|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1168-1177
Expression of  4-Integrin Defines the Earliest Precursor of
Hematopoietic Cell Lineage Diverged From Endothelial Cells
By
Minetaro Ogawa,
Masami Kizumoto,
Satomi Nishikawa,
Tetsuhiro Fujimoto,
Hiroaki Kodama, and
Shin-Ichi Nishikawa
From the Department of Molecular Genetics, Faculty of Medicine, Kyoto
University, Kyoto, Japan; and the Research Center Kyoto, Bayer Yakuhin,
Ltd, Kyoto, Japan.
 |
ABSTRACT |
Embryonic stem cells can differentiate in vitro into hematopoietic
cells through two intermediate stages; the first being FLK1+ E-cadherin proximal lateral mesoderm
and the second being CD45 VE-cadherin+
endothelial cells. To further dissect the CD45
VE-cadherin+ cells, we have examined distribution of
 4-integrin on this cell population, because 4-integrin is the
molecule expressed on hematopoietic stem cells. During culture of
FLK1+ E-cadherin cells,
CD45 VE-cadherin+
4-integrin cells differentiate first, followed by
4-integrin+ cells appearing in both
CD45 VE-cadherin+ and CD45
VE-cadherin cell populations. In the CD45
VE-cadherin+ cell population,
4-integrin+ subset but not
4-integrin subset had the potential to differentiate
to hematopoietic lineage cells, whereas endothelial cell progenitors
were present in both subsets. The CD45
VE-cadherin 4-integrin+ cells also
showed hematopoietic potential. Reverse transcription-polymerase chain
reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential
of the 4-integrin+ cells to give rise to hematopoietic
cell differentiation. Hematopoietic CD45
VE-cadherin+ 4-integrin+ cells were also
present in the yolk sac and embryonic body proper of 9.5 day postcoitum
mouse embryos. Our results suggest that the expression of 4-integrin
is a marker of the earliest precursor of hematopoietic cell lineage
that was diverged from endothelial progenitors.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE MAJOR SITE OF hematopoiesis changes
during the ontogeny of many vertebrate species. In the mouse embryo,
hematopoiesis begins in the extra-embryonic yolk sac at day 7.5 of
gestation.1 The visceral yolk sac mesodermal cells
aggregate into clusters to form blood islands that consist of an inner
core of blood cells and an external layer of endothelial cells. Large
nucleated primitive erythrocytes that express embryonic hemoglobin
develop in the blood islands and migrate into the embryo proper via
circulation that commences on day 9 of gestation. The major
hematopoietic site shifts to the fetal liver at day 10 of
gestation,2 where definitive erythrocytes containing
fetal/adult type hemoglobin, myeloid, and lymphoid cells first appear.
The site of hematopoiesis further shifts from the fetal liver to the
spleen and eventually settles in the bone marrow at day 15 to 16 of
gestation.3,4
Although the yolk sac is the first hematopoietic site and the only site
known to generate hematopoietic cells in situ, the first appearance of
erythroid cells in the blood vessels was staged to day 8 to 8.5 of
gestation.5 The mesodermal component of the para-aortic
splanchnopleura at day 8.5 of gestation was identified as the site that
contains hematopoietic precursors before liver colonization.6,7 At day 10 to 11 of gestation, the aorta, gonads, and mesonephros (AGM) region that originates from the para-aortic splanchnopleura contains multipotent hematopoietic cell
progenitors with long-term repopulating activity.8,9 Cytological analyses have detected two types of structures relevant to
hematopoiesis in the embryo proper before fetal liver hematopoiesis begins. The first are intra-arterial hematopoietic cell clusters present in the omphalomesenteric and umbilical arteries and dorsal aorta.10,11 The second are blood islands in the mesentery
of the hind gut, which is included in the para-aortic splanchnopleura of day 9.5 of gestation.11 The mesenteric blood island is
similar to the yolk sac blood island in that it consists of a core of hemocytoblasts circumscribed by an endothelial envelope.
Although the close association of development of hematopoietic and
endothelial cell lineages had raised a hypothesis that the two lineages
are derived from a common progenitor referred as the hemangioblast, so
far no direct evidence has been reported for the existence of a
bipotential cell that gives rise to only hematopoietic and endothelial
cell lineages. To understand the cellular basis underlying early
development of hematopoietic cell lineage, we need to dissect the
developmental pathway from lateral mesodermal cells to mature
hematopoietic cells. Recently, in vitro differentiation systems of
mouse embryonic stem (ES) cells identified early progenitors of
hematopoietic cell lineage. The first demonstration of the induction of
hematopoietic cells from ES cells in vitro was reported by Doetschman
et al.12 Thereafter, most attempts have been made to extend
the range of hematopoietic cell lineage induced from ES cells, to
detect hematopoietic progenitor cells in various stages of
differentiation, and to examine the kinetics of expression of the genes
relevant to hematopoiesis.13-22 An attempt to detect early
hematopoietic progenitors in the in vitro differentiation system of ES
cells also pointed to a common precursor for hematopoietic and
endothelial cells.23 FLK1, a tyrosine kinase receptor that is expressed on endothelial cells and their precursor, proximal lateral
mesoderm, was used as a cell surface marker to purify the earliest
progenitors of hematopoietic cell lineage from ES cell cultures as well
as developing mouse embryos.24,25 However, the induction of
in vitro hematopoiesis from ES cells normally required formation of
cell aggregates termed embryoid bodies (EB) or coculture with a stromal
cell layer, and this has largely restricted identification of cellular
intermediates between mesodermal cells and committed precursors for
hematopoietic and endothelial cell lineages.
Recently, we developed a novel culture system in which ES cells
differentiate into hematopoietic and endothelial cell lineages through
the proximal lateral mesoderm without formation of EB nor requirement
of feeder cells.26 In this culture system, ES cells
are cultured in a dish coated with type IV collagen so that progressive
separation of differentiated cells by fluorescence-activated cell
sorting (FACS) and reculture to induce further differentiation can be
easily achieved. This culture system identified FLK1+
VE-cadherin+ cells derived from FLK1+
VE-cadherin proximal lateral mesoderm as a
population that contains endothelial cell precursors endowed with
hematopoietic potential.26
The hemocytoblasts in the blood islands are interconnected to each
other and to circumscribing endothelial cells through cell junctions of
the zonula adherens and zonula occludens types.11 Cell
adhesion molecules of the cadherin family might be involved in the
zonula adherens type junctions. However, this type of cell junctions
should disappear and be replaced by other types of cell adhesion
machinery such as the integrin superfamily during development of
hematopoietic cells. The  4-integrin has been shown to be expressed by the hematopoietic stem cell and plays an important role in its
migration.27-29 Although several types of integrin
molecules are also found on broad range of the endothelial cells,
expression of 4-integrin is restricted to a small subset of
endothelium.30 To further dissect the hematopoietic
FLK1+ VE-cadherin+ cell population, we
recruited 4-integrin as an additional surface marker. In this
report, we examined expression of 4-integrin on the precursor cells
for hematopoietic and endothelial lineages that were derived from ES
cells and mouse embryonic tissues. Our results demonstrated that
4-integrin can be used as a valuable cell surface marker to focus on
a diverging point of endothelial and hematopoietic cell lineages.
| |
MATERIALS AND METHODS |
Cell lines.
CCE ES cell line, which was a gift from Dr M. Evans31 (Wellcome/CRC Institute, Cambridge,
UK), was maintained in a culture dish coated with gelatin (Type A from
porcine skin; Sigma, St Louis, MO) using Dulbecco's modified Eagle
medium (DMEM; GIBCO BRL, Grand Island, NY) supplemented with 15% fetal
calf serum (FCS; Whittaker Bioproducts, Walkersville, MD), 100 µmol/L
2-mercaptoethanol (2ME), 2 mmol/L L-glutamine, 10 mmol/L Minimum
Essential Medium nonessential amino acids (GIBCO BRL), and 5,000 U/mL
leukemia inhibitory factor (LIF; R&D Systems, Minneapolis, MN).
