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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3106-3112
HEMATOPOIESIS
In vitro differentiation of endothelial cells from AC133-positive
progenitor cells
Ursula M. Gehling,
Süleyman Ergün,
Udo Schumacher,
Christoph Wagener,
Klaus Pantel,
Marcus Otte,
Gunter Schuch,
Philippe Schafhausen,
Thorsten Mende,
Nerbil Kilic,
Katrin Kluge,
Birgit Schäfer,
Dieter K. Hossfeld, and
Walter Fiedler
From the Department of Hematology/Oncology, Department of Anatomy,
Department of Neuroanatomy, Department of Clinical Chemistry, and
Department of Gynecology, University Hospital Eppendorf, Hamburg,
Germany.
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Abstract |
Recent findings support the hypothesis that the
CD34+-cell population in bone marrow and peripheral blood
contains hematopoietic and endothelial progenitor and stem cells. In
this study, we report that human AC133+ cells from
granulocyte colony-stimulating factor-mobilized peripheral blood have
the capacity to differentiate into endothelial cells (ECs). When
cultured in the presence of vascular endothelial growth factor (VEGF)
and the novel cytokine stem cell growth factor (SCGF), AC133+ progenitors generate both adherent and
proliferating nonadherent cells. Phenotypic analysis of the cells
within the adherent population reveals that the majority display
endothelial features, including the expression of KDR, Tie-2, Ulex
europaeus agglutinin-1, and von Willebrand factor. Electron
microscopic studies of these cells show structures compatible with
Weibel-Palade bodies that are found exclusively in vascular
endothelium. AC133-derived nonadherent cells give rise to both
hematopoietic and endothelial colonies in semisolid medium. On transfer
to fresh liquid culture with VEGF and SCGF, nonadherent cells again
produce an adherent and a nonadherent population. In mice with severe
combined immunodeficiency, AC133-derived cells form new blood vessels
in vivo when injected subcutaneously together with A549 lung cancer
cells. These data indicate that the AC133+-cell
population consists of progenitor and stem cells not only with
hematopoietic potential but also with the capacity to differentiate into ECs. Whether these hematopoietic and endothelial progenitors develop from a common precursor, the hemangioblast will be studied at
the single-cell level.
(Blood. 2000;95:3106-3112)
© 2000 by The American Society of Hematology.
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Introduction |
The formation of new blood vessels can be due to two
different processes. The first process, vasculogenesis, implies the in situ differentiation of endothelial cells (ECs) from hemangioblasts and
their subsequent organization into a primary capillary
plexus.1,2 The second process, termed angiogenesis, is
defined as the formation of new vessels by sprouting from preexisting
blood vessels.3,4 At present, primary differentiation of
ECs from hemangioblasts or angioblasts seems to be a process that is
restricted to early embryogenesis, whereas angiogenesis occurs both
during development and postnatal life. The hemangioblast has recently
been identified and was shown to be a transient cell stage that
develops early and is lost quickly during embryonic
development.5 It is important to note that these findings
are based on in vitro experiments with murine embryoid bodies and may
not necessarily reflect developmental steps in humans. The possibility
that hemangioblasts or more mature endothelial progenitors persist into
adult life whereby they may circulate, differentiate, and contribute to
the formation of new blood vessels remains to be determined. In this
context, previous studies have demonstrated the existence of mature ECs
in the peripheral circulation.6-15 Recently, Asahara et
al16 reported the presence of CD34+ endothelial
progenitors in human peripheral blood. The investigators showed that
the progenitors differentiated into ECs in vitro and were incorporated
into sites of neovascularization in vivo. However, no data were
provided that definitely proved the CD34+ progenitors to be
the source of the generated ECs or the endothelial nature of the
cultured cells. Nevertheless, a similar study by Shi et
al17 provided strong evidence that CD34+ cells
isolated from human bone marrow, umbilical cord blood, and granulocyte
colony-stimulating factor (G-CSF)-mobilized peripheral blood contains
endothelial progenitors. In a canine bone marrow transplant model,
the investigators could demonstrate that bone marrow-derived
CD34+ endothelial progenitors had the capacity to
line an implanted vascular prosthesis.
