoietic development in the mouse embryo. TEK is
suggested to play an essential role in the development of hematopoiesis
in the AGM region. We have also shown that TEK and TIE are expressed in
the stem-cell fraction of fetal liver and adult bone
marrow.22
In this study, we established a novel culture system by using a stromal
cell line, OP9, which lacks macrophage colony-stimulating factor
(M-CSF).23 We showed that this culture system supports the
in vitro differentiation of hematopoietic cells and endothelial cells
from AGM cells. FACS analyses showed that TEK+ cells in the
AGM region were able to differentiate into hematopoietic and
endothelial lineages, suggesting that TEK+ cells in this
region are hemangioblasts.
| |
MATERIALS AND METHODS |
Cell preparation and culture conditions.
C57BL/6 mice were purchased from SCL (Hamamatsu, Japan). Embryos at
10.5 dpc were used to dissect the AGM region. The single-cell suspension was prepared after isolating with 2.4% dispase (GIBCO, Grand Island, NY) and passage of the selected tissues through a
26-gauge needle. OP9 stromal cells were plated in microtiter plates,
and cells from the AGM region were seeded on them. The culture media
were Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal
calf serum (FCS) (CSL Ltd, Victoria, Australia) and 100 U/mL murine
recombinant interleukin-3 (IL-3) (provided by T. Sudo, Toray Industries
Inc, Kamakura, Japan). Half of the medium was replaced every 5 days
with fresh medium containing IL-3. On day 14 of culture, nonadherent
cells were harvested. To stimulate the growth of endothelial cells from
single cells, we added 100 ng/mL human recombinant purified
angiopoietin-1 (provided by G. Yancopoulos, Regeneron Pharmaceuticals,
Inc, Tarrytown, NY) to the coculture system.24
Immunohistochemistry.
Immunohistochemistry was performed essentially as
described.25 AGM cells cultured on OP9 were fixed with 4%
paraformaldehyde at 4°C, and stained with a rat monoclonal
anti-mouse platelet endothelial cell adhesion molecule (PECAM-1)
antibody (1 µg/mL) (PharMingen, San Diego, CA) by the indirect
immunoperoxidase method using horseradish peroxidase-conjugated
antirat IgG. Peroxidase activity was visualized using
3,3'-diaminobenzidine (Dojindo, Kumamoto, Japan) and
nickel as substrates.
Uptake of acetylated low-density lipoprotein labeled with
DiI by endothelial cells.
Sorted TEK+ cells were cocultured with OP9 for 10 days.
After washing culture wells with phosphate-buffered saline (PBS) three times, 10 µg/mL of acetylated low-density lipoprotein labeled with
DiI (DiI-Ac-LDL) (Biomedical Technologies Inc, Stoughton, MA) was added, and adherent cells were incubated for 4 hours at 37°C. After removing the media containing DiI-Ac-LDL, cells were washed with PBS three times, and then observed under fluorescence microscopy. Uptake of DiI-Ac-LDL can be visualized using a standard rhodamine excitation emission filter.
Flow cytometric analysis and cell sorting.
Cell suspensions were stained for 30 minutes with the following
monoclonal antibodies (MoAbs): anti-TEK (Tek 4) was previously prepared
in our laboratory22; Sca-1 (E13-161.7), Gr-1 (RB6-8C5), Mac-1 (M1/70), TER119, B220 (RA3-6B2), CD4 (RM4-5), and CD8 (53-6.7), all of which were purchased from PharMingen; and anti-c-Kit
(ACK2), a gift from Dr S. Nishikawa (Kyoto University, Kyoto, Japan). Fluorescence-activated cell sorting (FACS) analysis and cell sorting were performed on a FACSvantage (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Progenitor assay by methylcellulose culture.
Sorted AGM cells or hematopoietic cells were embedded in 1 mL of
 -medium containing 1.3% methylcellulose (1,500 cp; Aldrich Chemical
Co, Milwaukee, WI), 30% FCS, 1% deionized bovine serum albumin (BSA)
(Sigma Chemical Co, St Louis, MO), 0.1 mmol/L 2-mercapto-ethanol (Sigma), 100 ng/mL stem-cell factor (SCF) (from Chemo-Sero-Therapeutic Co Ltd, Kumamoto, Japan), 200 U/mL recombinant mouse IL-3, 20 ng/mL
human recombinant IL-6 (provided by Ajinomoto, Kawasaki, Japan), and 2 U/mL recombinant human erythropoietin (Epo) (provided by Snow-Brand
Milk Product Co Ltd, Tochigi, Japan). The cells were cultured in a
35-mm culture dish and incubated at 37°C in a humidified atmosphere
with 5% CO2.