OP9 stromal cell line32 was maintained in MEM Alpha medium
(GIBCO BRL) supplemented with 20% FCS (HyClone Laboratories, Logan, UT).
Monoclonal antibodies (MoAbs), cell staining, and sorting.
The MoAb against E-cadherin, ECCD2,33 was a gift from Dr A. Nagafuchi (Kyoto University, Kyoto, Japan). The MoAbs AVAS12 (anti-FLK1),24 TER119 (erythroid lineage
marker),34 and VECD1 (anti-VE-cadherin)35 were
purified from hybridoma culture supernatants on a protein G-Sepharose
column (Pharmacia, Uppsala, Sweden). These MoAbs were labeled with
fluorescein isothiocyanate (FITC) or allophycocyanin (APC) by standard
methods. The FITC-labeled MoAbs 30-F11 (anti-CD45), M1/70
(anti-CD11b/Mac-1), and biotin-conjugated MoAbs TER119 and 9C10
(anti-CD49d/4-integrin) were purchased from PharMingen (San Diego,
CA). The phycoerythrin (PE)-conjugated streptavidin was purchased from
Southern Biotechnology Associates (Birmingham, AL).
Cells were blocked with normal mouse serum and stained with several
combinations of labeled MoAbs. The biotin-conjugated MoAbs were shown
by further staining with PE-streptavidin. Stained cells were
resuspended in Hanks' balanced salt solution (GIBCO BRL) containing
1% bovine serum albumin (Sigma) and 5 µg/mL propidium iodide (PI;
Sigma) to exclude dead cells. Cells were analyzed and sorted by FACS
Vantage (Becton Dickinson Immunocytometry Systems, San Jose, CA). Data
were analyzed and printed out by using software CellQuest (Becton
Dickinson Immunocytometry Systems).
In vitro differentiation of ES cells.
For induction of differentiation, 1 × 104 CCE cells
were transferred to each well of a type IV collagen-coated 6-well plate (BIOCOAT; Becton Dickinson Labware, Bedford, MA) and incubated for 4 days in MEM Alpha medium (GIBCO BRL) supplemented with 10% FCS (GIBCO
BRL) and 50 µmol/L 2ME (the induction medium) in the absence of LIF.
Cultured cells were harvested with cell dissociation buffer (GIBCO BRL)
and analyzed for expression of E-cadherin and FLK1 by flow cytometry.
Cells (1.5 to 2 × 106) were routinely recovered from
each well after 4 days of the first induction. We tested more than 30 batches of FCS by determining the percentages of FLK1+
cells induced after CCE cells were plated on type IV collagen-coated dish under the same conditions. There was an extreme variability between batches, and we selected a good lot in which we can induce a
high percentage of FLK1+ cells.
FLK1+ E-cadherin cells were sorted from
the harvested cells for second induction. Sorted cells (3 × 105) were transferred to each well of a 6-well plate
(Becton Dickinson) that was precoated with gelatin and incubated in the
induction medium for 3 days. Cultured cells were harvested with cell
dissociation buffer and analyzed for expression of VE-cadherin,
4-integrin, CD45, and TER119 by flow cytometry. Cells (3 to 5 × 105) were routinely recovered from each well after
3 days of the second induction. The CD45
TER119 cell population in the harvested cells was
fractionated into several fractions by cell sorting and cultured for
further induction of hematopoietic or endothelial cells.
For the induction of hematopoietic cells, sorted cells were transferred
into a 35-mm dish coated with type IV collagen (Becton Dickinson
Labware) and incubated for 7 days in the induction medium supplemented
with a mixture of recombinant growth factors containing 200 U/mL murine
interleukin-3 (IL-3), 2 U/mL human erythropoietin (Epo), 100 ng/mL
murine granulocyte colony-stimulating factor (G-CSF), and 100 ng/mL
murine mast cell growth factor (MGF). Recombinant Epo and G-CSF were
purchased from R&D Systems. Recombinant murine IL-3 and MGF were
prepared as described.36 Cultured cells were analyzed for
expression of surface markers by flow cytometry and for cell morphology
by May-Gruenwald Giemsa staining (Hemacolor; Merck, Darmstadt,
Germany). For measurement of frequency of hematopoietic precursors,
sorted cells were put into a 6-well plate that was preseeded with OP9
stromal cells and incubated in the induction medium supplemented with
the mixture of growth factors described above. After 24 hours, medium
was replaced with a fresh semisolid medium that consisted of the
induction medium, the mixture of growth factors, and 1.2%
methylcellulose (Muromachi Kagaku, Tokyo, Japan). Cells were further
cultured for 6 days and hematopoietic cell colonies were scored under a microscope.
For induction of endothelial cell growth, sorted cells were put into a
6-well plate that was preseeded with OP9 cells and incubated in the
induction medium. After 2 weeks, the cultures were fixed in situ by 4%
paraformaldehyde and stained with either the anti-FLK1 or
anti-VE-cadherin MoAbs and alkaline phosphatase-conjugated antirat IgG
antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).
FLK1+/VE-cadherin+ endothelial cell colonies
were shown by using NBT/BCIP substrate solution (Boehringer Mannheim,
Mannheim, Germany) and scored under a microscope.
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was prepared from sorted cells or cultured cells using ISOGEN
(Nippon Gene, Toyama, Japan). RNA was reverse-transcribed by using
Superscript II reverse transcriptase (GIBCO BRL) and oligo
(dT)12-18 primer (GIBCO BRL) according to the
manufacturer's instructions. PCR assays were performed in the reaction
mixture containing 1× ExTaq Buffer (Takara Shuzo, Osaka, Japan),
200 µmol/L dNTPs (Pharmacia), 25 U/mL ExTaq DNA polymerase (Takara
Shuzo), several dilutions of cDNA, and 2 µmol/L of the following
oligonucleotide primers: Gata1, 5' ACT CGT CAT ACC ACT
AAG GT 3', 5' AGT GTC TGT AGG CCT CAG CT 3';
Tal1, 5' CAT TGC AAG ATG TCT GTT GG 3', 5' GTG AAG CTG CAA AGC TGA TG 3'; Lmo2, 5' AGA ACA TAG
GGG ACC GCT AC 3', 5' GAT GAT CCC ATT GAT CTT GG 3';
Gapd (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]),
5' ATG GTG AAG GTC GGT GTG AAC GGA TTT GGC 3', 5' GCA
TCG AAG GTG GAA GAG TGG GAG TTG CTG 3'. Sequences of other
primers were described elsewhere: Gata2 and
Myb37; Hbb-bh1 (hemoglobin
Z)17; and Hbb-y (hemoglobin Y) and Hbb-b1 (hemoglobin  major).13 Amplification of the cDNA was
performed with 1 cycle at 95°C for 5 minutes, 70°C for 30 seconds, 72°C for 30 seconds, 3 cycles at 95°C for 1 minute,
66°C for 30 seconds, 72°C for 30 seconds, 3 cycles at 95°C
for 1 minute, 62°C for 30 seconds, 72°C for 30 seconds and 40 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and
72°C for 1 minute. RT-PCR products were electrophoresed through 1%
agarose gel and analyzed by staining with ethidium bromide.
Cell dissociation from embryos and colony formation analysis.
Pregnant ICR mice (9.5 day postcoitum) were purchased from Shimizu Co
Ltd (Kyoto, Japan). The yolk sac and embryonic body were separated as
described.3 Anterior and caudal parts were cut off from the
embryo as described,7 and the embryonic trunk lower than
the heart level was pooled. The pooled yolk sac and lower trunk were
incubated in dispase II (Boehringer Mannheim) at 37°C for 20 minutes. Cell clump was washed and further dissociated by incubation in
cell dissociation buffer (GIBCO BRL) at 37°C for 20 minutes.