The molecular mechanisms responsible for vasculogenesis and
angiogenesis are not completely understood.18 Several
growth factors are involved in regulation of endothelial
differentiation, proliferation, migration, and formation of functional
vessels. Previous studies19-23 have shown that vascular
endothelial growth factor (VEGF) is one of the major inducers of
vasculogenesis and angiogenesis. VEGF signaling is mediated by two
receptor tyrosine kinases, called VEGF-R1 (flt-1) and VEGF-R2
(flk-1/KDR), respectively.20,21 Gene targeting
experiments demonstrated that a functional flk-1/KDR receptor is essential for the development of hematopoiesis and vasculogenesis.20
Stem cell growth factor (SCGF), a novel cytokine, exerts its action on
primitive hematopoietic progenitor cells.24 In combination with other hematopoietic growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin, SCGF stimulates the formation of erythroid and granulocyte/macrophage colonies, whereas
SCGF alone cannot induce colony formation.24 Whether this
early-acting growth factor has an influence on endothelial progenitors,
or even on the common precursor of endothelial and hematopoietic cells,
is currently unknown. Here, we describe the influence of SCGF in
combination with VEGF on proliferation and differentiation of enriched
progenitor cells from G-CSF mobilized peripheral blood.
In this study, we wanted to test whether AC133+ progenitor
cells can be induced to differentiate into ECs in vitro.
AC133+ cells represent a subset of CD34+ stem
and progenitor cells with known hematopoietic
potential.25-27 Because anti-AC133 antibody recognizes all
of the noncomitted CD34+ cell population whereas it does
not bind to mature ECs,25 we suggested that
AC133+ cells could be an ideal starting population to
generate ECs.
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Materials and methods |
Patient characteristics and specimens
The study included aliquots of leukapheresis products from 9 patients (3 patients with breast cancer, 1 patient with ovarian cancer,
3 patients with testicular cancer, and 2 patients with sarcoma)
undergoing high-dose chemotherapy and autologous hematopoietic progenitor cell support. The median age of the study group was 38 years
(range 21-57 years). All patients were enrolled in University Hospital
Eppendorf Bone Marrow Transplant protocols, and all patients gave their
informed written consent to participate in these treatment protocols
and to use part of their leukapheresis products for experimental
purposes. Peripheral blood progenitor cells were collected by
leukapheresis after mobilization of hematopoietic progenitor cell with
recombinant human G-CSF (5 µg/kg/day s.c.) for 7 days. Leukaphereses
were performed on days 5, 6, and 7 of this administration course.
Positive selection of AC133+ cells by magnetic cell
sorting
Mononuclear cells were isolated by density gradient centrifugation
over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) for 25 minutes at
500g and washed 3 times in phosphate-buffered
saline (PBS; Life Technologies, Karlsruhe, Germany) sequentially at
200g, 300g, and 500g to remove platelets.
Mononuclear cells were then resuspended in PBS, incubated with AC133
(monoclonal antibody; MoAb) conjugated super paramagnetic microbeads
(AC133 Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany),
washed, and processed through a MACS magnetic separation column
(Miltenyi Biotec) to obtain purified AC133+ cells. An
aliquot of the AC133+ cell fraction was analyzed to assess purity.
Liquid cultures of human AC133+ cells
Isolated AC133+ cells were cultured in
fibronectin-coated chamber slides (fibronectin from Life Technologies;
chamber slides from Becton Dickinson GmbH, Heidelberg, Germany) at a
density of 2 × 106 cells/mL in IMDM supplemented
with 10% fetal bovine serum (FBS; Sigma, Deisenhofen, Germany), 10%
horse serum (Sigma), and 10-6 mol/L hydrocortisone (Sigma).
The following cytokines were added to the media: SCGF (100 ng/mL; TEBU,
Frankfurt, Germany) and VEGF (50 ng/mL; TEBU). Cells were incubated for
up to 14 days at 37°C in 5% CO2. Additional feeding
was performed, depending on cell proliferation. Then supernatant was
removed by gentle pipetting and replaced with fresh medium.
Proliferating cells in the supernatant were counted, adjusted to
2 × 106 cells/mL, and transferred to fresh culture
chambers to further study their developmental potential. Analysis of
adherent and nonadherent cells derived from freshly isolated
AC133+ cells as well as analysis of corresponding cell
populations generated from transferred AC133-derived nonadherent cells
was performed separately.
