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HEMATOPOIESIS
From the Institute of Molecular and Cellular
Biosciences, The University of Tokyo, and the Core Research for
Evolutional Science and Technology of Japan Science and Technology,
Japan.
Definitive hematopoietic stem cells arise in the
aorta-gonad-mesonephros (AGM) region from hemangioblasts, common
precursors for hematopoietic and endothelial cells. Previously, we
showed that multipotential hematopoietic progenitors and endothelial cells were massively produced in primary culture of the AGM region in
the presence of oncostatin M. Here we describe a role for
macrophage-colony-stimulating factor (M-CSF) in the development of
hematopoietic and endothelial cells in AGM culture. The number of
hematopoietic progenitors including multipotential cells was
significantly increased in the AGM culture of op/op
embryos. The addition of M-CSF to op/op AGM culture
decreased colony-forming unit (CFU)-GEMM, granulocyte macrophage-CFU, and erythroid-CFU, but it increased CFU-M. On the
other hand, the number of cells expressing endothelial markers, vascular endothelial-cadherin, intercellular adhesion molecule 2, and
Flk-1 was reduced in op/op AGM culture. The M-CSF receptor was expressed in PCLP1+CD45 Hematopoiesis is initiated sequentially in
different tissues during development. Primitive hematopoiesis occurs in
the yolk sac at mouse embryonic day 7.5 (E7.5), and definitive
hematopoiesis starts in the embryonic para-aortic splanchnopleural
region (pSp) at E8.5.1 Primitive hematopoiesis is
characterized by the production of fetal erythrocytes with the nucleus
and the lack of lymphocytes and myeloid cells except for macrophages.
Definitive hematopoiesis generates enucleated erythrocytes, various
kinds of myeloid and lymphoid cells, and long-term reconstituting
hematopoietic stem cells (LTR-HSCs). Although lymphocytes are found in
the E8.5 pSp region,2 LTR-HSCs first appear at E10.5 in
the aorta-gonad-mesonephros (AGM) region.3,4 Thereafter
hematopoiesis takes place in the fetal liver until bone marrow is formed.
Extensive studies on the early development of chick embryos indicate
that hematopoietic cells and endothelial cells arise from the common
precursor termed hemangioblast.5-7 A series of the
grafting experiments using chicks and quails demonstrated that the
splanchnopleural mesoderm generates hematopoietic cells and
endothelium.8 Cell marking studies using chick embryos indicated that intra-aortic hematopoietic clusters are derived from
endothelium and migrate to the para-aortic region.9,10 CD34+ cells in the dorsal aorta and vitelline artery of
human embryos were capable of generating hematopoietic cells in
vitro,11 and LTR-HSC activity was detected in the dorsal
aorta at E11.12 Disruption of the Cbfa2 (also
known as AML-1, Runx-1) gene in mice resulted in the lack of definitive
hematopoiesis in fetal liver13 and in the loss of
hematopoietic clusters in the dorsal aorta.14 These
studies indicate that endothelial cells in the dorsal aorta generate
hematopoietic cells.
Hematogenic activity is regulated by cytokines such as vascular
endothelial growth factor (VEGF). In the splanchnopleural mesoderm,
cells expressing VEGF receptor 2 (VEGF-R2) were shown to form
hematopoietic and endothelial colonies.15 Similarly, mouse
cells expressing Flk-1 (mouse counterpart of VEGF-R2) and vascular
endothelial cadherin (VE-cadherin) in the yolk sac and the pSp region
at E9.5 mouse embryos gave rise to lymphohematopoietic cells in
vitro.16 Flk-1 knockout mice exhibited severe defects in
hematopoiesis and vasculogenesis in the yolk sac,17,18 and a similar phenotype was found by gene disruption of the SCL/Tal-1 transcription factor.19-21 In addition, recent studies on
the in vitro differentiation of embryonic stem cells also indicate that hematopoietic cells and endothelial cells are derived from the same
origin in the presence of VEGF.22-24 Taken together, these studies establish that VEGF plays a key role in the development of
hematopoietic and endothelial cells.