Spleen colony assay.
The spleen colony formation capacity in primary and cultured cells was
assayed as described.26 Sorted cells were injected into
lethally irradiated mice (total body irradiation of 9.0 Gy). The
spleens were removed on day 12 after transplantation and fixed in
Bouin's solution, and then spleen colonies were counted by microscopical observation.
| |
RESULTS |
TEK+ cells in the AGM region.
We have cloned TEK, a receptor tyrosine kinase, from a cDNA library of
fetal liver and have shown that TEK is expressed in HSCs of fetal liver
and adult bone marrow cells.18,22 Immunohistochemistry showed TEK+ cells in the AGM region and vitelline artery at
10.5 dpc (Fig 1A). Since one embryo
contained approximately 1.4 × 104 cells in the AGM
region, AGM cells pooled from more than 15 embryos were analyzed in
each experiment. FACS analysis showed that 4.8% of these cells were
TEK+. Most of these cells coexpressed CD34 (74.1%) and
c-Kit (78.2%) (Fig 1B). Among c-Kit+ cells,
c-Kithigh (8.6%) and c-Kitlow (69.6%)
fractions were detected. By contrast, TEK cells
contained no CD34+ cells. To clarify the nature of AGM
primary cells defined by TEK, the ability to form hematopoietic
colonies in the presence of SCF, IL-3, IL-6, and Epo was analyzed
(Table 1). In contrast to AGM
TEK cells, TEK+ cells formed erythroid
burst (E)/mix (MIX), granulocyte-macrophage (GM), macrophage (M), and
megakaryocyte (Meg). These results suggested that hematopoietic
multipotential progenitor cells were included in the AGM
TEK+ population. The frequency of colony formation from AGM
TEK+ cells was similar to that of bone marrow
TEK+ cells, however, AGM TEK+ cells formed more
E/MIX colonies than bone marrow TEK+ cells.
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| Fig 1.
Expression of TEK in the AGM region. (A) Mouse embryo at
10.5 dpc was whole-mount-stained with TEK MoAb by
immunohistochemistry. The AGM region (arrow) and vitelline artery
(arrowhead) were strongly stained. hl, hind limb; h, heart; fl, fore
limb. (B) FACS analysis showed that ~5% of 10.5 dpc AGM cells were
TEK+. Most TEK+ cells coexpressed CD34 and
c-Kit.
|
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Establishment of a hematopoietic cell culture system from AGM cells.
To elucidate the mechanisms of hematopoietic development from AGM, we
established a novel AGM cell culture system by introducing a stromal
cell line, OP9. The AGM region was dissected from 10.5 dpc C57/BL6
embryo, and was isolated into single-cell suspensions by dispase
treatment. AGM cells were seeded on OP9 cells. Small round cells were
observed on OP9 cells in 14 days of culture in the presence of IL-3
(Fig 2). This proliferation of
hematopoietic cells was maintained for 4 months. Without OP9, no
hematopoietic cells were grown from AGM cells.
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| Fig 2.
Coculture of AGM cells with stromal cells. (A)
Embryo 10.5 dpc AGM cells were cocultured with the murine stromal cell
line, OP9, in the presence of IL-3. Small round cells were generated
for 14 days' culture. (B) Hematopoietic cells generated on OP9 cells
(middle). Without OP9 cells, fibroblast-like cells grew slowly to be
confluent in 4 weeks (right).
|
|
Hematopoietic cell development from AGM cells.
Hematopoietic cells at various stages of differentiation were detected
in this coculture of AGM with OP9 stromal cells. Sca-1+
and/or CD34+ cells were developed, while the
TEK+ fraction decreased (Fig
3A). As shown in Fig 3B, hematopoietic progenitor cells (Lin, c-Kit+,
Sca-1+) consisted of 3.2% of the cultured cells.
c-Kit+, Sca-1+ cells made up 73.1% of the
Lin fraction. From one embryo, 3 × 105 Lin, c-Kit+,
Sca-1+ cells developed in 14 days of culture. The in vitro
colony-forming activity of these progenitor cells was assayed in
methylcellulose medium containing IL-3, IL-6, SCF, and Epo. As shown in
Table 2, AGM-derived
hematopoietic progenitor cells formed a various kind of colonies. The
incidence of colony-forming cells in cultured AGM
Lin, c-Kit+, Sca-1+
cells was approximately 10%, which was lower than that of adult bone
marrow Lin, c-Kit+,
Sca-1+ cells. However, the variety of colony types formed
by cultured AGM cells was similar to that by bone marrow cells.
