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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 908-919
Hematopoietic Stem Cell Maintenance and Differentiation Are
Supported by Embryonic Aorta-Gonad-Mesonephros Region-Derived
Endothelium
By
Osamu Ohneda,
Christopher Fennie,
Zhong Zheng,
Christopher Donahue,
Hank La,
Ricardo Villacorta,
Belinda Cairns, and
Laurence A. Lasky
From the Departments of Molecular Oncology, Immunology, and
Pathology, Genentech, Inc, South San Francisco, CA.
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ABSTRACT |
Hematopoietic stem cells are capable of extensive self-renewal and
expansion, particularly during embryonic growth. Although the molecular
mechanisms involved with stem cell maintenance remain mysterious, it is
now clear that an intraembryonic location, the aorta-gonad-mesonephros
(AGM) region, is a site of residence and, potentially, amplification of
the definitive hematopoietic stem cells that eventually seed the fetal
liver and adult bone marrow. Because several studies suggested that
morphologically defined hematopoietic stem/progenitor cells in the AGM
region appeared to be attached in clusters to the ventrally located
endothelium of the dorsal aorta, we derived cell lines from this
intraembryonic site using an anti-CD34 antibody to select endothelial
cells. Analysis of two different AGM-derived CD34+ cell
lines revealed that one, DAS 104-8, efficiently induced fetal-liver
hematopoietic stem cells to differentiate down erythroid, myeloid, and
B-lymphoid pathways, but it did not mediate self-renewal of these
pluripotent cells. In contrast, a second cell line, DAS 104-4, was
relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early
hematopoietic progenitor cells of the
lin CD34+Sca-1+c-Kit+
phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone
marrow of lethally irradiated mice. These data bolster the hypothesis
that the endothelium of the AGM region acts to mediate the support and
differentiation of hematopoietic stem cells in vivo.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE HEMATOPOIETIC stem cell
dwells at a diversity of anatomic sites during vertebrate development
before it ultimately migrates to its final residence, the bone
marrow.1-3 Early evidence suggested that the fetal yolk sac
was the primordial locale of hematopoietic stem cells that seeded the
fetal liver,4 and recent studies have shown the ability of
yolk-sac-derived stem cells to reconstitute the bone marrow of newborn
recipients but not adult animals.5 Other data, however,
have suggested that an intraembryonic site, the para-aortic
splanchnopleur, may be the true initial location of hematopoietic stem
cell genesis and amplification. For example, based on studies of
chimeric avian embryos, it was shown that the hematopoietic stem cells
that seed the fetal liver and, ultimately, the bone marrow are derived
from an intraembryonic site and not the extraembryonic yolk
sac.6,7 The intraembryonic para-aortic splanchnopleur zone,
which goes on to differentiate into the aorta-gonad-mesonephros (AGM)
region, is therefore likely to be the site where definitive stem cells
are first generated.8,9 Other more recent data have
revealed that the para-aortic splanchnopleur/AGM region is of
significant interest to hematopoietic stem cell biologists for several
additional reasons. Godin and colleagues showed that the para-aortic
splanchnopleuric region of mice, in addition to birds, was the earliest
site of pluripotent hematopoietic stem cell
development.10-13 Other groups also showed that an
intraembryonic site in the mouse embryo gave rise to pluripotent stem
cells capable of multilineage reconsitution of irradiated
animals.14-16 Importantly, Medvinsky and
Dzierzak17 established that dissected AGM regions, when
placed in air/liquid organ cultures, initiated stem cell production in
the absence of the yolk sac and mediated an apparent amplification of
functional hematopoietic stem cells, consistent with the hypothesis
that this region produces a factor(s) involved with the genesis,
maintenance, and/or amplification of these pluripotent cells.
In addition to these important findings, morphological studies from a
number of investigators have implicated the endothelium of the
AGM-localized dorsal aorta as an in vivo site of hematopoietic progenitor cell residence and, potentially, amplification. Thus, using
the sialomucin CD34 as a marker for both the endothelium and the
hematopoietic stem/progenitor cell, these studies revealed that a
specific region of the dorsal aorta, contained within the AGM region,
harbored small clusters of CD34+ hematopoietic cells
attached to the endothelium in both human and murine embryos (for
example, see Fig 1B,C),18,19 similar to hematopoietic
clusters observed in the yolk-sac blood islands.19-21 Together with the in vitro genesis and expansion data, these
morphological studies implied that the endothelium of the AGM-localized
dorsal aorta might provide for the support of hematopoietic stem cells in vivo.
A number of laboratories have investigated the role of the embryonic
endothelium in hematopoiesis. Yoder and colleagues22 showed
that endothelial-like cell lines derived from the yolk sac induced the
amplification of mature myeloid cells and progenitors. Fennie and
associates 23 showed that CD34+ endothelial
cell lines derived from the yolk sac expanded both myeloid and
erythroid progenitors as well as mature cell lineages. Lu and
collaborators24 established that yolk-sac-derived
endothelial cells expanded mature and progenitor myeloid cells and
maintained cells capable of differentiating into both B- and T-cell
lineages. In addition, these investigators showed qualitative
maintenance of cells capable of repopulating all leukocyte lineages of
immunocompromised (scid) hosts. Finally, Wineman et al
25 and Moore et al26 showed maintenance of
competitively repopulating stem cells on a fetal liver-derived stromal
cell line for several weeks, although they did not examine the
endothelial nature of this stromal cell type. However, although many of
these studies attempted to address the ability of embryonic
endothelial-like stromal cells to maintain stem and progenitor cells in
vitro, they did not examine the proficiency of the endothelial cells derived from the AGM region, a known site of stem cell residence and
amplification, to induce the differentiation of progenitors and
maintenance of repopulating stem cells. Here we show that endothelial
cell lines derived from the AGM region are capable of mediating both
the differentiation of hematopoietic progenitor cells along myeloid,
erythroid, and lymphoid pathways as well as maintaining hematopoietic
stem cells capable of competitively repopulating lethally irradiated
adult bone marrow. These data support the hypothesis that the
endothelium of the AGM region is involved with embryonic hematopoietic
stem cell self-renewal and differentiation.
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MATERIALS AND METHODS |
Mice.
Timed pregnant mice (C57Bl/6) were purchased from
Simonsen laboratory (Gilroy, CA) or Harlan Sprague Dawley
laboratory (Dallas, TX). Congenic C57Bl/6, Ly5.2 male mice were
purchased from the National Cancer Institute (Frederick, MD). All mice
were housed at the Genentech, Inc. animal facility in autoclaved
microisolator cages on ventilated cage racks.
Generation of AGM-derived endothelial cell lines.