To investigate the mechanisms underlying the development of
hematopoietic and endothelial cells in the AGM region, we previously established a primary culture system of the AGM region.25
In this culture, hematopoietic cells and endothelial cells are
massively produced in the presence of stem cell factor (SCF), basic
fibroblast growth factor (bFGF), and oncostatin M (OSM), an interleukin
(IL)-6 family cytokine. Numerous endothelial-like cell clusters are
formed in AGM culture, and hematopoietic cells are generated from
endothelial-like cell clusters, indicating that the endothelial-like
cell clusters contain hemangioblasts. Using a hemangioblastlike cell
line derived from AGM culture, we identified podocalyxinlike protein 1 (PCLP1) as a marker for endothelial precursors.26
PCLP1+CD45 Among various cytokines that affect hematopoiesis,
macrophage-colony-stimulating factor (M-CSF) was initially
characterized as a cytokine that supports the survival, proliferation,
and differentiation of macrophages. M-CSF was also shown to affect
osteoclasts,27 neural cells,28 uterine and
placental cells,29 and smooth muscle cells.30
As osteoclasts are derived from monocytes and M-CSF induces their
differentiation, M-CSF-deficient mice (op/op) exhibit
osteopetrosis.27 We found that the number of immature hematopoietic progenitors in the AGM culture of op/op mice
was significantly larger than that of wild-type AGM culture and that the addition of M-CSF to op/op AGM culture reduced the
number of immature progenitors. We present evidence that M-CSF
modulates the development of hematopoiesis in AGM by promoting the
differentiation of PCLP-1+CD45 Primary culture of AGM
Expression of hematopoietic cytokines in AGM culture
Northern blot analysis of Flt-1 in AGM culture The expression of Flt-1 was detected by Northern blot analysis. Total RNA (10 µg) was electrophoresed on a 1.0% agarose gel, transferred to a positively charged nylon membrane, and hybridized with digoxin (DIG)-labeled Flt-1 probes. After overnight hybridization, the membrane was washed to remove nonspecific binding of the probe and was incubated with anti-DIG antibody. Hybridized fragments were detected according to the instructions supplied.DiI-Ac-LDL incorporation assay Cells prepared from the AGM region were cultured in 4-well chamber slides (Nalge Nunc International, Naperville, IL). After 8 days of culture, the adherent cells were incubated with 10 µg/mL 1,1'-dioctadecyl-3,3,3',3'-tetoramethylindo-carbocyanine perchlorate acetylated low-density lipoprotein (DiI-Ac-LDL) in culture medium (Biomedical Technologies, Cambridge, MA) for 4 hours at 37°C. These cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 minutes at room temperature. A confocal microscope with the appropriate filters was used to detect the emitted fluorescence.Flow cytometry of adherent cells in AGM culture Adherent cells in AGM culture were harvested with cell dissociation buffer (Gibco-BRL) and were filtered through a 70-µm nylon mesh (Cell Strainer; Falcon). Cells were incubated with mouse serum to prevent nonspecific binding of antibodies. Antibodies against Flk-1 and VE-cadherin were obtained from PharMingen (San Diego, CA). Anti-intercellular adhesion molecule 2 (ICAM-2) antibody was prepared in our laboratory (unpublished results, M. Tanaka and T. Hara, 1998), and an anti-PCLP1 antibody was described previously.26 Fluorescein isothiocyanate (FITC)-conjugated anti-rat immunoglobulin (Ig)G obtained from Pierce (Rockville, IL) was used as a secondary antibody, and FACSCalibur (Becton Dickinson) was used for flow cytometry. When AGM cells were cocultured with OP9 stromal cells, biotinylated antibodies against platelet endothelial cell adhesion molecule (PECAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin were used for flow cytometry after the blocking of nonspecific binding by anti-mouse CD16/CD32 antibody (PharMingen).Colony-forming unit assays Nonadherent cells in AGM culture were collected, and 1 × 104 cells were plated in a 35-mm plate with -minimal essential medium containing 0.8% methylcellulose, 30%
fetal calf serum, 1% deionized bovine serum albumin, 100 µM
2-mercaptoethanol, 10 ng/mL IL-3, 100 ng/mL IL-6, and 2 U/mL
erythropoietin (EPO). IL-3 was prepared as previously
described.31 EPO was kindly provided by Kirin Brewery, and
IL-6 was purchased from R&D Systems. Colonies were counted at the
seventh day of culture.