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| Fig 3.
The development of hematopoietic progenitor cells in
cultured AGM cells. (A) Primary and cultured AGM cells were analyzed
with TEK, CD34, and c-Kit MoAb by FACS. Embryo 10.5 dpc AGM cells were
cultured on OP9 cells and they developed Sca-1+ and
CD34+ cells in 14 days of culture. (B)
Lin, c-Kit+, Sca-1+ cells
consisted of 3.2% of the cultured AGM cell population.
|
|
To estimate the properties of stem-cell function, a spleen colony assay
was conducted. Five hundred sorted cells were injected into lethally
irradiated mice to define the frequency of CFU-S in each subpopulation
of HSCs. The incidence of day 12 CFU-S within cultured AGM cells was
7.4 ± 2.2 (n = 8), with the incidence being equivalent to that of
adult bone marrow (11.0 ± 3.0, n = 4). Differentiating hematopoietic cells on OP9 were also analyzed with FACS (Fig
4). It was confirmed that cultured AGM
cells were positive for Gr-1, Mac-1, TER119, and B220. However,
CD4+ or CD8+ cells were not detected.
May-Grünwald-Giemsa staining showed that the cultured cells
contained a large number of hematopoietic cells such as granulocytes,
monocytes/macrophages, erythroid cells, megakaryocytes, and lymphocytes
(data not shown). These results indicate that with this culture system,
one is able to promote hematopoietic differentiation from AGM cells.
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| Fig 4.
Analysis of cultured AGM cell surface expression with
hematopoietic differentiating markers. AGM cells cocultured with OP9
cells differentiated into Gr-1+ (granulocytes),
Mac-1+ (monocytes/macrophages), TER119+
(erythroid lineage cells), and B220+ cells (B
lymphocytes). CD4+ or CD8+ cells (T
lymphocytes) were not detected. For all fractions, 104
cells were counted for each analysis, and normal control was
<101.
|
|
Endothelial and hematopoietic differentiation potential of
TEK+ AGM cells.
To elucidate whether TEK+ AGM cells have an ability to
differentiate into both hematopoietic and endothelial cells, we applied coculture of AGM cells. AGM cells at 10.5 dpc were stained with TEK
MoAb, and 2,000 TEK+ and TEK cells were
plated on OP9 cells. Hematopoietic cells developed in 10 days' culture
from TEK+ cells, but did not develop from the
TEK cells (Fig 5A and
B). Methylcellulose colony assay showed that 6.4 ± 1.0 × 103 (n = 3) colony-forming cells were generated from
2,000 TEK+ cells after coculture with OP9 in the presence
of IL-3. After hematopoietic cells were removed, adherent cells on OP9
cell layers were immunostained with PECAM-1. PECAM-1+ cells
formed an endothelial cell network on the OP9 cell layer of
TEK+ wells, but not on that of TEK wells
(Fig 5C and D). Moreover, when the TEK ligand, angiopoietin-1 (100 ng/mL), was added, endothelial growth was enhanced and these cells
showed uptake of DiI-Ac-LDL (Fig
6, see page
1550). These results indicate that the TEK+ fraction among
AGM cells is able to differentiate into both hematopoietic and
endothelial cells in an OP9 coculture system.
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| Fig 5.
Hematopoietic and endothelial cell development from
TEK+ fraction of AGM cells at 10.5 dpc. Each number of
TEK+ or TEK cells (2,000) from AGM at 10.5 dpc was cocultured with OP9 cells. Hematopoietic cells were developed
from the TEK+ fraction for 10 days of culture (A). These
were not observed in the culture of the TEK fraction
(B). After cell suspensions were removed, the OP9 cell layer was
immunostained with PECAM-1 MoAb. PECAM-1+ endothelial
cells formed a network in the culture of the TEK+
fraction (C). No PECAM-1+ cells were detected in the
culture of the TEK fraction (D).
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| Fig 6.
Induction of endothelial cell growth by angiopoietin-1.
Two thousand TEK+ cells from the AGM region were seeded
onto OP9 cell layers in a 24-well plate. When angiopoietin-1 was added,
cord-like strutures of endothelial cell growth were observed (A) and
these cells show uptake of DiI-Ac-LDL (B).