Eleven-day C57BL/6 embryos (embryo age was determined starting with day
0 on the morning of vaginal plug discovery) were removed from uteri,
and 20 AGM regions were dissected with fine tungsten needles in L-15
medium (L-15) with heat-inactivated 5% fetal bovine serum (FBS; GIBCO,
Gaithersburg, MD) under the dissecting microscope. Tissues were treated
with 0.04% collagenase (Sigma, St Louis, MO) at 37°C for 1 hour
and pipetted gently to dissociate the cell clumps. Cells were cultured
in high glucose Dulbecco's modified Eagle's medium (DMEM; GIBCO)
supplemented with 10% FBS, 0.1 mmol/L nonessential amino acids, 2 mmol/L L-glutamine, penicillin-streptomycin, and 104
mol/L  -mercaptoethanol (HAVA medium) in 75-cm2 tissue
culture flasks (Corning 430720, Corning, NY) to allow for cell
adherence. To stimulate the growth of endothelial cells, 150-µg/mL
endothelial cell-growth supplement (ECGS; Becton Dickinson, Bedford,
MA) and 10 U/mL heparin (Sigma) were added to cultures. Cultures were
incubated at 37°C, 5% CO2 for 7 days. Cells were trypsinized and labeled with rabbit anti-murine CD34 polyclonal antibody (anti-CD34), and a CD34+ fraction was isolated
using MiniMACS magnetic bead system (Miltenyi Biotec, Auburn, CA) as
described previously.23 Subsequently, a CD34-enriched
fraction was sorted for CD34+ cells by
fluorescein-activating cell sorter (FACS; Coulter, Hialeah, FL) and
cultured in gelatin-coated (1.5%; Sigma) 20 × 100 mm dishes (Corning 25020) in HAVA medium23 with 150 µg/mL ECGS, 10 U/mL heparin, and 20% HAVA medium previously conditioned by incubation with adherent AGM-derived cells for 7 days (HAVA-S). After cell growth
reached 40% to 50% confluency, cells were infected with polyoma
middle T antigen/NEOr retrovirus as described
previously23,27 and cultured in HAVA-S with 800 µg/mL
G418. G418-resistant colonies appeared after 1-month cultivation, and
80 clones, termed DAS (dorsal aorta-derived stroma) cells, were
isolated. The DAS104 cell line was subcloned for highly CD34+ cells by FACS. Thirty DAS104-derived
CD34+ cells were cultured in 96-well plates (Falcon 3072)
in HAVA medium, and 18 subclones were obtained. Expression of CD34
antigen was examined in each subclone by FACS, and two subclones,
DAS104-4 and DAS104-8, were selected for further analysis. Analysis of capillary formation in Matrigel (Biocoat 40234C; Becton Dickinson) was
performed as per the manufacturer's instructions.
Antibodies.
The antibodies used in FACS were all obtained from Pharmigen (San
Diego, CA) except anti-CD34 polyclonal antibody28 and secondary reagents. The antibodies used in the lineage cocktail are
anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD5 (53-7.3), anti-CD8
(53-6.7), anti-B220 (6B2), anti-Gr-1 (8C5), and antierythroid (TER-119). Other antibodies include anti-Sca-1 (E13) labeled with phycoerythrin (PE), anti-c-Kit (2B8) labeled with biotin, fluorescein isothiocyanate- (FITC) or PE-conjugated anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-IgM, anti-Gr-1 and anti-Mac-1 antibody. Secondary reagents include goat antirat IgG labeled with cascade blue (Molecular Probes, Eugene, OR), 670-labeled streptavidin (GIBCO), and donkey antirabbit IgG-labeled FITC (Jackson Immunoresearch, West Grove, PA).
Isolation of fetal liver stem cells.
Fetal liver cells were prepared from day-13 embryos. Livers were
dissected and made into a single-cell suspension with a 22-G needle and
passed through a nylon mesh screen. Fetal liver mononuclear cells were
isolated by density centrifugation over Ficoll solution (1.077 g/mL;
Nycomed, Oslo, Norway ). Cells were cultured in 175-cm2
flasks (Falcon 3028) overnight, and nonadherent cells were obtained. Cells were suspended in L-15/5% FBS and incubated with CD16/CD32 (2.246) antibody (1:200) (Pharmigen) on ice for 15 minutes.
Subsequently, cells were stained with the lineage cocktail of unlabeled
antibodies for 30 minutes on ice. After washing twice, cells were
stained with goat antirat magnetic beads (Miltenyi Biotec, Auburn CA) and then stained with goat antirat cascade blue. Cells were applied to
BS type MACS column (Miltenyi Biotec), and nonadherent fraction was
collected. After incubation with normal rat serum (GIBCO) for 15 minutes on ice, cells were stained with anti-CD34 antibody, anti-Sca-1
antibody, and anti-c-Kit antibody. After final washes, cells were
suspended in L-15/5% FBS with 1 µg/mL propidium iodide (PI; Sigma)
and filtered through a nylon mesh screen. Viable cells were selected by
PI exclusion, and four-color FACS for lineage-negative, CD34+, Sca-1+, c-Kit+ cells was
performed on a dual-laser Epics Elite ESP cell sorter (Coulter
Immunology, Hialeah, FL). After cell sorting, over 95% purity was observed in sorted cells.
In vitro expansion of fetal liver cells on DAS cell lines.
All stromal cell lines were grown in HAVA medium at 37°C in 5%
CO2. Stem cells were also cultured on b-End3,27
YS Cl-71, or YS Cl-7223 and cocultured under the same
conditions as DAS cell lines. Stromal cells were irradiated (20 Gy,
137Cs, Gammacell 100; Nordin International Inc, Ontario,
Canada) 2 days before coculture and plated to confluence in
gelatin-coated 20 × 100-mm dishes (Corning). A total of 3 × 103 sorted fetal liver stem cells
(linCD34+Sca-1+c-Kit+)
was added onto each stromal cell line; cultures were maintained in HAVA
medium at 37°C, 5% CO2 in high humidity; and half
volume of the medium in each culture was changed every other day. After 7 or 10 days, cells were obtained with 0.04% trypsin-EDTA (GIBCO) or
2% collagenase H (Boehringer Mannheim, Indianapolis, IN), and single
cells were obtained using disruption by a 22-G needle and passage
through nylon mesh screen. Nonadherent cells were collected after
1-hour incubation on gelatin-coated dishes at 37°C. Numbers of
viable cells were scored under the microscope by Trypan blue exclusion,
and morphology was examined by Wright-Giemsa (Diff-Quik; Dade
Diagnostic, Aguada, Puerto Rico) staining of cytospin preparations.