Colony assays for multilineage hematopoietic and endothelial
progenitor cells
Purified AC133+ cells as well as cells cultured for 8 and 14 days were plated at
1 × 103-5 × 104 cells/mL in
semisolid growth media that consisted of 0.9% methylcellulose in IMDM,
30% FBS, 1% bovine serum albumin (BSA), 10-4 mol/L
mercaptoethanol, and 2 mmol/L L-glutamine (complete media from Cell Systems, St. Katharinen, Germany). In parallel experiments, cultures were stimulated either with a combination of hematopoietic growth factors, including stem cell factor (SCF; 50 ng/mL), interleukin 3 (IL-3; 20 ng/mL), IL-6 (20 ng/mL), G-CSF (20 ng/mL), GM-CSF (20 ng/mL), plus erythropoietin (3 U/mL; all from Cell
Systems) or with the combination of SCGF (100 ng/mL) and VEGF (50 ng/mL; both from TEBU). All cultures were performed in quadruplicate, incubated at 37°C in 5% CO2 and 95% humidity, and
scored after 14 days of culture using an inverted microscope.
Immunostaining of ECs
Freshly isolated AC133+ cells were spun at 500 rpm for 5 minutes onto glass slides by cytocentrifugation. Slides were air-dried for at least 24 hours and immunostained for the expression of the
following proteins: AC133 (Miltenyi Biotec); CD34, CD41a, CD41b (all
from Pharmingen, Hamburg, Germany); von Willebrand factor (vWF),
VE-Cadherin (Immunotech, Marseille, France); KDR (Sigma); P1H12 (MoAb
16 985; Chemicon International, Temecula, CA), and EN4 (Cell Systems).
All primary antibodies used were monoclonal. After blocking with 10%
AB-serum /PBS (AB-serum from Biotest, Dreieich, Germany) for 20 minutes, cytospins were incubated with the primary antibody for 45 minutes. This step was followed by incubation with a
biotinylated rabbit-anti-mouse secondary antibody (DAKO, Hamburg,
Germany) for 30 minutes and an additional incubation with a
streptavidine-alkaline phosphatase conjugate (DAKO) for 30 minutes,
combined with fast blue stain (Sigma) for visualizing positive
staining. The substrate reaction was performed in the presence of 1 mmol/L levamisole (Sigma).
Cultured cells in the chamber slides were washed twice with PBS and
fixed for 10 minutes with 4% paraformaldehyde. Specimens were then
incubated for 1 hour with the primary antibody as previously described.28,29 The following primary antibodies were used: anti-CD34, anti-CD31, anti-CD62E, anti-CD105, anti-CD106, anti-CD1a, anti-CD14 (all from Pharmingen), anti-VE-cadherin, anti-vWF (both from
Immunotech), anti-KDR, and anti-Tie-2 (both from Santa Cruz Biotechnology Inc, Heidelberg, Germany). With the exception of anti-Tie-2, all antibodies used were monoclonal. In brief, we used a
peroxidase anti-peroxidase complex. The peroxidase activity was
visualized by means of the Nickel-glucose oxidase technique. The
specimens were counterstained with Calcium Red. Controls included replacement of primary or secondary antibody with PBS, visualization of
peroxidase only, and incubation of cells with either normal rabbit
serum (for MoAbs; Sigma) or normal swine serum (for polyclonal antibodies; Sigma) in concentrations ranging from 0.1% to 0.01% instead of primary antiserum.
For the staining of putative endothelial colonies, semisolid medium
containing the colonies was overlayered with 4% paraformaldehyde for
10 minutes. Dissolved colonies were pipetted onto glass slides, air-dried for 24 hours, and analyzed for the expression of vWF (Immunotech), CD41a, and CD41b (Pharmingen). Immunostaining was performed, using either the fast blue stain or the peroxidase anti-peroxidase complex combined with the Nickel-glucose oxidase technique.