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Next, we examined the differentiation of AGM cells at the single-cell
level. Limiting dilution analysis of TEK+ AGM cells
predicted the frequency of hematopoietic colonies to be on the order of
one in 89 TEK+ AGM cells (Fig
7). The results of clonal analysis of
TEK+ AGM cells are shown in Table
3. When 1,752 TEK+ cells were
seeded onto microtiter wells with IL-3 by single-cell deposition in two
separate experiments, hematopoietic colonies were detected in 19 wells
after a 7-day culture. However, PECAM-1+ endothelial cells
were not detected in any wells by immunohistochemistry. Since
endothelial growth was poor at the single-cell level, we added
angiopoietin-1 in this culture system. When angiopoietin-1 (100 ng/mL)
was added to the coculture system, 10 of 811 wells were positive for
hematopoietic colonies after a 7-day culture (Fig 8A and B, see page
1550). Bipolar PECAM-1+ cells were detected in six
hematopoietic cell-positive wells by immunohistochemistry (Fig 8C).
Thus, the incidence of endothelial precursors in AGM cells was one per
135. These results indicate that hematopoietic and endothelial
cells differentiate from single AGM TEK+ cells, and that
the AGM TEK+ fraction is a candidate for hemangioblasts.
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| Fig 7.
Limiting dilution analysis of TEK+ AGM
cells. The frequency of colony-initiating progenitors at 37% of
negative wells corresponded to the Poisson analysis, 1/89 for
TEK+ AGM cells. This result is representative of
experiments done in triplicate.
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Table 3.
Incidence of Hematopoietic and Endothelial Cell
Differentiation From TEK+ Cells Derived From AGM Cells at
10.5 dpc
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| Fig 8.
Hematopoietic and endothelial cell development from
single TEK+ cells. Single TEK+ cells from
AGM at 10.5 dpc were seeded on OP9 in the presence of angiopoietin-1
and IL-3. (A) Hematopoietic cells developed on day 7 of culture in
96-well microtiter plates. (B) Hematopoietic blast cells (b) and OP9
cells (o) were revealed by cytospin preparation with Giemsa staining.
These cells were adhered to the stromal layer. (C)
PECAM-1+ endothelial cells were detected in the presence
of angiopoietin-1 (100 ng/mL) and IL-3 (100 U/mL). (D) No
PECAM-1+ cells were detected in the presence of IL-3 (100 U/mL) alone.
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| |
DISCUSSION |
AGM is the ontogenic source of various kinds of precursor cells,
including definitive HSCs. Since the number of HSCs in the AGM region
is small, it is difficult to elucidate the mechanisms of hematopoietic
cell development from AGM progenitor cells. We developed a culture
system by introducing a stromal cell line, OP9. OP9 cells were
established from newborn calvaria of the F2 (C57BL/6xC3H)-op/op mouse, which lacks
M-CSF23 by allowing various lineages to generate from
embryonic stem (ES) cells.27 In this system, we showed that
AGM cells at 10.5 dpc differentiated and proliferated into
hematopoietic cells. Proliferating hematopoietic cells contained a
significant number of colony-forming cells in culture and in spleen in
addition to mature multilineage cells. The ratio of
Lin, c-Kit+, Sca-1+ to
total nucleated cells was significantly larger (3.2%) than that of
marrow mononuclear cells (0.08%).28 Those hematopoietic cells had colony-forming ability (Table 2). While the colony-forming ability of AGM stem cells was lower than that of bone marrow stem cells, the incidence of day 12 CFU-S was almost equivalent. In addition, hematopoietic progenitor cells were maintained for long as 4 months on the same OP9 cells. OP9 cells supported the development of
hematopoietic cells from AGM cells, compared with the other stromal
cells. OP9 cells produce a key factor(s) for the development of
hematopoietic cells such as SCF, IL-6, and unknown factors. Recently,
Oncostatin M has been reported to play a role in the development of AGM hematopoietic cells.29 We detected the
expression of Oncostatin M mRNA in OP9 cells by the
reverse-transcriptase polymerase chain reaction (RT-PCR)
method. Since OP9 cells did not produce M-CSF, proliferation of
macrophages was not vigorous enough to inflict damage on stromal cells.
IL-3 was added to stimulate the proliferation of hematopoietic cells on
OP9 layer. Multilineage differentiation such as to granulocyte,
macrophages, erythrocytes, megakaryocytes, and B cells was observed on
the OP9 layer. However, CD4+ and/or
CD8+ cells were not detected in this culture system,
suggesting a thymic microenvironment is required. CD3+
natural killer T cells were detected (data not shown).