Irradiated DAS104-4 or DAS104-8 were cultured in gelatin-coated 6-well
plates (Falcon 3046), and 1 × 103 fetal liver stem
cells were added onto the stromal cells. Cultures were maintained
in HAVA medium in the presence of 3 U/mL erythropoietin (EPO; R&D
Systems, Minneapolis, MN) or 10 ng/mL interleukin-7 (IL-7; R&D Systems)
for 11 days or 12 days. Cells were obtained by vigorous pipetting, and
cell differentiation was identified by FACS. Myeloid cell
differentiation was based on expression of Gr-1 or Mac-1; B-cell
differentiation on B220 or IgM; erythroid differentiation on TER 119;
and T-cell differentiation on CD3, CD4, or CD8.
Irradiated DAS104-4 cells were plated to confluence on gelatin-coated
6-well plates (Biocoat 4057; Becton Dickinson) and 1 × 103 fetal liver stem cells were then either seeded directly
onto the stromal cells or into transwell apparatus (0.45 µmol/L pore size membrane; Biocoat 4057). Cultures were maintained for 7 days, and
cells were obtained with trypsin-EDTA or by vigorous pipetting. Numbers
of expanded cells were scored under the microscope in each well.
Long-term reconstitution assays.
All mice (C57Bl/6) used as donors in this experiment were Ly5.1.
C57Bl/6-Ly5.2 recipient mice were lethally irradiated with 1,050 rads
as a single dose from a 137Cs source. Freshly sorted stem
cells or progenitor cells cultured on stromal cells were injected into
the tail vein along with 1 × 106 whole bone marrow
cells from an Ly5.2 congenic source for radioprotection. Groups of 6 to
8 mice were used for each cell type injected. For analysis of
reconstitution, mice were bled from the retro-orbital sinus and assayed
for the presence of Ly5.1+ cells at 4 weeks or 8 weeks
after transplantation. Peripheral blood was collected in phosphate
buffered saline with 1 mmol/L EDTA and 10 U/mL heparin (Sigma). Red
cells were removed by sedimentation in 1% Dextran T-500 (Pharmacia
Biotech, Piscataway, NJ) and lysing in cold 0.15 mol/L
NH4Cl. Cells were stained with anti-Ly5.1 antibody (104;
Pharmingen) and then stained with PE-labeled streptavidin (Jackson
ImmunoResearch).
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of
cell line transcripts.
Total RNA was prepared from each stromal cell line using RNA isolation
reagent (STAT 60; TEL-TEST "B" Inc, Friendswood, TX). The
Perkin-Elmer (Foster City, CA) RT-PCR kit was used as
described previously.23 For each reaction, 1 µg of total
RNA was used for a total of 35 cycles in a Perkin-Elmer thermocycler
using the following conditions: 1 minute at 95°C, 1 minute at
56°C, and 2 minutes at 72°C. PCR reactions were electrophoresed
on 1.5% agarose gels, stained with ethidium bromide, and examined. The PCR primers were as follows: stem cell factor (SCF)-5 :
5-TCTTCCAAATGACTATATGATAACCCTC-3, SCF-3:
5-ATTCCTAAGGGAGCTGGCTGCAACAGGG-3; FLK2 ligand (FLK2/FLT3 L)-5: 5-CTGCTGTTGCTGCTGCTGAGTCCTTGC-3,
FLK2/FLT3L-3: 5-GTCCGGCTGGCACTGCACCTCCAGGC-3; vascular endothelial growth factor (VEGF)-5:
5-GTGATCAAGTTCATGGACGTCTACCAGCG-3, VEGF-3:
5-TACGTTCGTTTAACTCAAGCTGCCTCGC-3; macrophage
colony-stimulating factor (M-CSF)-5:
5-CAATGCTAACGCCACCGAGAGGCTCCAGG-3, M-CSF-3: 5-GGTACTCCTGGGTGGTCGCTGCTTGGC-3; granulocyte
colony-stimulating factor (G-CSF)-5:
5-AGTGACATATGGTCAGGACGAGAGGC-3,
G-CSF-3:5-GGGCCACCCCTAGGTTTTCCATCTGCT-3; transforming growth factor -1 (TGF-1)-5:
5-CCTTGCTGCTGCCTGTAGATGGGACTGAC-3, TGF-1-3:
5-GGTGCCCTGCCAGAAGACATGGCCTCC-3; CD34-5:
5-CTACCACGGAGACTTCTACACAAGG-3, CD34-3:5-CCAACCTCACTTCTCGGATTCCAGAGC-3;
CD31-5: 5-TGCGATGGTGTATAACGTCACCTCCA-3, CD31-3: 5-GCTTGGCAGCGAAACACTAACACGTG-3;
IL-3-5: 5-GTGGCCGGGATACCCACCGTTTAAC-3, IL-3-3: 5-GAGACGGAGCCAGATGCGGGCTGAGGTGG-3;
IL-6-5: 5-ATACCACTCCCAACAGACCTGTCTATACC-3, IL-6-3: 5-TGTGACTCCAGCTTATCTGTTAGGAGAGC-3;
thrombopoietin (TPO)-5: 5-CTTTGTCTATCCCTGTTCTGCTGCCTGCTG-3, TPO-3:
5-TGAGAAGTACTGCTTGGGACAGCTGTGG-3; EPO-5:
5-GAACGTCCCACCCTGCTGCTTTTACTCTCC-3, EPO-3:
5-CCCAGTACCCGAAGCAGTGAAGTGAGGC-3; granulocyte-macrophage colony-stimulating factor (GM-CSF)-5: 5-TCTACAGCCTCTCAGCACCCACCCGCTCA-3, GM-CSF-3:
5-GGCTGTCTATGAAATCCGCATAGGTGG-3; IL-1-5:
5-TGTCCAGATGAGAGCATCCAGCTTC-3, IL-1-3:
5-GATTCTTTCCTTTGAGGCCCAAGGCCA-3; IL-11-5:
5-AGATCTGGACAGCGCTGTTCTCTCCTAA-3, IL-11-3:
5-AGTCGAGTCTTTAACAACAGCAGGCC-3; LIF-5:
5-TCTCTTCATTTCCTATTACACAGCTC-3, LIF-3:
5-AGAAGGCCTGGACCACCACACTTAT-3; von Willebrand factor
(vWF)-5: 5-ATGATGGAGAGGTTACACATCTCTCAG-3, vWF-3: 5-CCAGCTCATCCACCCCACTGAGCAG-3;
TIE-2-5: 5-GAGAACATGTGAGAAAGCTTGTGAG-3, TIE-2-3: 5-CTAGGGTCATTTCTTCACTAGT-3;
FLK1-5: 5-CTTAGGTGCCTCCCCATACCCTGGG-3, FLK1-3: 5-TGGCCGGCTCTTTCGCTTACTGTTC-3;
FLT-1-5: 5-ATGATGCCAGCAAGTGGGAGTTTGC-3, FLT-1-3: 5-GGTTTCCATATTTGCAGTATTC-3; delta-like
protein (dlk)-5: 5-GAACCATGGCAGTGCATCTGCAAGGA-3,
dlk-3: 5-TTGCACAGACACTCGAAGCTCACCTG-3.