Flow cytometry
The purity of AC133-selected cells was determined for each
isolation. After lysis of erythrocytes with hemolytic buffer (0.155 mol/L NH4Cl, 0.012 mol/L NaHCO3, 0.1 mmol/L
EDTA, pH 7.2) for 2 minutes, 5 × 105 cells were
incubated with phycoerythrin (PE)-conjugated anti-AC133 MoAb (Miltenyi
Biotec). For two-color flow cytometry, FITC-anti-CD34 (Pharmingen) was
used as described.30 Isotype-matched mouse immunoglobulin
served as controls. All incubations were performed at 4°C. After
each incubation, cells were washed in PBS containing 0.1% BSA. Cells
were incubated with the MoAb for 30 minutes in the presence of normal
goat serum. Single- and two-color flow cytometric analyses were
performed, using a FACS SCAN flow cytometer (Becton Dickinson) and Cell
Quest software (Becton Dickinson). Each analysis included at least 5000 events. The percentage of AC133+ cells present was assessed
after correction for the percentage of cells reactive with an isotype
control. By using isotype-controls for PE and FITC, gates for
phenotypic analysis of CD34+ cells were set so that the
lower left panel contained at least 98% of the total cells analyzed.
Transmission electron microscopy
After 14 days of culture, adherent cells in the chamber slides were
trypsinized, fixed in 5.5% glutaraldehyde for 16 hours, postfixed for
2 hours in 1% OsO4, and dehydrated in 35% ethanol for 15 minutes. One percent of gelatin solution was then added to the cell
sediment, and the mixture of gelatin and cell sediment was placed at
-20°C for 4 minutes before dehydration in 50% ethanol. The cells
were further prepared for embedding in Epon 812 (Serva, Heidelberg,
Germany) as previously described.31 Thin sections were cut,
stained with uranyl acetate and lead citrate, and then examined, using
a Philips EM 300 (Einthoven, Netherlands) transmission electron microscope.
Culture and preparation of A549 lung cancer cells
The lung cancer cell line A549 used for in vivo studies was obtained
from the Institute of Molecular Biology and Cancer Research, University
of Marburg, Germany (a gift from K Havemann) and maintained in DMEM
with 10% FBS. A549 cells were harvested by trypsinization and washed
with PBS before subcutaneous injection into mice.
In vivo studies
After 14 days of liquid culture in the presence of VEGF and SCGF,
AC133-derived adherent cells were harvested by trypsinization and mixed
with AC133-derived nonadherent cells that were obtained by pipetting
the supernatant of the culture. Additionally, cultured tumor cells of
the lung cancer cell line A549 were prepared as described above. In
parallel experiments, SCID mice (bred at the University Hospital
Eppendorf) were injected subcutaneously with either
1 × 106 tumor cells, with
1 × 106 AC133-derived putative ECs, or with a
mixture of 1 × 106 tumor cells and
1 × 106 AC133-derived cells. All recipients
survived the procedure. Tumor growth in the different groups of mice
was measured weekly. At week 5 posttransplantation, all mice were
killed for histocytochemical analysis of the grown tumors.
Immunohistocytochemical staining of mouse tumors
Tissue blocks of tumors grown in SCID mice following either
subcutaneous injection of 1 × 106 A549 lung cancer
cells (10 tissue blocks) or subcutaneous injection of
1 × 106 A549 cells plus 1 × 106
AC133-derived putative ECs (10 tissue blocks) were fixed for 24 hours
with Bouin fixative at room temperature. After dehydration in ascending
alcohol concentrations, tissue blocks were embedded in
paraffin. Sections 6 µm in size were mounted onto
chrome-gelatin precoated slides. The sections were deparaffined,
rehydrated, and further processed for the visualization of CEACAM1 with
the MoAb 4D1/C2 (1:200, a gift from C. Wagener). We used an
amplification combination of the peroxidase anti-peroxidase and the
avidin-biotin-peroxidase complex technique. The peroxidase activity was
visualized by use of the Nickel-glucose technique. The specimens were
counterstained with Calcium Red.
The following controls were performed: (1) The primary, secondary, and
tertiary antibodies were replaced by PBS; (2) only the peroxidase was
visualized; and (3) instead of the primary antiserum, sections were
incubated with normal rabbit serum (Sigma) in concentrations ranging
between 0.1% and 0.01%.
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Results |
Characterization of AC133+-enriched cell fractions
As determined by flow cytometry, the median purity of positively
selected AC133+ cells after magnetic cell sorting was 72%
(range 62%-98%). A small portion of enriched AC133+ cells
did not coexpress CD34 (median 0.92%, range 0%-17.1%), whereas all
CD34+ cells coexpressed AC133. The obtained purities showed
a positive correlation with the initial content of AC133+
cells in the leukapheresis products.