Finally, single-cell suspension was prepared by gentle pipetting.
Cells were stained with anti-CD45, anti-TER119, anti-VE-cadherin, and
anti-4-integrin MoAbs and CD45
TER119 population was fractionated into several
fractions by cell sorting. Sorted cells were suspended in a matrix gel
consisted of the induction medium, 0.08% type I collagen (Nitta
Gelatin Co, Osaka, Japan), and the mixture of growth factors containing
IL-3, Epo, G-CSF, and MGF and incubated in a 35-mm dish precoated with
type IV collagen. Hematopoietic cell colonies were scored after 7 days.
The culture was dehydrated by capillary action and stained with
May-Gruenwald Giemsa solution for morphological examination of colonies.
| |
RESULTS |
Differentiation of 4-integrin+ cells
from FLK1+ mesodermal cells induced in the ES cell
culture.
ES cells that were maintained in a gelatin-coated dish in the presence
of LIF were transferred into a type IV collagen-coated dish and
cultured in the absence of LIF to induce differentiation of mesodermal
cells. FACS analysis of the cells induced for 4 days showed that nearly
half of the cells downregulated the expression of E-cadherin and
upregulated FLK1 (Fig 1A). Previous studies demonstrated that the FLK1 is a marker of proximal lateral
mesoderm.24,26,38 The FLK1+
E-cadherin mesodermal cells were sorted by FACS as
shown in Fig 1A and recultured in the same condition but in a
gelatin-coated dish. The second culture induced the proliferation of
VE-cadherin+ endothelial cells and CD45+ or
TER119+ hematopoietic cells. FACS analysis of the cells
cultured for 3 days showed that all CD45+ or
TER119+ cells induced in the culture were
4-integrin+ (Fig 1B). Thirty percent of the cell
population that was devoid of CD45+/TER119+
committed blood cells also expressed 4-integrin on the surface (Fig
1B). Expression of VE-cadherin was found on 13% to 17% of the
CD45 TER119 cells. In the
VE-cadherin+ CD45
TER119 cell fraction, nearly half of the cells were
4-integrin+ (Fig 1B). VE-cadherin
4-integrin+ cells were also present in the
CD45 TER119 population. We
examined the kinetics of appearance of 4-integrin+ cells
in the CD45 TER119 cell
population in the culture induced from FLK1+
E-cadherin cells. Differentiation of
VE-cadherin+ cells was already evident 1 day after the
initiation of the culture, whereas only a few cells in the
CD45 TER119 cell population
expressed 4-integrin (Fig 2). On the
second day, both the VE-cadherin+
4-integrin+ and VE-cadherin
4-integrin+ cells appeared simultaneously in the
CD45 TER119 cell fraction and the
number of the 4-integrin+ cells continuously increased
in the culture (Fig 2). We did not observe any situation in which the
appearance of either the VE-cadherin+
4-integrin+ or VE-cadherin
4-integrin+ cells precedes that of the other.
View larger version (38K):
[in this window]
[in a new window]
| Fig 1.
Induction of differentiation of ES cells in vitro. (A)
CCE ES cells were allowed to differentiate in a type IV collagen-coated
dish for 4 days. Expression of FLK1 and E-cadherin on the CCE cells
before (left panel, day 0) and after (middle panel, day 4) the
induction was analyzed by flow cytometry. FLK1+
E-cadherin cells were sorted and reanalyzed (right
panel). (B) The ES cell-derived FLK1+
E-cadherin cells were cultured in a gelatin-coated dish
for 3 days. Expression of CD45, TER119, and 4-integrin on the total
cells (left panel) and VE-cadherin and 4-integrin on the
CD45 TER119 cells (right panel) was
analyzed. The numbers indicate the percentage of cells that appeared in
each quadrant. The result shown is a representative of more than five
independent experiments.
|
|
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
Kinetics of appearance of 4-integrin+
cells in the culture initiated from ES cell-derived FLK1+
cells. FLK1+ cells sorted from differentiating CCE cells
were cultured in a gelatin-coated dish for 1 to 3 days and analyzed
for expression of VE-cadherin and 4-integrin by flow
cytometry. CD45+ cells and TER119+ cells
were excluded from the analyses. The numbers indicate the percentage of
cells that appeared in each quadrant. The result shown is a
representative of three independent experiments.
|
|
Hematopoietic potential of VE-cadherin+
4-integrin+ and
VE-cadherin
4-integrin+ cell fractions.
We previously showed that the CD45
TER119 VE-cadherin+ cells that were
derived from ES cells in vitro included endothelial precursors with
hematopoietic potential.26 To investigate whether the
4-integrin+ cells induced from ES cells have a capacity
to generate hematopoietic lineage cells, the CD45
TER119 cells in the 3-day culture of
FLK1+ cells were fractionated into VE-cadherin+
4-integrin, VE-cadherin+
4-integrin+, VE-cadherin
4-integrin, and VE-cadherin
4-integrin+ populations by FACS
(Fig 3A). The sorted cells were put into a
culture that contains OP9 stromal cell layer and were maintained for 7 days in the presence of recombinant IL-3, Epo, G-CSF, and MGF. To
measure the frequency of the progenitors that give rise to clonal
expansion of hematopoietic cells, we covered the cultures with a
semisolid medium containing methylcellulose. One in 40 cells in the
VE-cadherin+ 4-integrin+ fraction
proliferated to form a hematopoietic cell colony (Fig 3B).
Approximately 70% of the colonies contained hemoglobinized erythrocytes (Fig 3C). In contrast to the VE-cadherin+
4-integrin+ fraction, the VE-cadherin+
4-integrin fraction contained much fewer
hematopoietic cell progenitors (1 in 1,000 cells; Fig 3B). One in 100 cells in the VE-cadherin 4-integrin+
fraction also gave rise to a hematopoietic cell colony (Fig 3B). The
hematopoietic cell colonies derived from the
VE-cadherin 4-integrin+ fraction
could not be morphologically distinguished from the colonies obtained
in the VE-cadherin+ 4-integrin+ cell culture
(data not shown). The VE-cadherin
4-integrin fraction did not give rise to any
hematopoietic cell colonies in the same culture condition (Fig 3B).
These results demonstrated that both VE-cadherin+ and
VE-cadherin fractions derived from FLK1+
mesodermal cells contained precursors of hematopoietic cell lineage and
that those progenitors exclusively express 4-integrin.
View larger version (36K):
[in this window]
[in a new window]
| Fig 3.
Hematopoietic potential of the CD45
TER119 4-integrin+ cells induced from
ES cell-derived FLK1+ cells. FLK1+ cells
sorted from differentiating CCE cells were cultured in a gelatin-coated
dish for 3 days. Cultured cells were fractionated by FACS into
VE-cadherin+ 4-integrin,
VE-cadherin+ 4-integrin+,
VE-cadherin 4-integrin, and
VE-cadherin 4-integrin+ cells.
CD45+ cells and TER119+ cells were excluded
from the sorting gates. Sorted cells were cultured on OP9 stromal cell
layer in the presence of IL-3, Epo, G-CSF, and MGF for 7 days. (A)
Reanalyses of the sorted cells. A representative result of more than
five independent experiments is shown. (B) Frequency of hematopoietic
colony-forming cells in the indicated fractions. The arrow indicates
that no colony-forming cell was detected. Error bars indicate standard
deviations for four independent determinations. (C) Morphology of a
hematopoietic cell colony formed in the culture of
VE-cadherin+ 4-integrin+ cells.