Analysis of fetal liver stem cell division on DAS104-4 cells.
Sorted
linCD34+Sca-1+c-Kit+
fetal liver stem cells were stained with 8 × 106 mol/L PKH67 (Sigma)29 for 3 minutes
at room temperature. Cells were washed three times and fluorescence
intensity of PKH67-GL was analyzed by FACS. A total of 5 × 103 PKH67+ cells were plated onto irradiated
DAS104-4 cells in 10-cm dishes and cultured for 3 days, 4 days, or 5 days. Cells were obtained by 2% collagenase H treatment, and cell
aggregates were dissociated by gentle passage through syringe with a
22-G needle. Cells were suspended in L-15/5% FBS and incubated with
CD16/CD32 antibody for 15 minutes on ice. Subsequently, cells were
stained with CD45 (Ly-5, 30-F11) antibody conjugated with PE
(Pharmingen) for 30 minutes on ice. After washing twice, cells
were suspended in L-15/5% FBS with 1 µg/mL PI and passed through a
nylon mesh screen. Cells were gated for live cells
(PI) and CD45 (PE+ hematopoietic cells),
and PKH67-GL fluorescence intensity was analyzed by FACS. Fluorescence
intensity of stem cells in PKH67+ cells was obtained by
gating cells for high expression of PKH67. Mean ± SD was evaluated
from triplicate dishes. Cells in PKH67 high or low gates were isolated
by cell sorting and used for reconstitution as described above.
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RESULTS |
Generation of AGM-derived CD34+ endothelial cell
lines.
We and others have previously shown the utility of the anti-CD34
polyclonal antibody for the isolation of both embryonic endothelium as
well as hematopoietic progenitor cells.5,16,23,30 This antibody is particularly useful for endothelial cell isolation and
characterization, because global analyses of the adult and embryo
revealed that only hematopoietic progenitor cells, endothelial cells,
and a small subset of neurons express this antigen.20,21,28 Other groups have found clusters of CD34+ hematopoietic
progenitor cells attached to the ventral endothelium of the dorsal
aorta using anti-CD34 antibodies to stain the AGM region in both human
and murine embryos, and these results are confirmed in
Fig 1.18,19 At least a portion
of these CD34+ hematopoietic cells are stem cells capable
of repopulating irradiated bone marrow, consistent with the proposal
that AGM-derived endothelium supports hematopoietic stem
cells.16,31 To obtain these endothelial cells, the AGM
region of day-11 murine embryos was isolated by dissection (Fig 1A).
Staining of this isolated tissue with anti-CD34 antibody showed
positively reacting blast-like hematopoietic progenitor cells, a subset
of which were presumably stem cells, and elongated, endothelial-like
cells that also expressed CD34 (Fig 1C). Because endothelial cells are
adherent and the numbers of cells obtained were low, isolated AGMs were
enzymatically dissociated and the adherent cells were expanded in
tissue culture for 7 days. CD34+ adherent cells, presumably
of endothelial origin, were isolated by FACS, and the resultant cells
were transformed using a polyoma virus middle-T-expressing retrovirus.
Earlier we and others had shown that this oncogene appears to transform
endothelial cells and maintain these cells in an endothelial-like
state.23,27,32 CD34+ (ie, endothelial),
middle-T-transformed, G418-resistant cells were isolated by FACS, and
80 cell lines were established. Thirty lines were tested for their
ability to mediate the expansion of differentiated hematopoietic cells,
the morphologies of hematopoietic cell colonies, and the expansion of
high proliferative potential mixed colonies (HPP-CFC) in methyl
cellulose. One cell line, termed DAS 104, was chosen for its ability to
mediate high-level expansion of differentiated hematopoietic cells as
well as HPP-CFC, and this line was sorted again for CD34 expression by
FACS (Fig 1D). Two morphologically similar CD34+ adherent
cell lines, DAS 104-4 and DAS 104-8 (Fig 1E), were selected for further
analysis. The endothelial cell nature of these lines was additionally
tested using RT-PCR. As Table 1 shows, a
number of endothelial cell markers, including CD34, FLT1, and FLK1
(both VEGF receptors), vWF, and CD31 (platelet endothelial cell
adhesion molecule [PECAM]), were found to be expressed by both cell
lines. Finally, both the DAS 104-4 and DAS 104-8 cell lines efficiently formed capillary-like tubes when placed in Matrigel
(Fig 2). Together, these data are
consistent with the conclusion that these two lines represent
endothelium derived from the AGM region.
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| Fig 1.
Isolation and characterization of AGM-derived endothelial
cells. (A) A day-11 murine embryo highlighting (rectangle) the AGM region that was dissected and used as a source for CD34+
endothelial cells. (B) Transverse section through 11-day murine embryo
AGM region stained with anti-CD34 antibody. Note the positively stained
endothelium and adherent hematopoietic progenitor cells attached to the
dorsal aorta (arrow), similar to those previously observed in the blood
islands of the yolk sac and the AGM region of murine and human
embryos.18-21 (C) Dissected AGM region stained with
anti-CD34 antibody. Note the positively stained endothelial cells and
adherent hematopoietic progenitor cells attached to the dorsal aorta
(arrow). (D) A progenitor DAS 104 cell line was generated by polyoma
virus middle-T transformation and was stained with anti-CD34 antibody.
The CD34+ cells were isolated by FACS sorting. (E) DAS
104-4 and DAS 104-8 cell lines were stained with anti-CD34 antibody and
analyzed by FACS. Note that both cell lines are positive for the CD34
antigen. Morphology of DAS 104-4 and DAS 104-8 cell lines showing
typical flattented, endothelial-like structures.
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| Fig 2.
Capillary formation in vitro. Murine 3T3 fibroblasts,
brain-derived capillary endothelial cell line b-End-3, and DAS 104-4 or
DAS 104-8 endothelial cell lines were plated in Matrigel, grown for 2 days, and photographed.
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Expansion of fetal liver stem cells on AGM-derived cell lines.