Immunocytochemical analysis of the AC133+ cells revealed
that 1.0% ± 0.3% were KDR-positive. Less than 0.1% of the
AC133+ cells were found to express the endothelial markers
vWF, VE-Cadherin, and P1H12, respectively. These findings suggest
that the AC133+ population did not contain
notable numbers of mature ECs.
Development of AC133+ cells in liquid culture
AC133+ cells were grown for 14 days on
fibronectin-coated chamber slides in the presence of SCGF and VEGF.
Within 1-2 hours of culture under these conditions, cells became
adherent. During the first days of culture, adherent cells formed a
monolayer, consisting predominantly of small-sized cells. Single, large
cells with endothelial morphology were observed. No significant
proliferation was noted during this culture period. On day 6-7, a
proliferating population of round nonadherent cells occurred. These
cells were transferred to fresh chambers in which they again became
adherent and started to proliferate. Adherent cells in the initial
chambers also continued to expand so that proliferating cells needed to be transferred on average every third day. Growth curves showed an up
to 8-fold increase in the cell number after 14 days of culture (Figure
1). Morphological analysis of the adherent
cells at this time point revealed a heterogeneous cell population,
comprising small-sized round cells, large-sized round cells with
cytoplasmic granules, and large flat cells with endothelial morphology
that formed clusters in some areas of the chamber (Figure
2). In a few experiments, cultures were
continued for up to 3 months. Following stimulation with SCGF and VEGF
for 14 days, proliferation could then be maintained in the presence of
VEGF alone (data not shown), whereas VEGF alone could not induce
significant expansion of freshly isolated AC133+ cells.
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| Fig 1.
Growth curves of nonadherent cells generated from
AC133+ progenitor cells in liquid cultures with stem cell
growth factor and vascular endothelial growth factor.
Proliferating nonadherent cells were obtained with the supernatant, and
the number of cells per mL was counted. Three independent experiments
are shown. Evaluation of the cell count was performed at various time
points, depending on the individual growth pattern in each experiment.
Results of 3 independent experiments are shown. Abbreviations: Pat.
1-3, patient 1-3.
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| Fig 2.
Morphology of adherent cells cultured for 14 days in
liquid cultures in the presence of stem cell growth factor and vascular
endothelial growth factor.
All photographs are taken from the same experiment. (A) shows an area
with predominant small-sized round cells and flat elongated cells, (B)
cluster formation, (C) large-sized round cells with cytoplasmic
granules, and (D) area with exclusively small-sized round
cells. Original magnification ×100.
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Clonogenic potential of cultured cells
Purified AC133+ cells and proliferating cells were
plated in semisolid medium that was supplemented either with a
combination of hematopoietic growth factors known to support
multilineage colony formation or with SCGF and VEGF to stimulate the
formation of endothelial colonies. As shown in Figure
3, a linear relationship between input of
cells and number of colonies grown could be demonstrated. Interestingly, nonadherent cells cultured for 8 days (d8 cells) and
even for 14 days (d14 cells) in the presence of the recombinant growth
factors VEGF and SCGF still had clonogenic potential. Furthermore, day
8 cells were capable of inducing the growth of burst-forming unit
erythroid under the influence of a combination of hematopoitic growth
factors, indicating that this population still contained immature
progenitors. In comparison to freshly isolated AC133+
cells, day 8 cells as well as day 14 cells produced higher numbers of
putative endothelial colonies when stimulated with SCGF and VEGF
(Figure 3). Independent of the developmental stage of input cells,
colonies grown under the influence of SCGF and VEGF showed a morphology
(Figure 4B and D) that was different from
the morphology of the hematopoietic colonies (Figure 4A and C). To
determine whether these loosely packed colonies composed of very small
cells were of the endothelial lineage (so called colony-forming unit endothelial cell), colonies were analyzed by immunocytochemistry (see
below).
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| Fig 3.
Morphology of colonies generated from AC133-derived
cells.