Hemoglobinized erythrocytes are observed. The bar represents 100 µm.
|
|
To characterize the hematopoietic cells derived from the
VE-cadherin+ 4-integrin+ and
VE-cadherin 4-integrin+ fractions,
the cells sorted from these fractions were cultured for 7 days in a
type IV collagen-coated dish in the presence of recombinant IL-3, Epo,
G-CSF, and MGF. The number of cells per culture increased 20-fold and
fourfold in the cultures initiated from the VE-cadherin+
4-integrin+ and VE-cadherin
4-integrin+ fractions, respectively. May-Gruenwald
Giemsa staining of the harvested cells showed that erythroblasts,
polymorphonuclear cells, and monocytes/macrophages were induced in the
cultures initiated from both fractions (Fig
4A through C). Essentially the same frequencies of TER119+
and Mac-1+ cells were detected in both cultures by FACS
analyses (Fig 4D and E). We analyzed expression of the hemoglobin genes
in the harvested cells by RT-PCR. The transcripts of the embryonic type Hbb-bh1 (hemoglobin Z), the embryonic/fetal type Hbb-y
(hemoglobin Y), and the fetal/adult type Hbb-b1 (hemoglobin major) genes39 were comparably detected in the
hematopoietic cells derived from both fractions (data not shown),
indicating that the 4-integrin+ fractions have a
potential to produce both the primitive and definitive erythroid
lineages.
View larger version (89K):
[in this window]
[in a new window]
| Fig 4.
Phenotype of hematopoietic cells differentiated from the
VE-cadherin+ 4-integrin+ and
VE-cadherin 4-integrin+ cell fractions.
The two fractions were sorted from differentiating CCE cells as shown
in Fig 3A and cultured in a type IV collagen-coated dish in the
presence of IL-3, Epo, G-CSF, and MGF for 7 days. (A through C)
May-Gruenwald Giemsa staining of cytospots prepared from the cultured
cells initiated from the VE-cadherin+
4-integrin+ fraction. Erythroblasts (A),
monocytes/macrophages (B), and polymorphonuclear cells (C) are
observed. The bars represent 25 µm. (D and E) Expression of Mac-1 and
TER119 on the cultured cells initiated from the
VE-cadherin+ 4-integrin+ (D) and
VE-cadherin 4-integrin+ (E) cell
fractions analyzed by flow cytometry. The numbers indicate the
percentage of cells that appeared in each quadrant. The result shown is
a representative of two independent experiments.
|
|
Clonal proliferation of endothelial cells derived from
VE-cadherin+ cell fractions.
We previously reported that the VE-cadherin+ cells derived
either from embryonic tissues or ES cell culture proliferated in response to stromal cells to form endothelial cell
colonies.26 The colonies consisted of FLK1+
VE-cadherin+ CD31(PECAM-1)+ cells that have the
capacity to take up acetylated low-density lipoprotein (Ac-LDL),
indicating that the VE-cadherin+ cell fraction contains
clonogenic precursors of endothelial cell lineage (Hirashima et al,
manuscript submitted). To compare the capacity of the
VE-cadherin+ 4-integrin and
VE-cadherin+ 4-integrin+ cells to form
endothelial cell colonies, these cell fractions that were induced from
FLK1+ cells in the ES cell culture were purified by FACS
and cultured in the presence of OP9 stromal cell layer for 2 weeks. The
formation of colonies that consist of FLK1+ endothelial
cells was shown by immunohistochemical staining of the whole cultures
with an anti-FLK1 MoAb (Fig 5A). Both the
VE-cadherin+ 4-integrin and
VE-cadherin+ 4-integrin+ cell fractions gave
rise to endothelial cell colonies. The frequency of colony-forming
cells was almost comparable in both fractions (1 in 35 cells; Fig 5B).
The VE-cadherin 4-integrin+ fraction
contained much fewer colony-forming cells (1 in 1,000 cells; Fig 5B).
We obtained a comparable result by staining with the anti-VE-cadherin
MoAb (data not shown).
View larger version (42K):
[in this window]
[in a new window]
| Fig 5.
Potential of CD45 TER119
VE-cadherin+ cells to form endothelial cell colonies.
VE-cadherin+ 4-integrin,
VE-cadherin+ 4-integrin+, and
VE-cadherin 4-integrin+ cells were
sorted from differentiating CCE cells as shown in Fig 3A and cultured
on OP9 stromal cell layer for 2 weeks. The cultures were stained in
situ with either anti-FLK1 or anti-VE-cadherin MoAbs. (A) Morphology
of a FLK1+ endothelial cell colony formed in the culture
of VE-cadherin+ 4-integrin+ cells.
The bar represents 400 µm. (B) Frequency of cells capable of
formation of endothelial cell colony in the indicated fractions. A
representative result of three independent experiments is shown.
|
|
Expression of transcription factors in the cells differentiated from
ES cell-derived FLK1+ mesoderm.
We next examined the expression of transcription factors that are known
to be involved in regulation of hematopoietic cell development in the
cell fractions differentiated from ES cell-derived FLK1+
cells by RT-PCR. Figure 6 shows that
expression level of the Gata2 gene that is essential for
hematopoietic cell development was higher in the
4-integrin+ fractions that have a hematopoietic
potential than in others. On the contrary, transcript of the
Lmo2 gene, whose product physically associates with GATA2, was
abundantly detected in all fractions tested. The transcripts of the
Tal1 gene, whose product also associates with Lmo2, was
detected in the VE-cadherin+
4-integrin, VE-cadherin+
4-integrin+, and VE-cadherin
4-integrin+ fractions that have a potential of either
hematopoietic or endothelial cell differentiation. Transcripts of the
Gata1 gene that is essential for erythroid lineage development
were abundantly detected in all fractions. Higher expression of the
Myb gene that is essential for the development of definitive
hematopoiesis in fetal liver was also detected in the
4-integrin+ fractions than in others.
View larger version (41K):
[in this window]
[in a new window]
| Fig 6.
Expression of mRNA of transcription factors in the
VE-cadherin+ 4-integrin,
VE-cadherin+ 4-integrin+,
VE-cadherin 4-integrin, and
VE-cadherin 4-integrin+ cells induced
in vitro from ES cell-derived FLK1+ cells. These
fractions were all CD45 TER119. Different
dilutions of cDNA prepared from sorted cells were subjected to PCR
amplification specific for Gata1, Gata2, Tal1,
Lmo2, Myb, and Gapd transcripts. PCR products
were separated on 1% agarose gel stained with ethidium bromide. The
result shown is a representative of two independent experiments.
|
|
Reanalyses of sorted fractions did not detect any CD45/TER119
fluorescence (data not shown). However, because the expression level of
TER119 on primitive erythrocytes is low as compared with that on
definitive erythrocytes,40 some mature primitive
erythrocytes may contaminate into sorted fractions and might result in
the overall detection of Gata1 and Lmo2 transcripts. To
test this possibility, cytospots were prepared from the sorted cells
and presence of mature primitive erythrocytes was examined by staining with an antiembryonic hemoglobin antibody. Substantial number of mature
primitive erythrocytes were detected in all the fractions examined
(10% in VE-cadherin+ 4-integrin, 2%
in VE-cadherin+ 4-integrin+, 10% in
VE-cadherin 4-integrin+, and 33% in
VE-cadherin 4-integrin
fraction). Thus, it is possible that the overall expression of the
Gata1 and Lmo2 genes is attributed to the contaminated
primitive erythrocytes. Nevertheless, the highest frequency observed in the VE-cadherin 4-integrin
fraction also indicates that these mature primitive erythrocytes contributed to neither the expression of the Gata2,
Tal1, and Myb genes detected by RT-PCR nor the
formation of hematopoietic cell colonies on stromal cells.
Hematopoietic potential of CD45
VE-cadherin+
4-integrin+ cells in the yolk sac and
embryonic body of 9.5 dpc.