Initial studies were done to examine the ability of each AGM-derived
cell line to mediate the expansion and differentiation of
linCD34+Sca-1+c-Kit+
hematopoietic stem cells isolated from fetal liver.33
Figure 3A illustrates that DAS 104-4 and
DAS 104-8 cells were capable of inducing several thousand-fold
increases in hematopoietic cell number after only 7 days of coculture,
whereas the polyoma middle-T-transformed endothelial cell line, b-End
3,27 showed only modest expansion of these progenitor
cells. Examination of expanded hematopoietic cells by Wright-Giemsa
staining revealed that the amplified cells appeared to be less mature
when grown on DAS 104-4 versus DAS 104-8 (Fig 4). However, the cells expanded on
both cell lines appeared to have a predominately myeloid appearance.
Rare blast-like cells were also observed in the cytospin analyses of
expanded cells, and examination of the colony morphology of
hematopoietic cells incubated on either AGM-derived cell line revealed
both large macrophage-like cells and small blast-like cells in
"cobblestone"-type hematopoietic colonies (Fig 3C). In addition,
c-Kit+ cells isolated from both the AGM and
linCD34loSca-1+c-Kit+
cells from adult bone marrow are also dramatically expanded on both
endothelial cell lines (data not shown). These data suggested that both
AGM-derived endothelial cell lines were capable of mediating the
dramatic and rapid expansion and differentiation of fetal liver, AGM,
and adult bone marrow hematopoietic stem cells along at least the
myeloid pathway.
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| Fig 3.
Expansion of fetal liver hematopoietic stem cells on
AGM-derived endothelial cell lines. (A) Three thousand fetal liver stem cells
(linCD34+Sca-1+c-Kit+)
were plated on each stromal cell line and cultured for 7 days. Cells
were trypsinized, and nonadherent cells were collected and counted. (B)
Cell contact is required for efficient expansion of fetal liver stem
cells on DAS 104-4. One thousand fetal liver stem cells
(lin
CD34+Sca-1+c-Kit+) were
plated either directly onto the DAS 104-4 cell line or in a transwell
(insert) immersed in media conditioned by this cell line. After 1 week,
nonadherent hematopoietic cells were isolated and counted. (C)
Hematopoietic colony morphologies of fetal liver stem cells plated on
the DAS 104-4 cell lines after 5 days in culture. Panels a (20×) and
c (200×) show a "cobblestone"-type structure containing
blast-like cells (arrow in panel a). Panels b (20×)and d (200×)
show macrophage-like cells that predominate in the cultures.
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| Fig 4.
Wright-Giemsa staining of hematopoietic cells expanded on
DAS 104-4 and DAS 104-8. (A) Hematopoietic cells expanded for 7 days on
DAS 104-4. The large cells are stromal cells and the small cells are
hematopoietic cells. Note the relatively immature morphology of the
hematopoietic cells. (B) Hematopoietic cells expanded for 7 days on DAS
104-8. Note the mature macrophage-like appearance of the smaller
hematopoietic cells. The bar corresponds to 100 µm.
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Fetal liver stem cell expansion requires direct contact.
Because morphological analyses of embryos showed direct contact between
CD34 hematopoietic progenitor cells and the yolk sac and AGM
endothelium,18-20 we investigated if direct contact was required for hematopoietic cell amplification by the AGM-derived stromal cells. Fetal liver stem cells
(linCD34+Sca-1+c-Kit+)
were incubated with the DAS 104-4 cell line either directly or using a
transwell apparatus that inhibited physical contact between the
hematopoietic and stromal cells. Nonadherent (hematopoietic) cell
numbers were determined 1 week after inoculation of the cultures with
the fetal liver stem cells. As Fig 3B illustrates, cells that were in
direct contact with the stromal cell line were amplified approximately
2,600-fold, while cells separated from the stromal cells by the
transwell apparatus showed only an approximately 70-fold amplification.
Hematopoietic reconstitution assays of lethally irradiated animals (see
below) revealed that hematopoietic cells derived from transwell
cultures were incapable of mediating bone marrow reconstitution,
whereas cells derived from direct contact cultures mediated long-term
reconstitution (data not shown). These data show that direct physical
contact between the stromal cells and the hematopoietic progenitors is
required for maximal amplification and differentiation, consistent with
the in vivo morphological analyses showing clusters of
CD34+ hematopoietic cells attached to the endothelium of
the dorsal aorta.18,19
Differentiation of fetal liver stem cells along erythroid and
B-lymphoid pathways.
Previous analysis of yolk-sac-derived endothelial cell lines showed
that the addition of EPO to cultures containing yolk-sac CD34+ hematopoietic progenitor cells induced a fraction of
these cells to differentiate along the erythroid pathway.23
Other studies showed the ability of a different yolk-sac-derived
endothelial cell line to induce B-cell formation.24 To
examine the ability of AGM-derived endothelial cells to induce both
erythroid and B-lymphoid differentiation, fetal liver stem cells were
incubated with each cell line in the presence of EPO or IL-7,
respectively. Expanded cells were then analyzed for the erythroid cell
surface marker, TER-119, or the B-cell marker, B 220, using FACS. As
Table 2 illustrates, the majority of cells
expanded in the absence of any exogenous differentiation factors
expressed the myeloid-specific markers Mac-1 and Gr-1, consistent with
the cytospin analysis described above. In the presence of EPO, the DAS
104-8 cell line induced fetal liver stem cells to differentiate
predominately down the erythroid lineage pathway, with approximately
72% of the cells expressing the TER-119 marker. Even more striking
were results obtained with IL-7 on the DAS 104-8 cell line, where
approximately 93% of the mature cells expressed the B220 B cell
marker. These data together suggested that the DAS 104-8 cell line was
capable of efficiently inducing differentiation of fetal liver stem
cells down the myeloid, erythroid, and B-lymphoid pathways. In contrast to DAS 104-8, DAS 104-4 appeared to be far less effective at provoking differentiation down the erythroid and B-lymphoid pathways. Thus, this
cell line induced only approximately 24% of differentiated cells down
the erythroid path in the presence of EPO and was essentially incapable
of mediating the formation of B cells in the presence of IL-7 (Table
2). These data argue that, although the AGM-derived endothelial cell
lines are heterogeneous in their effects on the growth and
differentiation of hematopoietic stem cells, they are capable of
efficiently inducing the diverse differentiation of fetal liver stem
cells.
Stem cell maintenance on AGM-derived endothelial cells.