In parallel experiments, cells cultured for 8 days (day 8 cells, A and
B) and 14 days (day 14 cells, C and D) in liquid cultures with stem
cell growth factor (SCGF) and vascular endothelial growth factor (VEGF)
were transferred to methylcellulose and stimulated either with the
hematopoietic growth factor combination SCF, interleukin 3 (IL-3), IL-6, granulocyte colony-stimulating factor,
granulocyte-macrophage colony-stimulating factor plus erythropoietin,
or with the combination of SCGF and VEGF. In response to the
hematopoietic growth factors, day 8 cells formed multilineage colonies,
including the formation of burst-forming unit erythroid (A); whereas
under the same culture conditions, day 14 cells produced colonies that
were restricted to the granulocyte-macrophage lineage (C). Colonies
grown from day 8 cells and day 14 cells, respectively, under the
influence of SCGF and VEGF displayed a morphology that was different of
the hematopoietic colonies and were assumed to represent endothelial
colonies. Original magnification ×100.
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| Fig 4.
Comparison of the clonogenic potential of freshly
isolated AC133+ cells (day 0 cells) and AC133-derived
proliferating cells cultured for 8 days (day 8 cells) and 14 days (day
14 cells), respectively, in liquid culture with stem cell growth factor
(SCGF) and vascular endothelial growth factor (VEGF).
As demonstrated in the 3 left panels of bars, a different number of day
0 input cells were tested to evaluate their capacity to form
endothelial colonies (colony-forming unit endothelial cell, CFU-EC).
The input of 5 × 104 AC133+ progenitor
cells was found to produce reliable numbers of putative CFU-EC and was
then compared with the input of 5 × 104 day 8 cells
and day 14 cells, respectively. The results of 4 independent
experiments are shown, each experiment was performed in quadruplicate.
Abbreviations: d0 = day 0 cells, freshly isolated AC133+
progenitor cells; d8 = day 8 cells, AC133-derived cells cultured for 8 days in liquid culture; d14 = day 14 cells, AC133-derived cells
cultured for 14 days in liquid culture; BFU-E = burst-forming unit
erythrocyte; CFU-GEMM, colony-forming unit
granulocyte-erythrocyte-macrophage-megakaryocyte; CFU-GM = colony-forming unit granulocyte-macrophage.
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Identification of ECs
The percentages and the staining patterns of cells expressing the EC
markers CD34, CD31, VE-Cadherin, KDR, Tie-2, Ulex europaeus agglutinin-1, and vWF, respectively, as determined by immunostaining of
adherent cells from day 14, are shown in Table
1 and Figure 5.
All of the cultured cells were positive for KDR, Tie-2, Ulex europaeus agglutinin-1, and vWF, whereas CD34, CD31, and
VE-Cadherin were expressed by a subset of 30%-50% of the cells.
Similar results were observed in 3 independent experiments. Because a
close developmental association might exist between the endothelial
lineage and macrophages/dendritic cells (data not shown), adherent
cells were examined for their reactivity with MoAbs against CD1a
(dendritic cells) and CD14 (monocytes). No staining for these markers
was observed. In addition, immunocytochemical analysis of the colonies
derived from day 8 and day 14 cells in the presence of SCGF and VEGF
revealed that all of the stained colony-forming cells expressed vWF
(Figure 6). Because the expression of vWF
is not specific for cells of the endothelial lineage, but also found on
early megakaryocytes, additional immunostaining with anti-CD41a MoAb
and anti-CD41b MoAb, targeting megakaryocytic antigens, was performed.
Colonies grown in the presence of VEGF and SCGF were found neither to
express CD41a nor to be positive for CD41b. The findings suggest that the colonies generated from the AC133-derived nonadherent cell population as well as the AC133-derived adherent cells in liquid cultures are of endothelial lineage.
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Table 1.
Percentages of positive cells in the adherent cell
fraction derived from AC133+ cells in liquid culture for
14 days in the presence of VEGF and SCGF
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| Fig 5.
Immunocytochemical analysis of AC133-derived adherent
cells using the nickel-glucose peroxidase anti-peroxidase technique.
(A) Negative control (AC133-derived adherent cells cultured for 14 days
in the presence of stem cell growth factor [SCGF] and vascular
endothelial growth factor [VEGF]). (B-G) AC133-derived adherent cells
after 14 days of culture with SCGF and VEGF and labeled with anti-CD34
monoclonal antibody (MoAb), anti-VE-Cadherin MoAb, anti-KDR MoAb,
anti-Tie-2 polyclonal antibody, anti-Ulex europaeus
agglutinin-1 MoAb, and anti-von Willebrand factor MoAb,
respectively. Original magnification
×100.
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| Fig 6.