Retroviral infection of AGM cells Murine-soluble Flt-1 cDNA was provided by Dr M. Shibuya (Institute of Medical Science, University of Tokyo, Japan),32 and the coding region of sFlt-1 was inserted into the retroviral vector, pMIG, which carries internal ribosomal entry site-enhanced green fluorescent protein (EGFP) after the multicloning sites. BOSC23 packaging cells were transfected with the plasmid constructs using the lipofectamine plus reagent (Gibco-BRL), and the supernatants were harvested at 48 hours after transfection. AGM cells were cultured with the supernatants for 5 days and used for CFU-C assays and fluorescence-activated cell sorter (FACS) analysis.Matrigel assay Cell suspension was passed through a 40-µm cell strainer (Falcon) to remove cell aggregates, and 2.0 × 105 cells were replated onto Biocoat Matrigel basement membrane (Becton Dickinson) in a 6-well plate with 1% fetal calf serum. After 12 hours of culture, cell morphology was observed through a microscope.
Suppression of hematopoiesis by M-CSF in AGM culture To investigate the hematopoietic activity in AGM cultures derived from wild-type and op/op mutant mice, nonadherent cells were harvested after 8 days of incubation, and colony-forming activity was evaluated (Figure 1A). Inspection of colonies indicated that the numbers of erythroid blast-forming units (BFU-E) and mixed CFU (CFU-Mix) were increased significantly in op/op AGM culture. As a result, more culture CFU (CFU-C) were found in op/op AGM culture than in wild-type AGM culture. Consistent with the increased number of granulocyte, erythrocyte, macrophage, and megakaryocyte (GEMM) in op/op AGM culture, we previously showed that more c-Kit+ cells were present in nonadherent cells in op/op AGM culture than in wild-type AGM culture.25 These results suggested that M-CSF deficiency altered the development of myeloid cells, leading to the accumulation of immature hematopoietic cells in AGM culture. We therefore added M-CSF to op/op AGM culture to test whether exogenous M-CSF could reduce colony formation. The number of hematopoietic cells grown in op/op AGM culture with M-CSF was slightly smaller than that in op/op AGM culture without M-CSF (data not shown). The numbers of BFU-E, CFU-GM, and CFU-GEMM were all reduced by the addition of M-CSF, whereas the number of CFU-M was increased by M-CSF (Figure 1B). These results suggest that M-CSF promotes hematopoietic differentiation at an early stage in AGM culture. Because M-CSF stimulates mainly the monocyte-macrophage lineage but not other hematopoietic lineages, it is likely that it affects hematopoiesis through monocyte-macrophages or nonhematopoietic cells.
Reduced production of cytokines in op/op AGM culture We considered the possibility that the altered hematopoiesis in op/op AGM resulted from the production of hematopoietic cytokines in culture because we observed that various cytokines including IL-6, OSM, LIF, G-CSF, and VEGF-B, -C, and
-Dwere expressed in AGM culture (see below). To test this
possibility, adherent cells in the wild-type, op/+, and
op/op AGM culture were harvested, and the expression of
various cytokines was analyzed by reverse transcription (RT)-PCR.
Although no significant differences were observed in the expression of
VEGFs (Figure 2) or of IL-3, IL-7, and
IL-11 (data not shown) between wild-type and op/op AGM
cells, the expression of OSM, LIF, IL-6, and G-CSF was significantly reduced in op/op AGM cells (Figure 2). These results suggest
that M-CSF and these cytokines may modulate the differentiation of hematopoietic cells. It is also possible that M-CSF regulates the
expression of cytokines in other types of cells.