Although the above data indicated that one of the AGM-derived
endothelial cell lines was capable of inducing differentiation along
three mature hematopoietic lineages, they did not address the ability
of these endothelial cells to maintain or expand hematopoietic stem
cells. This question was particularly important in light of in vitro
studies that suggested that isolated AGM regions appeared to expand
repopulating hematopoietic stem cells when placed in organ culture for
3 days.17 To investigate this question, we purified
linCD34+Sca-1+c-Kit+
hematopoietic stem cells from fetal liver using FACS and examined whether DAS 104-4, DAS 104-8, and a previously described
yolk-sac-derived endothelial cell line, YS CL72,23 could
maintain these cells in an undifferentiated state in culture.
Table 3 illustrates that the DAS 104-4 cell
line expanded this population of fetal liver stem cells after 7 days of
incubation in two separate experiments, whereas the DAS 104-8 cell line
was unable to mediate expansion of these progenitor cells. Although
these data suggest that DAS 104-4 may support fetal liver stem cells
more efficiently than DAS 104-8, the ability of such progenitor cells
to mediate reconstitution of the bone marrow of lethally irradiated
animals must be shown to prove this conjecture.34,35 To
explore this question, competitive repopulation assays were performed
with lin
CD34+Sca-1+c-Kit+ fetal liver stem
cells. Table 4 shows that as few as 1,000 uncultured linCD34+Sca-1+c-Kit+
fetal liver stem cells could substantially contribute (66% to 72%) to
circulating hematopoietic cells, including mature T-cell, B-cell, and
myeloid lineages, at 15 weeks after transfer to lethally irradiated
animals using the competitive repopulation assay. Animals transplanted
with the equivalent of 1,000 linCD34+Sca-1+c-Kit+
fetal liver stem cells cultured for 7 days on the DAS 104-8 cell lines
showed only approximately 1% contribution of these cultured cells to
mature hematopoietic lineages, consistent with the supposition that
this cell line is incapable of maintaining these stem cells in an
undifferentiated state. In sharp contrast to these results, Table 4
shows that incubation of these fetal liver stem cells on DAS 104-4 for
7 days resulted in little apparent loss of hematopoietic reconstituting
ability. Thus, the equivalent of 1,000 fetal liver stem cells incubated
on this cell line for 7 days gave similar levels of hematopoietic
reconstitution (approximately 63% to 85%) at 15 weeks post
transplantation as 1,000 primary fetal liver stem cells, suggesting
that this cell line is able to maintain these hematopoietic cells in an
undifferentiated state in spite of the fact that these progenitor cells
are also dramatically expanded down the myeloid pathway of
differentiation on this cell line. Finally, fetal liver stem cells
incubated on the YS CL 72 cell line showed a modest degree of stem cell
maintenance (approximately 4% to 30% reconstitution) as compared with
the AGM-derived DAS 104-4 cell line. These data thus show that the DAS
104-4 cell line provides an efficient supportive environment for the
maintenance or self-renewal of fetal liver-derived hematopoietic stem
cells. In addition, the results are consistent with the conclusion that the endothelium of the AGM region provides a milieu in vivo for both
the support and differentiation of these pluripotent progenitor cells
as well.
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|
Table 4.
Lineage Reconstitution in Lethally Irradiated Mice
Transplanted With Uncultured or Cultured Fetal Liver Stem Cells
|
|
Stem cell self-renewal on DAS 104-4 stromal cells.
As the two models in Fig 5 illustrate, the
maintenance of hematopoietic stem cells on the DAS 104-4 cell line
could be explained by either the self-renewal of the stem cell after
each cell division (model A) or the induction of a quiescent state
without division of the stem cell (model B). To examine which of these
two possible modes of stem cell maintenance was occurring in this in
vitro system, highly purified stem cells were labeled with PKH67, a highly fluorescent dye that becomes impermeable after stem cell uptake.
If model A is correct then the level of fluorescence in the entire stem
cell population should decrease with time in culture, and there should
be virtually no highly fluorescent cells remaining after 7 days in
vitro. Alternatively, if model B is correct then a fraction of the
initial stem cells, which should be significant in view of the purity
of the starting population, should maintain a high level of
fluorescence, whereas the remaining fraction of more differentiated
cells, which divide and ultimately give rise to the mature
hematopoietic cells, should show a decreasing level of fluorescence
with time in culture. As Fig 6 illustrates,
the vast majority of fetal liver-derived stem cells labeled with the fluorescent dye PKH67-GL29 rapidly lose fluorescence as
they are cultured on the DAS 104-4 cell line, consistent with the
self-renewal model A. While it might be argued that only a very small
fraction of the initial linCD34+Sca-1+c-Kit+
were actual stem cells, analysis of the extremely small numbers of
cells (<0.5%) remaining in the highly fluorescent gate after 3, 5, or 7 days of culture on DAS 104-4 reveals that these cells also lose
fluorescence with increasing time in culture (Fig 6). To further
address the question of self-renewal versus quiescence, we examined the
reconstituting ability of cells in the PKH67-low versus -high gates. As
Fig 7 shows, all of the reconsituting
activity was found in the PKH67-high region, where the cells showed a
mean fluorescence intensity of 8.45 versus 279.4 for the starting
population. These data suggest that the stem cells divide and
self-renew on the DAS 104-4 cell line, but their proliferation rate is
less than that for the committed cell population. Together with the hematopoietic reconstitution experiments in Table 4, these data argue
for a self-renewal process as the mechanism for stem cell maintenance
in this system.
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| Fig 5.
Models of stem cell maintenance. Illustrated are two
models for stem cell maintenance. In both models, the starting
population is assumed to contain both stem cells (S) and more
differentiated progenitor cells (D). Model A illustrates a self-renewal
process where the stem cell divides asymmetrically to give rise to a
committed and a pluripotent daughter cell, thus maintaining stem cell
numbers at each generation. Model B illustrates a quiescence process
where stem cells are maintained in a nonproliferative state and the committed progenitors divide to give rise to mature lineages.
|
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| Fig 6.
Analysis of fluorescently labeled stem cells in vitro.
linCD34+Sca-1+c-Kit+
fetal liver stem cells were stained with the fluorescent dye PKH67.29 PKH67+ cells were plated onto
DAS104-4 cells and cultured for 3, 4, or 5 days. Cells were obtained,
gated for live cells (PI) and CD45 (hematopoietic
cells), and PKH67-GL fluorescence intensity was analyzed by FACS.
Fluorescence intensity of stem cells in the PKH67high
region was obtained by gating cells for high expression of PKH67 and
analyzing the fluorescence intensity of these cells. Note that the
fluorescence intensity of cells in the PKH67high region
goes down with time, although at a slower rate than the bulk of
cells.
|
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View larger version (16K):
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| Fig 7.