The next question we addressed was whether the VE-cadherin+
4-integrin+ and VE-cadherin
4-integrin+ cells that were induced in the ES cell
culture represent intermediate stages of the developmental pathway of
hematopoietic lineage in the mouse embryo. Cells were dissociated from
the yolk sac and lower trunk of embryo proper of 9.5 dpc mouse embryos
(see Materials and Methods) and analyzed by flow cytometry. The
VE-cadherin+ 4-integrin,
VE-cadherin+ 4-integrin+, and
VE-cadherin 4-integrin+ cells were
all detected in the CD45 TER119
cell fraction dissociated from both the yolk sac and embryo proper (Fig 7A). To examine the hematopoietic
potential of these cell populations, the cells were sorted by FACS as
shown in Fig 7B and cultured in a type I collagen gel in the presence
of recombinant IL-3, Epo, G-CSF, and MGF. The VE-cadherin+
4-integrin+ fraction but not the
VE-cadherin+ 4-integrin fraction
sorted from both the yolk sac and embryo proper gave rise to
hematopoietic cell colonies (Fig 8A and B).
This result indicates that most of the precursors of hematopoietic cell
lineage in the CD45 VE-cadherin+ cells
in the embryos express 4-integrin on the surface. Although the
VE-cadherin 4-integrin+ cells were
able to be dissociated from both the yolk sac and embryo proper, only
the cells derived from the yolk sac gave rise to hematopoietic cell
colonies (Fig 8B). May-Gruenwald Giemsa staining showed no
morphological difference on the hematopoietic cell colonies derived
from the VE-cadherin+ 4-integrin+ and
VE-cadherin 4-integrin+ fractions in
the yolk sac (data not shown).
View larger version (34K):
[in this window]
[in a new window]
| Fig 7.
Expression of VE-cadherin and 4-integrin on the cells
dissociated from 9.5 dpc mouse embryos. The yolk sacs and embryonic
bodies (lower trunk) were separated from 9.5 dpc mouse embryos and a
single-cell suspension was prepared by using dispase and cell
dissociation buffer. Cells were analyzed for expression of VE-cadherin
and 4-integrin by flow cytometry (A). CD45+ cells and
TER119+ cells were excluded from the analyses. The
VE-cadherin+ 4-integrin,
VE-cadherin+ 4-integrin+, and
VE-cadherin 4-integrin+ cells were
sorted and reanalyzed (B). The numbers indicate the percentage of cells
that appeared in each quadrant. The result shown is a representative of
three independent experiments.
|
|
View larger version (44K):
[in this window]
[in a new window]
| Fig 8.
Hematopoietic potential of VE-cadherin+
4-integrin+ cells sorted from yolk sac and embryonic
body proper of 9.5 dpc mouse embryo. The VE-cadherin+
4-integrin, VE-cadherin+
4-integrin+, and VE-cadherin
4-integrin+ cells were sorted from yolk sac and embryo
proper of 9.5 dpc mouse embryos as shown in Fig 7. CD45+
and TER119+ cells were excluded from the sorting gates.
Sorted cells were cultured for 7 days in a matrix gel containing type I
collagen in the presence of IL-3, Epo, G-CSF, and MGF. Morphology of
hematopoietic cell colonies were examined by May-Gruenwald Giemsa
staining (A). Colonies consisted of granulocytes (g) and erythrocytes
(e) are observed. The bar represents 100 µm. (B) Frequency of
hematopoietic colony-forming cells in the indicated cell fractions
derived from yolk sac and embryonic body proper. Error bars indicate
standard deviations for three independent determinations.
|
|
| |
DISCUSSION |
By using an in vitro differentiation system of ES cells,
VE-cadherin+ endothelial cells that have a hematopoietic
potential can be induced from FLK1+ proximal lateral
mesodermal cells.26 Among the surface markers that have
been used to label endothelial cells (including FLK1, CD31, and CD34),
only VE-cadherin is exclusive for endothelial cells.35,41,42 VE-cadherin+ cells generated in
vitro from ES cells give rise to VE-cadherin+
FLK1+ CD31+ sheet-like structures on stromal
cells, and these sheets incorporate Ac-LDL (Hirashima et al, manuscript
submitted). Thus, the VE-cadherin+ cells
differentiated from ES cells are most likely to represent the
endothelial cell lineage. In this report, we showed that a subset of
this VE-cadherin+ endothelial cell population expresses
4-integrin on the surface (Figs 1 and 2). The integrins that have
been detected on resting endothelial cells in vivo include 61,
51, 31, 21, v3, and 64, whereas the
distribution of 4-integrin is restricted to the capillary of
placenta.30 However, 4-integrin has been shown to be
present on the endothelium of newly forming vessels during angiogenesis
in the lung of mouse embryos.43 We also detected
VE-cadherin+ 4-integrin+ cells in the yolk
sac and embryo proper of 9.5 dpc mouse embryos (Fig 7). All the
VE-cadherin+ cells isolated from 9.5 dpc embryos possess
the capacity to incorporate Ac-LDL,41 indicating that they
are endothelial cells. Thus, it is possible that 4-integrin is
expressed transiently by a subset of developing endothelium in the
mouse embryos. Our analyses demonstrated that the endothelial cells
that have hematopoietic potential were found exclusively in the
4-integrin+ subset (Figs 3 and 8). However, because the
VE-cadherin+ 4-integrin+ fraction also
contains cells that give rise to endothelial cell colonies (Fig 5),
expression of 4-integrin on an endothelial cell does not necessarily
indicate that the cell is fully committed to hematopoietic cell
lineage. A part of the 4-integrin+ endothelial cells may
retain a capacity to form an endothelial cell sheet when cultured in an
appropriate condition in vitro. However, it is still unknown whether a
single VE-cadherin+ 4-integrin+ progenitor
produces both hematopoietic cells and mature endothelium in vivo. In
vivo labeling of single progenitors in the endothelial lining by an
appropriate marker (ie, retroviral vector or green fluorescent protein)
should be required to solve this question. Although 4-integrin can
be used as a marker of the hematopoitic endothelium, lack of this
molecule did not affect early development of hematopoietic cell
lineage,44,45 which indicates that expression of
4-integrin is not essential for cell specification to hematopoietic lineage.
It is generally thought that different sets of transcription factors
should be found in cell populations that have the same origin but
different potential.46 Gene disruption studies have shown
that several transcription factors play essential roles on the
development of hematopoietic cells in mouse embryos. Loss of either
Gata2, Lmo2, or Tal1 profoundly affects
development of all hematopoietic cell lineages.47-50 It has
been proposed that the products of these three genes physically
interact to establish a transcriptional transactivating
complex.51 Although Gata1 gene product also can be
integrated into the transactivating complex, loss of Gata1
specifically affected erythroid lineage.52,53 Our RT-PCR
analyses on the expression of these genes in the
CD45 TER119
VE-cadherin+ fractions indicated that expression level of
the Gata2 gene was higher in the VE-cadherin+
4-integrin+ fraction that has hematopoietic potential
than in the nonhematopoietic VE-cadherin+
4-integrin fraction (Fig 6). Although expression
levels of the Lmo2 and Tal1 genes were the same in the
two fractions, this result might be consistent with the hypothesis that
the molecular complex formed by GATA2, Lmo2, and TAL1 is involved in
early hematopoiesis.51 Furthermore, the expression of the
Tal1 gene in the VE-cadherin+
4-integrin fraction that has a capacity to form
endothelial cell colonies agrees with the previous report showing that
TAL1 is present in endothelial cells in the developing mouse
embryo54 (Figs 5 and 6). The Myb gene is essential
for definitive erythropoiesis in the fetal liver but not for primitive
erythropoiesis in the yolk sac.55 Expression of
4-integrin in the fetal liver was abrogated in the
Myb-deficient mice, suggesting an important role for
Myb in the regulation of the Itga4 (4-integrin) gene
in, at least, the hematopoietic lineage.56 We detected
higher expression of the Myb gene in the
VE-cadherin+ 4-integrin+ fraction than in
the VE-cadherin+ 4-integrin fraction
(Fig 6), which is again consistent with the previous observations.
Consequently, our results suggest that differential expression of some
of the transcription factors, such as Gata2 and
Myb, correlates with the potential of VE-cadherin+
4-integrin+ cells to give rise to hematopoietic cell
differentiation. However, it should be noted that our analyses on the
expression of transcription factors were not based on single cells.