Immunocytochemical analysis of colonies grown from day 8 cells in semisolid media.
Day 8 cells were cultured for 14 days in methycellulose supplemented
with stem cell growth factor and vascular endothelial growth factor.
Resulting colonies were then dissolved by fixation, transferred to
glass slides, and stained with anti-von Willebrand factor monoclonal
antibody. Original magnification ×100.
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To further confirm these results, transmission electron microscopy was
performed. As shown in Figure 7, adherent
cells cultured for 14 days displayed organelles morphologically
corresponding to Weibel-Palade bodies that are unique for vascular
endothelium.32 These findings from immunostaining together
with transmission electron microscopy analysis provide strong evidence
for the endothelial nature of AC133+-derived cells.
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| Fig 7.
Transmission electron microscopy photomicrographs of
adherent cells cultured in liquid cultures with stem cell growth factor
and vascular endothelial growth factor for 14 days.
Single-membrane bound structures, identified as Weibel-Palade bodies,
are indicated by arrows. Magnification × 30,000.
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In vivo studies
Xenogeneic transplantation studies were performed, using the SCID
mouse model. In this set of experiments, SCID mice were injected
subcutaneously with either 1 × 106 tumor cells from
the lung cancer cell line A549, with 1 × 106
AC133-derived putative ECs, or with a mixture of
1 × 106 A549 cells and 1 × 106
AC133-derived cells. Beginning at week 2 posttransplantation, a
different kinetic of tumor growth could be observed within the 3 groups
of mice. Animals injected with AC133-derived putative ECs alone did not
develop any tumors. Mice that were inoculated with A549 cells alone
developed tumors that reached half the size of tumors initiated with
both A549 cells and AC133-derived putative ECs.
Histochemical staining clearly showed a different morphology of
A549-induced tumor cell growth in comparison to the growth pattern of
tumors initiated with A549 cells together with AC133-derived putative
ECs. Subcutaneous tumors generated from A549 cells showed central
necrosis and less blood vessel formation (as quantified by counting the
number of blood vessels per tumor tissue section) than tumors grown in
the presence of A549 cells combined with AC133-derived cells. In tumors
initiated by injection of AC133-derived cells with A549 cells, no areas
of necrosis could be observed. Additional histocytochemical analysis,
using the human specific MoAb 4D1/C2 (anti-CEACAM1, a gift from C. Wagener), demonstrated positive staining of ECs in tumor blood vessels
generated with AC133-derived cells and A549 cells (Figure
8B and C), whereas staining of blood
vessels in tumors initiated with A549 cells alone was negative (Figure
8A).
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| Fig 8.
Histochemical staining of tumor grown in mice with severe
combined immunodeficiency with the human-specific anti-CEACAM1
monoclonal antibody 4D1/C2.
Panel (A) shows negative staining with 4D1/C2 in tumors grown after
subcutaneous injection of A549 lung cancer cells. Panels (B) and (C)
show positive staining of endothelial cells lining blood vessels in
tumors initiated with A549 lung cancer cells plus AC133-derived
putative endothelial cells. (D) Negative control (tumor tissue of mice
injected subcutaneously with A549 lung cancer cells and AC133-derived
putative endothelial cells). Tumor cells are indicated by arrowheads.
Endothelial cells are indicated by arrows. Original
magnification ×100.