Reconstituting activity of cells in the PHK67-low versus
PHK67-high region. PKH67-labeled cells were either directly injected into lethally irradiated animals or incubated for 7 days on DAS 104-4, sorted for PKH-67-low versus -high fluorescence, and then injected into
lethally irradiated animals. As can be seen, all of the reconstituting
activity is found in the PKH67-high fraction with a mean fluorescence
of 8.45 versus a starting fluorescence of 279.4, consistent with
self-renewal and maintenance of stem cell phenotype of hematopoietic
cells proliferating on DAS 104-4.
|
|
Cytokine profiles in the DAS 104-4 and 104-8 cell lines.
Cytokine and growth factor expression were compared in the DAS 104-4 and DAS 104-8 cell lines using RT-PCR to examine if differences in
synthesis of known factors could account for the differential ability
of these lines to maintain repopulating stem cells, although it should
be noted that a caveat to this procedure is its lack of quantitation.
Table 1 illustrates that both cell lines expressed the same diversity
of hematopoietic regulatory molecules. This includes dlk, the recently
described delta-like molecule that was proposed to mediate stem cell
self-renewal or maintenance in a fetal liver-derived stromal cell
line.26,36 In addition, both cell lines express IL-11, a
cytokine previously proposed to maintain stem cells in culture when
used together with either stem cell factor or FLK2
ligand.37 Because DAS 104-8 does not maintain stem cells
yet appears to express the same types of hematopoietic factors as DAS
104-4, these data argue for the possibility that a novel protein(s)
expressed by the DAS 104-4 cell line may be involved with the
self-renewal of fetal liver stem cells.
| |
DISCUSSION |
The hematopoietic microenvironment must provide both for the
differentiation as well as the self-renewal of the hematopoietic stem
cell.1,3,38 A great deal of data have accumulated that suggest that these events are accomplished by physical interactions between hematopoietic stem/progenitor cells and stromal elements in the
various hematopoietic sites.25,26,39 Although the bone marrow and fetal liver are commonly used as sources for these stromal
elements, the morphological complexity of these tissues makes the
isolation of the appropriate hematopoietic-supportive stromal cell type
formidable. In addition, it is not clear if hematopoietic stem cells
are expanded in adult bone marrow, because the majority of these cells
seem to be in a nondividing state.40 In contrast, the
morphologies of both the yolk-sac and the AGM region of the early
mammalian embryo are relatively simple, especially with respect to
hematopoietic elements. In both cases, these regions show clusters of
CD34+ hematopoietic progenitor cells attached to the
endothelium of either the yolk-sac blood islands or the AGM-localized
dorsal aorta.18-20 In addition, it appears that both of
these regions contain CD34+ hematopoietic progenitor cells
that are capable of repopulating the bone marrow of irradiated
recipients, although the yolk-sac-derived stem cells may be deficient
in trafficking to the adult bone marrow compartment.16,31,41 Perhaps most important is the finding that hematopoietic stem cells in the AGM region are capable of a modest
level of expansion in vitro, suggesting that if these cells are the
ones observed to be attached to the endothelium, this endothelial site
may produce a factor(s) capable of stem cell expansion.17
Together, these results suggest that AGM-derived endothelium may be an
important supportive cell for hematopoietic development and stem cell
maintenance in the early embryo, and the data reported here are in
large part consistent with this hypothesis.
Because the major conclusion of this paper is that the endothelium of
the AGM region, like that of the yolk sac,23 appears to be
involved with the regulation of hematopoietic stem cell self-renewal
and growth, it is important to emphasize the methodologies and results
that indicate that the DAS cell lines are in fact endothelial cells. A
number of laboratories have conclusively shown that the
CD34+ cells in the embryo and adult appear to be
predominately either endothelial or hematopoietic in
origin.19-21 In addition, endothelial cells, but not
hematopoietic progenitor cells, are adherent under tissue culture
conditions. Thus, the combination of using anti-CD34 antibody to purify
cells from the AGM region, together with the use of a tissue culture
incubation step to isolate adherent CD34+ cells, should
result in an enriched population of endothelial cells. In addition, the
polyoma middle-T oncogene has been shown to be relatively specific for
endothelial cells,32 and earlier studies showed that
inoculation of retroviruses expressing this oncogene into murine
embryos resulted in the production of only endothelial cell tumors that
could be used as a source for endothelial cell lines.27
Thus, the use of this oncogene to transform the isolated AGM-derived
endothelial cells should result in cell lines that maintain at least a
fraction of their endothelial characteristics. This latter hypothesis
has been shown in the case of the DAS cell lines that express the
endothelial markers CD34, FLT1, FLK1, vWF, and CD31 (PECAM). In
addition, these cells produce the endothelial growth factor VEGF, which
may allow for autocrine-mediated proliferation of these cell lines by
activation of the FLT1 or FLK1 receptors. The formation of
capillary-like structures when these cells are grown in Matrigel also
strongly supports the contention that DAS 104-4 and 104-8 are
endothelial cells. Together, these data argue that these cell lines are
endothelial in origin and phenotype.
The endothelial cell lines decribed here fullfill a number of criteria
that would be expected from an in vitro representation of hematopoiesis
in the AGM region. For example, both cell lines are capable of
mediating the dramatic expansion of progenitor cells along the myeloid
pathway, and the DAS 104-8 line is also capable of expansion along the
erythroid and B-lymphoid pathways in the presence of EPO or IL-7,
respectively. In addition, direct physical contact is required for this
high level of expansion and differentiation, suggesting that the growth
factor(s) involved with hematopoietic expansion in this system is
likely to be immobilized on the stromal cell surface. Both this ability
to expand progenitor cells along all three mature hematopoietic
lineages as well as the requirement for direct cell contact are likely
to represent phenomena found in the intraembryonic hematopoietic site
(Fig 1).18,19 In addition, the DAS 104-4 cell line is also
capable of maintaining fetal liver stem cells in a pluripotent state in spite of the fact that this line contains transcripts for a diversity of growth and differentiation factors (Table 1). This latter result
partially mimics the in vitro organ culture of isolated AGM
regions,17 where hematopoietic stem cells are not only
maintained in an undifferentiated state but are also modestly expanded.
The reasons for the apparent lack of amplification of these pluripotent cells in our in vitro system are not apparent but may be due to changes
in the AGM-derived endothelial cells during oncogenic transformation or
prolonged cell culture. An additional possibility is that other,
non-endothelial cell-type interactions may be required for
amplification, but not maintenance, of the pluripotent stem cell.
Alternatively, the amplification observed in the AGM organ culture
studies17 may be conversion of a constant number of stem
cells to a state that allows them to more efficiently home to the bone
marrow, a modulation that may not occur under our culture conditions.