PECAM-1 is a specific marker for endothelial cells and platelets. In
this culture system, PECAM-1+ cells were detected on OP9
cells after 7 to 10 days' culture of TEK+ AGM cells.
Moreover, we confirmed the endothelial cells by the uptake of
DiI-Ac-LDL. Since fibroblasts do not show the uptake LDL, some
elongated cells are endothelial cells.
We have previously shown that TEK+ cells are present in the
stem-cell fraction, Lin, c-Kit+,
Sca-1+ cells.22 Primary AGM cells at 10.5 dpc
were clearly divided into two fractions, TEK+ and
TEK cells. Interestingly, most TEK+
cells express c-Kit and CD34, but not Sca-1. TEK+ cells
were stained among the hematopoietic cells and endothelial cells in the
vitelline artery connecting the AGM and yolk sac. This expression
profile is similar to that of CD34, which was examined by means of in
situ hybridization.30
Endothelial and hematopoietic cells in blood islands were proposed to
originate from a common precursor, termed the hemangioblast, based on
their simultaneous emergence. However, the existence of this cell
remains to be proven. To elucidate whether hemangioblasts existed in
the TEK+ fraction of AGM cells at 10.5 dpc, single AGM
cells were plated on the OP9 stromal layer containing IL-3 by an
automatic single-cell deposition system. Hematopoietic colonies did
develop, however, no proliferation of endothelial cells was detected
even after 14 days of this culture. Since 2,000 TEK+ cells
formed on the endothelial network on the stromal layer, a minimal
number of cells or certain cytokines may be required for the detection
of endothelial cells from single AGM cells. In our recent study,
production of angiopoietin-1, but not angiopoietin-2, was detected in
OP9 by RT-PCR.31 When 100 ng/mL angiopoietin-1 was added in
this culture system, PECAM-1+ bipolar endothelial cells
were generated from single cells (Table 3 and Fig 8). These cells were
thought to form a vascular bed. Bipolar cell development would be the
first step in the formation of the vascular endothelial cell network.
Thus, it is concluded that the development of endothelial cells, as
well as of hematopoietic cells, is supported by OP9 cells.
Recently, ligands for the TEK receptor, termed angiopoietin-1 and -2, were cloned by secretion-trap expression cloning.24 Mice
lacking angiopoietin-1 display angiogenic deficits,32 and this finding supports putative roles for TEK and angiopoietin-1 in
angiogenesis. It is now considered that TEK controls the ability of
endothelial cells to recruit periendothelial support cells to stabilize
the structure of blood vessels and modulate their function. In our
recent study, angiopoietin-1 promoted cells expressing TEK to adhere to
fibronectin.31 Moreover, angiopoietin-1 acts on endothelial
cells synergistically with VEGF. Asahara et al showed that cells
isolated with CD34 or anti-Flk-1 antibody can differentiate into
endothelial cells.33 Eichmann et al showed that early
mesodermal Flk-1+ cells give rise to hematopoietic cell
colonies and that the ligand VEGF supports the growth of endothelial
colonies in chicken embryo.10 These results indicate that
the adhesion between endothelial and hematopoietic cells is mediated by
TEK and required for the intravascular hematopoiesis of the AGM region.
In contrast to primary TEK+ AGM cells, TEK+
cells developed from culturing AGM cells on OP9 did not have the
ability to differentiate into endothelial cells (data not shown),
indicating that hemangioblasts of AGM primary cells differentiated into
a hematopoietic lineage, losing the ability of endothelial cell
development. To enrich the hemangioblasts, multiparameters such as
CD34, c-Kit, and Flk-1 should be introduced. It might be possible to
examine more precisely the incidence of endothelial cell and/or
hematopoietic progenitors in the AGM region. Access to these early
developing precursors will enable us to further define lineage
relationships and molecular commitment steps within the embryonic AGM region.
In conclusion, we established a coculture system for AGM with a stromal
cell line, OP9. This in vitro coculture system shows that
TEK+ cells are candidates for hemangioblasts, which can
differentiate into hematopoietic cells and endothelial cells.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted October 22, 1998.
Supported in part by Grants-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan. I.H. is supported
by Research Fellowships of the Japan Society for the Promotion of
Science for Young Scientists.
I.H. and X.-L.H. contributed equally to this work.
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 Toshio Suda, MD, Department of Cell
Differentiation, Institute of Molecular Embryology and Genetics,
Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto,
860-0811, Japan.
| |
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Abstract
Introduction