Investigations at the single-cell level on a fate of a cell that
expresses a certain set of transcription factors should be required to
clarify the relationship between expression of transcription factors
and cell specification.
In addition to the CD45 TER119
VE-cadherin+ 4-integrin+ fraction, we
detected another fraction, CD45
TER119 VE-cadherin
4-integrin+, that also showed a potential to give rise
to hematopoietic cells (Fig 3). Because we excluded CD45+
cells from the analyses, it is unlikely that the hematopoietic potential of the VE-cadherin
4-integrin+ fraction is attributed to hematopoietic stem
cells that are known to be CD45+.57 We detected
no significant difference between the hematopoietic cells derived from
the VE-cadherin+ 4-integrin+ and
VE-cadherin 4-integrin+ fractions in
cell morphology (Fig 4), in surface marker expression (Fig 4), and in
transcription of the -hemoglobin genes (data not shown). We also
failed to detect any difference between two cell fractions in the
expression levels of transcription factors that are involved in
hematopoietic cell development (Fig 6). It is still unknown whether
VE-cadherin 4-integrin+ cells are
immediate progeny of VE-cadherin+
4-integrin+ cells. Because hematopoietic stem cells
express 4-integrin and mature hematopoietic cells do not express
VE-cadherin on the surface, expression of VE-cadherin should be shut
off and expression of 4-integrin should be turned on upon
differentiation of the hematopoietic endothelial cells to hematopoietic
cells. However, our results demonstrate that the
VE-cadherin+ 4-integrin+ and
VE-cadherin 4-integrin+ fractions
appeared simultaneously in the culture of ES-derived FLK1+
cells (Fig 2). Thus, although some of the
VE-cadherin 4-integrin+ cells that
have hematopoietic potential might differentiate from the
VE-cadherin+ 4-integrin+ cells, it is
equally possible that the VE-cadherin
4-integrin+ cells are also derived directly from
FLK1+ VE-cadherin mesodermal cells.
Both CD45 TER119
VE-cadherin+ 4-integrin+ and
CD45 TER119
VE-cadherin 4-integrin+ fractions
directly sorted from 9.5 dpc yolk sac had hematopoietic potential,
whereas only the CD45 TER119
VE-cadherin+ 4-integrin+ fraction in the
embryo proper at the same embryonic stage had the same potential (Fig
8). This indicates that the VE-cadherin
4-integrin+ fraction is not homogeneous and is likely to
contain other lineages, such as smooth muscle cells.43 It
is possible that our procedure simply failed to dissociate the
VE-cadherin 4-integrin+ cells that
have hematopoietic potential from embryonic body proper. Alternatively,
there may be two alternative pathways in early development of
hematopoietic cell lineage: one path goes through the
VE-cadherin+ stage, whereas the other does
not. Both pathways might be taken by the progenitors
present in the yolk sac, whereas the progenitors in the embryo proper
follow only the VE-cadherin+ pathway. This hypothesis may
be consistent with the previous model that the primitive hematopoiesis
is generated in the yolk sac from hemangioblasts, whereas the
definitive hematopoiesis is generated from endothelial cells in the
embryo proper.58 Moreover, it should be remembered that
hematopoietic endothelial cells are generated not only in the embryo
proper, but also in the yolk sac.41 We previously reported
that VE-cadherin+ endothelial cells derived from embryonic
tissues have a potential to differentiate to lymphoid cell
lineages.41 Thus, we currently hypothesize that the
VE-cadherin+ 4-integrin+ progenitors
represent precursors of definitive hematopoietic lineage that give rise
to multiple types of blood cells.
In conclusion, the 4-Integrin+ subset of endothelial
cells encompasses cells that are in the earliest step, so far
identified, of diversification of the vascular endothelium toward the
hematopoietic cell lineage. 4-integrin may provide a valuable tool
that should allow the elucidation of the cellular and molecular basis
underlying the developmental process of the hematopoietic cell lineage.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs M. Evans for the CCE cell line and A. Nagafuchi for the ECCD2 MoAb. We thank to Dr S. Fraser for critical
reading of this manuscript.
| |
FOOTNOTES |
Submitted July 6, 1998; accepted October 6, 1998.
Supported by grant from the Ministry of Education, Science and Culture
of Japan (Grant No. 10770139).
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 Minetaro Ogawa, PhD, Department of
Molecular Genetics, Faculty of Medicine, Kyoto University,
Shogoin-Kawaharacho 53, Sakyo-ku, Kyoto 606-8507, Japan; e-mail:
mogawa{at}virus.kyoto-u.ac.jp.
| |
REFERENCES |
1.
Moore MA, Metcalf D:
Ontogeny of the haemopoietic system: Yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo.
Br J Haematol
18:279, 1970[Medline]
[Order article via Infotrieve]
2.
Houssaint E:
Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line.
Cell Differ
10:243, 1981[Medline]
[Order article via Infotrieve]
3.
Ogawa M, Nishikawa S, Ikuta K, Yamamura F, Naito M, Takahashi K, Nishikawa S:
B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac.
EMBO J
7:1337, 1988[Medline]
[Order article via Infotrieve]
4.
Delassus S, Cumano A:
Circulation of hematopoietic progenitors in the mouse embryo.
Immunity
4:97, 1996[Medline]
[Order article via Infotrieve]
5.
Godin I, Dieterlen Lievre F, Cumano A:
B-lymphoid potential in pre-liver mouse embryo.
Semin Immunol
7:131, 1995[Medline]
[Order article via Infotrieve]
6.
Godin IE, Garcia Porrero JA, Coutinho A, Dieterlen Lievre F, Marcos MA:
Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors.
Nature
364:67, 1993[Medline]
[Order article via Infotrieve]
7.
Godin I, Dieterlen Lievre F, Cumano A:
Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus.
Proc Natl Acad Sci USA
92:773, 1995[Abstract/Free Full Text]
8.
Medvinsky AL, Samoylina NL, Muller AM, Dzierzak EA:
An early pre-liver intraembryonic source of CFU-S in the developing mouse.
Nature
364:64, 1993[Medline]
[Order article via Infotrieve]
9.
Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E:
Development of hematopoietic stem cell activity in the mouse embryo.
Immunity
1:291, 1994[Medline]
[Order article via Infotrieve]
10.
Smith RA, Glomski CA:
"Hemogenic endothelium" of the embryonic aorta: Does it exist?
Dev Comp Immunol
6:359, 1982[Medline]
[Order article via Infotrieve]
11.
Garcia Porrero JA, Godin IE, Dieterlen Lievre F:
Potential intraembryonic hemogenic sites at pre-liver stages in the mouse.
Anat Embryol Berl
192:425, 1995[Medline]
[Order article via Infotrieve]
12.
Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R:
The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.
J Embryol Exp Morphol
87:27, 1985[Medline]
[Order article via Infotrieve]
13.
Schmitt RM, Bruyns E, Snodgrass HR:
Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor gene expression.
Genes Dev
5:728, 1991[Abstract/Free Full Text]
14.
Burkert U, von Ruden T, Wagner EF:
Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.
New Biol
3:698, 1991[Medline]
[Order article via Infotrieve]
15.
Wiles MV, Keller G:
Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture.
Development
111:259, 1991[Abstract]
16.
Gutierrez Ramos JC, Palacios R:
In vitro differentiation of embryonic stem cells into lymphocyte precursors able to generate T and B lymphocytes in vivo.
Proc Natl Acad Sci USA
89:9171, 1992[Abstract/Free Full Text]
17.
Keller G, Kennedy M, Papayannopoulou T, Wiles MV:
Hematopoietic commitment during embryonic stem cell differentiation in culture.
Mol Cell Biol
13:473, 1993[Abstract/Free Full Text]
18.
Nakano T, Kodama H, Honjo T:
Generation of lymphohematopoietic cells from embryonic stem cells in culture.