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Discussion |
This study examined the capacity of AC133+ cells to
differentiate into the endothelial lineage. Hypothesizing from previous studies with CD34+ cells that the peripheral blood
progenitors may require an early-acting growth factor to induce
proliferation and that a lineage-restricted growth factor could then
trigger differentiation of proliferating cells into a certain
lineage,30 we stimulated the cells with the combination of
SCGF and VEGF. It is important to note that the addition of FBS and
horse serum was found to be essential for endothelial development in
vitro, indicating that serum contains further unidentified endothelial
growth stimuli. By using these culture conditions, we demonstrated that
enriched AC133+ cells continued to generate ECs. Adherent
cells displayed endothelial characteristics, including the expression
of CD34, CD31, VE-Cadherin, KDR, Tie-2, Ulex
europaeus-agglutinin-1, and vWF, respectively, and the presence
of Weibel-Palade bodies. Theoretically, endothelial development
observed in our study could have resulted from contaminating ECs within
the AC133+ population. However, the following
considerations argue against this possibility. First, peripheral blood
usually contains very low numbers of ECs.11 Second, no
AC133-CD34+ cells were found in the isolated
population, as revealed by flow cytometric analysis. Additionally,
<0.1% AC133+ cells stained positive for markers that are found on
mature ECs, such as VE-Cadherin, Tie-2, Ulex
europaeus-agglutinin-1, and vWF, respectively. Furthermore, it
is implausible that contaminating mature ECs give rise to endothelial colonies in semisolid medium.11,12
It has been discussed for a long time whether hematopoietic and ECs
arise from a common precursor, the hemangioblast. In a recent study,
Choi et al5 identified the hemangioblast within a murine
embryonic stem cell-derived precursor population. Although not proven,
it can be hypothesized that this finding in general is transferable to
the human system. Hence, it is no longer a question whether the
hemangioblast exists, but rather whether it can persist into adult
life. In our experiments, we show that, depending on the culture
conditions used, AC133+ cells from G-CSF-mobilized
peripheral blood can be differentiated along the endothelial or the
hematopoietic pathway. In our opinion, these AC133+ cells
fulfill many criteria of true hemangioblasts. Whether the enriched AC133+-cell population contains separate
progenitors for endothelial and hematopoietic cells or a common
precursor cannot be answered by the present study. Further
investigation of this question will require analysis at the single-cell
level, using multiparameter cell sorting. The coexpression of AC133,
Flk-1/KDR, and c-kit could serve as an ideal
marker combination for the selection of bipotent precursors.
We showed that, in the presence of SCGF and VEGF, progenitor cells
could be maintained for at least 14 days in vitro. Even after this
extended period of culture, proliferating cells still had the capacity
to form hematopoietic and endothelial colonies in semisolid media. Our
culture system may have great potential for the ex vivo expansion of
endothelial and hematopoietic cells (eg, to provide autologous ECs for
in vivo studies). Therefore, the observation that the
AC133+-cell population includes endothelial precursors may
not only help improve our understanding of the developmental
association of the hematopoietic and endothelial lineages in the adult
but also be of clinical relevance (eg, in the field of ischemic
disorders). In addition, several studies33-35 have shown
that tumor growth depends on the formation of new blood vessels in the
tumor tissue. It is currently believed that the underlying mechanisms
of tumor-induced blood vessel formation is angiogenesis.36
However, it cannot be excluded that malignant tumors are capable of
incorporating circulating ECs or their precursors for tumor
neovascularization. Peripheral blood stem cells that are increasingly
used for autologous and allogeneic transplantation following high-dose
chemotherapy may, in part, differentiate into ECs in vivo and so may
contribute to enhanced tumor growth.
We have studied tumor growth-promoting activity of AC133-derived
putative ECs in SCID mice that were injected subcutaneously with and
without tumor cells. We could demonstrate enhanced tumor growth when
tumor cells were injected together with AC133-derived cells.
Furthermore, we could show that injected AC133-derived cells formed the
endothelial layer of new blood vessels in these tumors. These findings
from the in vivo studies suggest that the AC133-derived population
contains ECs.
In summary, we have described the developmental potential of
AC133+ precursors from G-CSF-mobilized peripheral blood. We
showed that AC133+ cells, when cultured in the presence of
SCGF and VEGF, give rise to both hematopoietic and EC lineages. Further
identification and characterization of the AC133+-cell
subset with endothelial potential provides an unique approach to prove
the existence of the hemangioblast in the adult and its role in
postnatal blood vessel formation.
| |
Acknowledgments |
We thank P. Kühnl and C. Löliger for providing aliquots
from leukapheresis products. The authors would also like to thank D. Oestreich for assistance with photography. We are grateful to K. Havemann for providing the A549 lung cancer line.
| |
Footnotes |
Submitted June 29, 1999; accepted January 12, 2000.
Supported by grants from the Eppendorfer Krebs-und
Leukämiehilfe-Foundation, Hamburg, Germany, and by grant No. Fi
389/4-1 from the Deutsche Forschungsgemeinschaft (DFG).
Reprints: Ursula M. Gehling, Department of
Hematology/Oncology, University Hospital Eppendorf, Martinistraße 52, 20 246 Hamburg, Germany; e-mail: gehling{at}uke.uni-hamburg.de.
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.
| |
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Top
Abstract
Introduction