We then examined by RT-PCR whether M-CSF increased the expression of these cytokines in op/op AGM culture. We found that the expression of IL-6, OSM, LIF, and G-CSF was up-regulated in op/op AGM culture by M-CSF (Figure 2). These results suggest that the increased numbers of immature hematopoietic cells in op/op AGM culture can be partly explained by a modulation of cytokine production by M-CSF in AGM culture. Alteration of endothelial cell differentiation in op/op AGM culture Although M-CSF may modulate the differentiation of committed hematopoietic progenitors by regulating cytokine production, it is also possible that it affects hematopoiesis at a much earlier stage. Given that hematopoietic progenitors are derived from hemangioblasts, the differentiation of endothelial cells might be altered by the lack of M-CSF in AGM culture. Because hematopoietic cells are generated from endothelial-like cell clusters in AGM culture, these cell clusters are thought to contain hemangioblasts. However, there was no significant difference in the number of such cell clusters between wild-type and op/op AGM cultures (data not shown). Therefore, we examined the expression of endothelial cell markers in adherent cells in AGM culture by flow cytometry, semiquantitative RT-PCR, and Northern blot analysis. There was no apparent difference in morphology and viability of cells between cultures (Figure 3A), indicating that the proliferation and viability of cells was not altered by the lack of M-CSF. The expression of PCLP-1, which was previously identified as a marker for immature endothelial cells,26 was not altered in op/op AGM cells (Figure 3B). Immunostaining of the AGM cells in plates showed that endothelial-like cell clusters in wild-type and op/op AGM culture were equally stained with anti-PCLP1 antibody (data not shown). PCLP1+CD45 cells from op/op AGM
culture exhibited an endothelial cell morphology in gelatin-coated
plates similar to that from wild-type AGM culture (data not shown).
Moreover, the expression of Flt-1, a receptor for VEGF, was unchanged
in the op/op AGM culture by Northern blot analysis (Figure
3C), and the endothelial-like cells in op/op AGM culture
incorporated DiI-Ac-LDL at a level similar to that in wild-type AGM
culture (Figure 3D). These results suggest that the numbers of
endothelial precursors were not different between op/op and
wild-type AGM culture. In contrast, flow cytometric analysis revealed
that the expressions of Flk-1, VE-cadherin, and ICAM-2 were decreased
in nonadherent cells of op/op AGM culture compared with
those in wild-type culture (Figure 3B). Furthermore, the expression of
mature endothelial markers such as PECAM-1 and von Willebrand factor
analyzed by semiquantitative RT-PCR was also decreased in the
op/op AGM culture (data not shown). These results suggest
that most of the endothelial-like cells in op/op AGM culture
were already committed to the endothelial cell lineage but failed to
differentiate to the stage that wild-type AGM cells differentiated.
To investigate the role of M-CSF in endothelial differentiation, M-CSF
was added to op/op AGM culture, and the expression of
endothelial markers was examined after 8 days of incubation in the
presence or absence of M-CSF. The addition of M-CSF to op/op
AGM culture increased the expression of Flk-1, VE-cadherin, and ICAM-2
to the levels found in wild-type AGM culture (Figure 4), indicating that M-CSF is involved in
the expression of these endothelial cell markers.
Role of VEGF for hematopoiesis in AGM culture Because endothelial differentiation was halted and simultaneously the expression of Flk-1 (VEGF receptor 2) was reduced in op/op AGM culture, we examined the role of VEGF in the development of hematopoietic and endothelial cells. To inhibit endogenous VEGF in AGM culture, we used soluble Flt-1. Flt-1 is a tyrosine kinase receptor that binds VEGF-A and VEGF-C more strongly than Flk-1. However, because Flt-1 fails to induce proliferation signals, it functions as an antagonist of VEGF.33 It was demonstrated that a soluble form of Flt-1, sFlt-1, is produced in the human peripheral blood and inhibits VEGF signaling through Flk-1 by competing the VEGF binding.34 We used the retrovirus vector to overexpress sFlt-1 in AGM culture (Figure 5). The number of immature hematopoietic progenitors produced in AGM culture infected with sFlt-1 virus was larger than that in culture with the empty vector, though the total number of colonies was unchanged.