Science
265:1098, 1994[Abstract/Free Full Text]
19.
Potocnik AJ, Nielsen PJ, Eichmann K:
In vitro generation of lymphoid precursors from embryonic stem cells.
EMBO J
13:5274, 1994[Medline]
[Order article via Infotrieve]
20.
Palacios R, Golunski E, Samaridis J:
In vitro generation of hematopoietic stem cells from an embryonic stem cell line.
Proc Natl Acad Sci USA
92:7530, 1995[Abstract/Free Full Text]
21.
Kennedy M, Firpo M, Choi K, Wall C, Robertson S, Kabrun N, Keller G:
A common precursor for primitive erythropoiesis and definitive haematopoiesis.
Nature
386:488, 1997[Medline]
[Order article via Infotrieve]
22.
Yamane T, Kunisada T, Yamazaki H, Era T, Nakano T, Hayashi SI:
Development of osteoclasts from embryonic stem cells through a pathway that is c-fms but not c-kit dependent.
Blood
90:3516, 1997[Abstract/Free Full Text]
23.
Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G:
A common precursor for hematopoietic and endothelial cells.
Development
125:725, 1998[Abstract]
24.
Kataoka H, Takakura N, Nishikawa S, Tsuchida K, Kodama H, Kunisada T, Risau W, Kita T, Nishikawa SI:
Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells.
Dev Growth Differ
39:729, 1997[Medline]
[Order article via Infotrieve]
25.
Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, Keller G:
Flk-1 expression defines a population of early embryonic hematopoietic precursors.
Development
124:2039, 1997[Abstract]
26.
Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H:
Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemapoietic lineages.
Development
125:1747, 1998[Abstract]
27.
Williams DA, Rios M, Stephens C, Patel VP:
Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions.
Nature
352:438, 1991[Medline]
[Order article via Infotrieve]
28.
Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R:
Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins.
Nature
380:171, 1996[Medline]
[Order article via Infotrieve]
29.
Craddock CF, Nakamoto B, Andrews RG, Priestley GV, Papayannopoulou T:
Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice.
Blood
90:4779, 1997[Abstract/Free Full Text]
30.
Mechtersheimer G, Barth T, Hartschuh W, Lehnert T, Moller P:
In situ expression of beta 1, beta 3 and beta 4 integrin subunits in non-neoplastic endothelium and vascular tumours.
Virchows Arch
425:375, 1994[Medline]
[Order article via Infotrieve]
31.
Robertson E, Bradley A, Kuehn M, Evans M:
Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.
Nature
323:445, 1986[Medline]
[Order article via Infotrieve]
32.
Kodama H, Nose M, Niida S, Nishikawa S, Nishikawa S:
Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells.
Exp Hematol
22:979, 1994[Medline]
[Order article via Infotrieve]
33.
Shirayoshi Y, Nose A, Iwasaki K, Takeichi M:
N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin.
Cell Struct Funct
11:245, 1986[Medline]
[Order article via Infotrieve]
34.
Ikuta K, Kina T, MacNeil I, Uchida N, Peault B, Chien YH, Weissman IL:
A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells.
Cell
62:863, 1990[Medline]
[Order article via Infotrieve]
35.
Matsuyoshi N, Toda K, Horiguchi Y, Tanaka T, Nakagawa S, Takeichi M, Imamura S:
In vivo evidence of the critical role of cadherin-5 in murine vascular integrity.
Proc Assoc Am Phys
109:362, 1997[Medline]
[Order article via Infotrieve]
36.
Sudo T, Nishikawa S, Ogawa M, Kataoka H, Ohno N, Izawa A, Hayashi S, Nishikawa S:
Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M.
Oncogene
11:2469, 1995[Medline]
[Order article via Infotrieve]
37.
Weiss MJ, Keller G, Orkin SH:
Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells.
Genes Dev
8:1184, 1994[Abstract/Free Full Text]
38.
Yamaguchi TP, Dumont DJ, Conlon RA, Breitman ML, Rossant J:
flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors.
Development
118:489, 1993[Abstract]
39.
Whitelaw E, Tsai SF, Hogben P, Orkin SH:
Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse.
Mol Cell Biol
10:6596, 1990[Abstract/Free Full Text]
40.
Ogawa M, Nishikawa S, Yoshinaga K, Hayashi S, Kunisada T, Nakao J, Kina T, Sudo T, Kodama H, Nishikawa S:
Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo.
Development
117:1089, 1993[Abstract]
41.
Nishikawa SI, Nishikawa S, Kawamoto H, Yoshida H, Kizumoto M, Kataoka H, Katsura Y:
In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.
Immunity
8:761, 1998[Medline]
[Order article via Infotrieve]
42.
Breier G, Breviario F, Caveda L, Berthier R, Schnurch H, Gotsch U, Vestweber D, Risau W, Dejana E:
Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.
Blood
87:630, 1996[Abstract/Free Full Text]
43.
Sheppard AM, Onken MD, Rosen GD, Noakes PG, Dean DC:
Expanding roles for alpha 4 integrin and its ligands in development.
Cell Adhes Commun
2:27, 1994[Medline]
[Order article via Infotrieve]
44.
Yang JT, Rayburn H, Hynes RO:
Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development.
Development
121:549, 1995[Abstract]
45.
Arroyo AG, Yang JT, Rayburn H, Hynes RO:
Differential requirements for alpha 4 integrins during fetal and adult hematopoiesis.
Cell
85:997, 1996[Medline]
[Order article via Infotrieve]
46.
Cross MA, Enver T:
The lineage commitment of haemopoietic progenitor cells.
Curr Opin Genet Dev
7:609, 1997[Medline]
[Order article via Infotrieve]
47.
Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, Alt FW, Orkin SH:
An early haematopoietic defect in mice lacking the transcription factor GATA-2.
Nature
371:221, 1994[Medline]
[Order article via Infotrieve]
48.
Warren AJ, Colledge WH, Carlton MB, Evans MJ, Smith AJ, Rabbitts TH:
The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development.
Cell
78:45, 1994[Medline]
[Order article via Infotrieve]
49.
Shivdasani RA, Mayer EL, Orkin SH:
Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL.
Nature
373:432, 1995[Medline]
[Order article via Infotrieve]
50.
Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH:
The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages.
Cell
86:47, 1996[Medline]
[Order article via Infotrieve]
51.
Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H, Forster A, Rabbitts TH:
The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins.
EMBO J
16:3145, 1997[Medline]
[Order article via Infotrieve]
52.
Pevny L, Simon MC, Robertson E, Klein WH, Tsai SF, D'Agati V, Orkin SH, Costantini F:
Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1.
Nature
349:257, 1991[Medline]
[Order article via Infotrieve]
53.
Simon MC, Pevny L, Wiles MV, Keller G, Costantini F, Orkin SH:
Rescue of erythroid development in gene targeted GATA-1-mouse embryonic stem cells.
Nat Genet
1:92, 1992[Medline]
[Order article via Infotrieve]
54.
Kallianpur AR, Jordan JE, Brandt SJ:
The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis.
Blood
83:1200, 1994[Abstract/Free Full Text]
55.
Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott WJ Jr, Potter SS:
A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.
Cell
65:677, 1991[Medline]
[Order article via Infotrieve]
56.
Postigo AA, Sheppard AM, Mucenski ML, Dean DC:
c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB.
EMBO J
16:3924, 1997[Medline]
[Order article via Infotrieve]
57.
Morrison SJ, Uchida N, Weissman IL:
The biology of hematopoietic stem cells.
Annu Rev Cell Dev Biol
11:35, 1995[Medline]
[Order article via Infotrieve]
58.
Dieterlen Lievre F, Godin I, Pardanaud L:
Where do hematopoietic stem cells come from?