In spite of this lack of stem cell expansion, the DAS 104-4 cell still
remains one of the few stromal cell lines capable of maintaining
competitively repopulating progenitor cells for several days in
vitro,25,26 and this result thus makes this cell line a
potentially interesting source for a factor(s) involved with stem cell
maintenance.
The heterogeneous hematopoietic activity of the DAS 104-4 and 104-8 cell lines may have relevance with respect to the choice between stem
cell maintenance and differentiation.39 To recapitulate, the DAS 104-8 cell line was highly effective at inducing stem cells to
differentiate along all three hematopoietic pathways, but the line was
ineffective at mediating stem cell self-renewal. In contrast, the DAS
104-4 cell line was poor at inducing, especially, the differentiation
of cells along the lymphoid pathway, but was highly effective at
maintaining repopulating stem cells. Interestingly, morphological
examination of hematopoietic cells expanded on these two stromal lines
showed a less mature population derived from DAS 104-4 versus DAS
104-8. These data are consistent with a hypothesis suggesting that
maintenance of the stem cell phenotype on the DAS 104-4 cell line may
be due to the lack of a factor that is necessary to induce these
pluripotent cells to become responsive to differentiation factors such
as EPO or, especially, IL-7. This conjecture seems unlikely, however,
because myeloid differentiation seems to readily occur on this cell
line. Alternatively, the DAS 104-8 cell line may lack a factor,
expressed by the DAS 104-4 line, which instructs the stem cell to
maintain a pluripotent state in the presence of these differentiation
factors. Of interest is the fact that repopulating stem cells are
maintained on the DAS 104-4 cell line despite the significant expansion
down the myeloid pathway of development. This suggests either that a
fraction of the initial cells are maintained in a quiescent, yet
pluripotent, state or that the initial undifferentiated cells produce
both committed and pluripotent cells after they divide. However, if the
fetal liver stem cells are maintained in a quiescent state on DAS
104-4, then this state must be induced, because stem cells from this
embryonic site are thought to be an actively dividing population.35,40 This has important implications for the
mechanisms of stem cell maintenance,42 and the system
described here may allow for an analysis of this important question.
Analysis of hematopoietic stem cell proliferation in the DAS 104-4 culture system indicated that these cells divided yet maintained a
pluripotent state (Table 4; Figs 6 and 7). This result is compatible with the self-renewal model (model A) illustrated in Fig 5, and it
suggests that stem cell division produces a pluripotent as well as a
differentiated progenitor offspring. These data are thus consistent
with two recent reports,29,43 which both showed that bone
marrow stem cells appeared to maintain a pluripotent state in spite of
the fact that they were in a nonquiescent (ie, cycling) situation. The
potential mechanism that might allow for the maintenance of
pluripotentiality after cell division can only be speculated upon at
this point, but it seems likely that some type of asymmetric
distribution of cell components is involved. Recent data in the
Drosophila system indicate that an asymmetric distribution of the
Prospero transcription factor by the Miranda protein is involved with
stem cell maintenance.44 It will be interesting to
determine if a similar mechanism is involved with the stem cell
maintenance observed here.
Examination of hematopoietic cytokine expression by RT-PCR revealed
that a number of these factors were significantly expressed in both
AGM-derived endothelial lines. Thus, both lines showed clear expression
of transcripts for stem cell factor, FLK2 ligand, M-CSF, IL-6, IL-11,
TPO, and LIF, consistent with the suggestion that these factors alone
may not be involved with stem cell maintenance on the DAS 104-4 cell
line, although the RT-PCR method does not detect quantitative
differences between expression of these factors. Although many studies
have attempted to use a diversity of known hematopoietic growth factors
to maintain repopulating stem cells in culture, only a recent study by
Ogawa and colleagues has shown, using the stringent competitive
repopulation assay, that stem cells incubated with either stem cell
factor or FLK2 ligand plus IL-11 are maintained in a pluripotent
state.37 Although these data suggest that these cytokine
combinations might explain the DAS 104-4 stromal cell maintenance of
repopulating stem cells, the fact that both the DAS 104-4 and 104-8 lines make all of these factors argues for a more novel combination of
factors to explain the differing stem cell maintenance abilities of
these two stromal cell lines. In support of this conjecture are data
showing that the cytokine transcripts found in yolk-sac-derived
endothelial cell lines, including YS CL72, which modestly maintains
repopulating stem cells, show a remarkably similar pattern to those
found for the two AGM-derived cell lines.23 The same
argument can be made for the delta-like protein, dlk. This epidermal
growth factor repeat-containing protein was shown to be expressed in a
fetal liver-derived stromal line that was capable of maintaining
competitively repopulating stem cells for several
months.26,36 In addition, it was shown that expression of
this protein in a stromal cell line incapable of mediating in vitro
hematopoiesis endowed the line with the ability to expand cobblestone
areas and maintain colony-forming activity and a low level of
repopulating stem cells. These data were thus consistent with the
suggestion that dlk was involved with the maintenance of early
progenitor cells, including stem cells.36 Although the data
reported here are not inconsistent with the ability of this factor to
maintain progenitor cell colony-forming activity in vitro, the fact
that both the DAS 104-4 and DAS 104-8 cell lines express the transcript
encoding this factor is inconsistent with the possibility that dlk
maintains repopulating stem cells, assuming the dlk transcript is
translated in both cell lines. The RT-PCR transcript analysis reported
here is thus consistent with the possibility that a novel factor(s) is
involved with the maintenance of competitively repopulating stem cells
by the DAS 104-4 cell line.
In summary, the results reported here show that endothelium derived
from the AGM region of day-11 murine embryos is capable of mediating
hematopoietic expansion along erythroid, myeloid, and lymphoid pathways
as well as maintaining competitively repopulating hematopoietic stem
cells, apparently by a self-renewal mechanism. These data concur with
morphological studies suggesting a role for the endothelium of the
dorsal aorta in regulating hematopoiesis in the AGM region, and they
are consistent with previous analyses showing a similar role for the
endothelium of the yolk-sac blood islands. It is thus likely, at least
in the case of embryonic hematopoiesis, that blood cell development
requires an interaction between hematopoietic progenitor cells and a
subset of the endothelium in a diversity of hematopoietic sites. The
cell lines described here may now provide for an opportunity to dissect
the molecules involved with the maintenance and differentiation of
hematopoietic stem cells during embryonic blood cell formation.
| |
FOOTNOTES |
Submitted February 4, 1998;
accepted April 1, 1998.
Address reprint requests to Laurence A. Lasky, PhD,
Department of Molecular Oncology, Genentech, Inc, 460 Pt San Bruno
Blvd, South San Francisco, CA 94080; e-mail: lal{at}gene.com.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Charles Hoffman for help with figures.
| |
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Abstract
Introduction
Abstract