To further elucidate the role of VEGF in AGM hematopoiesis, we tested
whether exogenous VEGF affects hematopoiesis. After 10 days of
incubation of AGM cells in the presence or absence of VEGF-A, the
colony-forming activity of nonadherent cells was examined (Figure
6). The addition of VEGF-A reduced the
number of colony-forming cells of immature progenitors but not CFU-M in
wild-type AGM culture. Although the reason for the increase of CFU-M by
VEGF-A is unknown, it was reported that VEGF induced the migration of
monocytes and differentiation to macrophagelike cells from human cord
blood CD34+ cells, suggesting that VEGF has the potential
to stimulate the monocytic lineage.35 Previously we showed
that adherent cells, which had incorporated DiI-Ac-LDL and formed
clusters of endothelial-like cells, gave rise to DiI-Ac-LDL-positive
hematopoietic cells in AGM culture.26 These results
suggest that the production of hematopoietic progenitors from
hemangioblasts or the differentiation of hematopoietic progenitors is
negatively regulated by VEGF in AGM culture. Alternatively, VEGF may
play a role in lineage commitment and differentiation of hematopoietic
cells or colony-forming cells. In contrast, neither sFlt-1 nor
VEGF had a significant effect on the expression of Flk-1 and
VE-cadherin.
M-CSF promotes endothelial differentiation of
PCLP-1+CD45
population in the AGM region gave rise to hematopoietic and endothelial cells in coculture with OP9 stromal cells, it is likely that M-CSF acts
on PCLP1+CD45 cells. If this is the case, the
M-CSF receptor encoded by the c-fms gene must be present on
PCLP1+CD45 cells. To test the expression of
the M-CSF receptor, adherent cells in AGM culture were analyzed by
using anti-M-CSF receptor antibody, and 48% were found to express the
M-CSF receptor (Figure 7A). We next
isolated the PCLP-1+CD45 cells from AGM
culture by flow cytometry and analyzed the expression of
c-fms by RT-PCR. The specific fragment of c-fms
was amplified by RT-PCR (Figure 7B), indicating that the M-CSF receptor
was expressed on PCLP-1+CD45 cells in AGM
culture. On the other hand, the expression of M-CSF was detected in the
AGM region at 11.5 days postcoitum (Figure 7B) and AGM culture
(data not shown). Finally, immunocytochemistry using the anti-M-CSF
receptor antibody revealed positive signals within clusters of
endothelial-like cells and within large and flattened cells surrounding
the clusters, which looked like smooth muscle cells (Figure
7C).
We then examined whether M-CSF had any effect on
PCLP1+CD45
In this report, we present evidence that M-CSF has the potential
to modulate the development of hematopoietic and endothelial cells in
the AGM region. A lack of M-CSF results in the accumulation of immature
hematopoietic progenitors in AGM culture, as evidenced by increased
numbers of CFU-GEMM, CFU-GM, and BFU-E. Consistent with this, the
addition of M-CSF to op/op AGM culture decreased the number
of colony-forming cells. M-CSF also affects the differentiation of
endothelial cells, as the expression of endothelial cell markers, such
as Flk-1, VE-cadherin, and ICAM-2, was decreased in op/op endothelial-like cells in AGM culture. The results in this study can be summarized as shown in Figure
9.
Because M-CSF does not act directly on most hematopoietic cells, it is
likely that its effects on CFU-GEMM, CFU-GM, and BFU-E are indirect. In
addition, because BFU-E and CFU-GEMM numbers were increased in
op/op AGM culture, M-CSF affects hematopoiesis at a very
early stage. We found that the production of cytokines such as IL-6,
G-CSF, and LIF was decreased in op/op AGM culture and was
increased by the addition of M-CSF in op/op AGM culture, suggesting that these cytokines regulate hematopoiesis at an early stage. Although the production of these cytokines may not solely account for the development of hematopoietic cells in AGM culture, it
likely contributes to hematopoiesis in the AGM region.
Monocytes-macrophages are a major target of M-CSF and have the
potential to produce various cytokines. M-CSF may modulate
hematopoiesis by enhancing cytokine production through the stimulation
of monocytes-macrophages. On the other hand, given that smooth muscle
cells in the bone marrow are known to contribute to hematopoiesis and
smooth muscle cells were also found at the periphery of
endothelial-like cell clusters in AGM culture as We show that the addition of M-CSF recovered the expression of Flk1,
ICAM-2, and VE-cadherin in op/op AGM culture, suggesting that their expression also requires signaling through the M-CSF receptor in AGM culture. Promoter analysis revealed that binding sites
for SCL, GATA, and Ets are present in the promoter regions of the
flk-1 gene.37 The mutational analysis of the
flk-1 promoters indicated that SCL, GATA, and Ets are
required for the endothelial cell-specific expression.38
In addition, it was reported that Ets-binding sites are present in the
promoter region of the VE-cadherin gene39 and that the
overexpression of Ets1 induces the expression of
VE-cadherin.40 M-CSF was shown to activate Ets family
proteins by phosphorylation,41,42 and it may induce the
expression of Flk-1 and VE-cadherin in endothelial cells by regulating
those transcription factors. Consistent with this idea, the expression of transcription factors SCL, GATA, and Ets was detected in the PCLP1+CD45 It is known that op/op mice exhibit excessive bone
formation, and this abnormality can be corrected by either M-CSF or
VEGF.44-46 Toothless rats exhibit the same phenotype as
op/op mice and show an abnormal microvasculature of the
distal femoral chondro-osseous junction.47 Administration
of M-CSF in toothless rats ameliorated not only excessive bone
formation but also the defect of the microvasculature.47 We showed that the addition of VEGF-A increased the number of CFU-M in
wild-type AGM culture, indicating that VEGF affects the development of
the monocytic lineage. We also observed that the M-CSF receptor was
expressed in PCLP1+CD45 Expression of Flk-1, VE-cadherin, and ICAM-2 was decreased in op/op nonadherent AGM cells and the reduction in Flk-1 expression in op/op mice may lead to a reduced response to VEGF, resulting in unfavorable conditions for endothelial differentiation. Although M-CSF likely stimulates differentiation of endothelial cells through the modulation of Flk-1 expression, it remains possible that M-CSF directly stimulates the differentiation of endothelial cells. Because M-CSF itself is a growth and differentiation factor for macrophages, the lack of M-CSF may lead to the accumulation of immature hematopoietic cells. These results suggest that the accumulation of immature hematopoietic cells in op/op AGM culture is caused by the block or delay of endothelial differentiation from hemangioblasts and the reduced differentiation of monocytes.
We thank Drs M. Shibuya (The University of Tokyo) and S. Hayashi (Tottori University) for providing us with sFlt-1 cDNA and op/+ mice, respectively.
Submitted July 19, 2001; accepted November 26, 2001.
Supported in part by Grants-in-Aid for Scientific Research and Special Coordination Funds from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a research grant from Core Research for Evolutionary Science and Technology, and a grant from the Organization for Pharmaceutical Safety and Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Atsushi Miyajima, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; e-mail: miyajima{at}ims.u-tokyo.ac.jp.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
), and
adherent cells in AGM culture were harvested for synthesis of ssDNAs.
PCR was performed to examine the expression of hematopoietic cytokine
using a specific set of primers. Lane 1, wild-type; lane 2, op/+; lane 3, op/op; lane 4, op/op + M-CSF.
-actin
promoter,