Int Arch Allergy Immunol
112:3, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
X. Huang, H. Sakamoto, and M. Ogawa
Thrombopoietin controls proliferation of embryonic multipotent hematopoietic progenitors
Genes Cells,
July 1, 2009;
14(7):
851 - 860.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-J. Yoon, B.-K. Koo, R. Song, H.-W. Jeong, J. Shin, Y.-W. Kim, Y.-Y. Kong, and P.-G. Suh
Mind bomb-1 Is Essential for Intraembryonic Hematopoiesis in the Aortic Endothelium and the Subaortic Patches
Mol. Cell. Biol.,
August 1, 2008;
28(15):
4794 - 4804.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-S. Huang, J.-Y. Hsieh, Y.-H. Wu, C.-H. Jen, Y.-H. Tsuang, S.-H. Chiou, J. Partanen, H. Anderson, T. Jaatinen, Y.-H. Yu, et al.
Functional Network Reconstruction Reveals Somatic Stemness Genetic Maps and Dedifferentiation-Like Transcriptome Reprogramming Induced by GATA2
Stem Cells,
May 1, 2008;
26(5):
1186 - 1201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Era, N. Izumi, M. Hayashi, S. Tada, S. Nishikawa, and S.-I. Nishikawa
Multiple Mesoderm Subsets Give Rise to Endothelial Cells, Whereas Hematopoietic Cells Are Differentiated Only from a Restricted Subset in Embryonic Stem Cell Differentiation Culture
Stem Cells,
February 1, 2008;
26(2):
401 - 411.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Senju, H. Suemori, H. Zembutsu, Y. Uemura, S. Hirata, D. Fukuma, H. Matsuyoshi, M. Shimomura, M. Haruta, S. Fukushima, et al.
Genetically Manipulated Human Embryonic Stem Cell-Derived Dendritic Cells with Immune Regulatory Function
Stem Cells,
November 1, 2007;
25(11):
2720 - 2729.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Baba, T. Heike, M. Yoshimoto, K. Umeda, H. Doi, T. Iwasa, X. Lin, S. Matsuoka, M. Komeda, and T. Nakahata
Flk1+ cardiac stem/progenitor cells derived from embryonic stem cells improve cardiac function in a dilated cardiomyopathy mouse model
Cardiovasc Res,
October 1, 2007;
76(1):
119 - 131.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Baba, T. Heike, K. Umeda, T. Iwasa, S. Kaichi, Y. Hiraumi, H. Doi, M. Yoshimoto, M. Kanatsu-Shinohara, T. Shinohara, et al.
Generation of Cardiac and Endothelial Cells from Neonatal Mouse Testis-Derived Multipotent Germline Stem Cells
Stem Cells,
June 1, 2007;
25(6):
1375 - 1383.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. K. Bone and M. J. Welham
Phosphoinositide 3-kinase signalling regulates early development and developmental haemopoiesis
J. Cell Sci.,
May 15, 2007;
120(10):
1752 - 1762.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Shinoda, K. Umeda, T. Heike, M. Arai, A. Niwa, F. Ma, H. Suemori, H. Y. Luo, D. H. K. Chui, R. Torii, et al.
{alpha}4-Integrin+ endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells
Blood,
March 15, 2007;
109(6):
2406 - 2415.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Dai, H. Sakamoto, Y. Shimoda, T. Fujimoto, S.-I. Nishikawa, and M. Ogawa
Over-expression of c-Myb increases the frequency of hemogenic precursors in the endothelial cell population.
Genes Cells,
August 1, 2006;
11(8):
859 - 870.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Sakamoto, G. Dai, K. Tsujino, K. Hashimoto, X. Huang, T. Fujimoto, M. Mucenski, J. Frampton, and M. Ogawa
Proper levels of c-Myb are discretely defined at distinct steps of hematopoietic cell development
Blood,
August 1, 2006;
108(3):
896 - 903.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Taoudi, A. M. Morrison, H. Inoue, R. Gribi, J. Ure, and A. Medvinsky
Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver
Development,
September 15, 2005;
132(18):
4179 - 4191.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hirai, I. M Samokhvalov, T. Fujimoto, S. Nishikawa, J. Imanishi, and S.-I. Nishikawa
Involvement of Runx1 in the down-regulation of fetal liver kinase-1 expression during transition of endothelial cells to hematopoietic cells
Blood,
September 15, 2005;
106(6):
1948 - 1955.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Li, S. A. Johnson, W. C. Shelley, M. Ferkowicz, P. Morrison, Y. Li, and M. C. Yoder
Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro
Blood,
December 15, 2003;
102(13):
4345 - 4353.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Nobuhisa, M. Takizawa, S. Takaki, H. Inoue, K. Okita, M. Ueno, K. Takatsu, and T. Taga
Regulation of Hematopoietic Development in the Aorta-Gonad-Mesonephros Region Mediated by Lnk Adaptor Protein
Mol. Cell. Biol.,
December 1, 2003;
23(23):
8486 - 8494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-T. Fujimoto, S. Kohata, H. Suzuki, H. Miyazaki, and K. Fujimura
Production of functional platelets by differentiated embryonic stem (ES) cells in vitro
Blood,
December 1, 2003;
102(12):
4044 - 4051.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. J. Favre, M. Mancuso, K. Maas, J. W. McLean, P. Baluk, and D. M. McDonald
Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung
Am J Physiol Heart Circ Physiol,
November 1, 2003;
285(5):
H1917 - H1938.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Nakayama, D. Duryea, R. Manoukian, G. Chow, and C.-y. E. Han
Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells
J. Cell Sci.,
May 15, 2003;
116(10):
2015 - 2028.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Matsumura, M. Hirashima, M. Ogawa, H. Kubo, H. Hisatsune, N. Kondo, S. Nishikawa, T. Chiba, and S.-I. Nishikawa
Modulation of VEGFR-2-mediated endothelial-cell activity by VEGF-C/VEGFR-3
Blood,
February 15, 2003;
101(4):
1367 - 1374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hirai, M. Ogawa, N. Suzuki, M. Yamamoto, G. Breier, O. Mazda, J. Imanishi, and S.-I. Nishikawa
Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)-1 promoter/enhancer during mouse embryogenesis
Blood,
February 1, 2003;
101(3):
886 - 893.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. K. A. Mikkola, Y. Fujiwara, T. M. Schlaeger, D. Traver, and S. H. Orkin
Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo
Blood,
January 15, 2003;
101(2):
508 - 516.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Salven, S. Mustjoki, R. Alitalo, K. Alitalo, and S. Rafii
VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells
Blood,
January 1, 2003;
101(1):
168 - 172.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. E. Burger, S. Coetzee, W. L. McKeehan, M. Kan, P. Cook, Y. Fan, T. Suda, R. P. Hebbel, N. Novitzky, W. A. Muller, et al.
Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells
Blood,
November 15, 2002;
100(10):
3527 - 3535.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Papayannopoulou, G. V. Priestley, B. Nakamoto, V. Zafiropoulos, and L. M. Scott
Molecular pathways in bone marrow homing: dominant role of {alpha}4{beta}1 over {beta}2-integrins and selectins
Blood,
October 15, 2001;
98(8):
2403 - 2411.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. S. Kaufman, E. T. Hanson, R. L. Lewis, R. Auerbach, and J. A. Thomson
Hematopoietic colony-forming cells derived from human embryonic stem cells
PNAS,
September 4, 2001;
(2001)
191362598.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M Mahlapuu, M Ormestad, S Enerback, and P Carlsson
The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm
Development,
January 1, 2001;
128(2):
155 - 166.
[Abstract]
[PDF]
|
|
|
|
|
|
|
N. Nakayama, J. Lee, and L. Chiu
Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro
Blood,
April 1, 2000;
95(7):
2275 - 2283.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. S. Kaufman, E. T. Hanson, R. L. Lewis, R. Auerbach, and J. A. Thomson
Hematopoietic colony-forming cells derived from human embryonic stem cells
PNAS,
September 11, 2001;
98(19):
10716 - 10721.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction