1 Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR
6543, Centre Antoine Lacassagne, 33 avenue de Valombrose, Nice, France
2 Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research,
Haartman Institute, University of Helsinki, Helsinki, Finland
* These authors contributed equally to this work
Author for correspondence (e-mail:
gpages{at}unice.fr
)
Accepted 7 March 2002
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Summary |
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Key words: Embryonic stem cells, Endothelial cells, Differentiation, Selection, Angiogenesis
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Introduction |
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In different situations, including advanced age, diabetes or
hypercholesterolemia, such neovascularization is impaired, due to the
reduction of vascular endothelial growth factor (VEGF) expression and to
endothelial cell dysfunction (Couffinhal et
al., 1999; Rivard et al.,
1999a
; Rivard et al.,
1999b
; Van Belle et al.,
1997
). Consequently, endothelial cell transplantation as a
potential method for treatment of patients with vascular defects has become
attractive and challenging in the past few years. This approach could also be
used to improve tissue grafting after injury or reperfusion of ischemic
tissues.
Several studies have described protocols to isolate and expand the
population of circulating EPCs from blood
(Asahara et al., 1997;
Kalka et al., 2000
;
Shi et al., 1998
). In
different animal models of neovascularization, EPCs were shown to participate
and enhance the formation of new blood vessels, and consequently increase
tissue salvage (Asahara et al.,
1999a
; Asahara et al.,
1999b
; Kawamoto et al.,
2001
; Takahashi et al.,
1999
). However, EPCs represent a small proportion (0.1-0.5%) of
circulating blood cells, and their expansion ex-vivo after harvest takes a
considerable amount of time. This aspect is a concern for treating
life-threatening conditions in patients. In this context, the development of a
strategy for large scale purification of vascular endothelial cells (ECs) is
of great interest.
Embryonic stem (ES) cells are derived from the inner cell mass of mouse
blastocysts and have the potential of generating all embryonic cell lineages,
including endothelial cells (Doetschman et
al., 1985; Risau et al.,
1988
). ES-derived endothelial cells express most known endothelial
cell markers, including CD31, VEGFR-2 (Flk-1), VE-cadherin, Tie-1 and Tie-2,
and have the potential to form pseudo-vascular structures when differentiated
into embryoid bodies (Vittet et al.,
1996
).
ES cells have a high proliferation rate and can easily be modified to
express transgenes. In this respect, they could be used to genetically select
ES-derived endothelial cells for grafting. Approaches employing genetic
selection have already been used in ES cells in order to select for
cardiomyocytes and neural-precursor cells
(Klug et al., 1996;
Li et al., 1998
). Importantly,
ES-derived cardiomyocytes were shown to form stable intracardiac grafts after
injection into mice (Klug et al.,
1996
).
In this report, we used a similar strategy to yield large numbers of ECs
from genetically modified ES cells. To this end, undifferentiated ES cells
were transfected with a fusion gene consisting of the tie-1 promoter,
specifically expressed in endothelial cells
(Iljin et al., 1999;
Korhonen et al., 1995
),
followed by sequences encoding the selection marker puromycin. Expression of
the puromycin-resistance gene in ES-derived endothelial cells facilitated
their purification during their in vitro differentiation. In addition, we
observed that the release of puromycin selection was accompanied by the
occurrence of cells positive for
-smooth muscle actin. We also show
that purified endothelial cells participate in tumor angiogenesis in vivo.
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Materials and Methods |
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The luciferase gene construct containing the VE-cadherin promoter
was generated by subcloning the SalI(blunted)-XhoI fragment
of the mouse VE-cadherin promoter [a gift from P. Huber, CEA,
Grenoble, France (Gory et al.,
1999)] into the SmaI-XhoI sites of the pGL2
basic vector.
A BamHI-KpnI fragment corresponding to the
puromycin-N-acetyl-transferase (puror) cDNA was introduced into the
same sites of the pGEM-3Zf(+/-) vector containing the
HindIII-ApaI fragment of the mouse tie-1 promoter
(Korhonen et al., 1995).
Antibodies
Rat antibodies against mouse CD31 (clone MEC 13.3), mouse CD34 (clone
RAM34) and mouse VE-cadherin (clone 75) were from BD Pharmingen (Los Angeles,
CA). Rat anti-VEGFR-2 was purchased from Clinisciences (France).
Anti--smooth muscle actin was purchased from Sigma (Saint-Louis,
MO).
Alexa Fluor-conjugated goat anti-mouse antibody, FITC and Alexa Fluor-conjugated streptavidin were from Molecular Probes (Eugene, OR). The biotin-conjugated donkey anti-rat antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). Biotin-conjugated anti-rabbit antibodies were from Amersham Pharmacia Biotech (Sweden). The FITC-conjugated rabbit anti-goat antibody was from Dako (Denmark).
Cell culture
OLA 129 ES cells, a gift from A. Smith (Centre for Genome Research,
Edinburgh, UK) were maintained in Dulbecco's modified Eagle's medium (DMEM)
with Glutamax-1 and NaPyruvate (Gibco BRL, Germany) containing 10% fetal calf
serum (FCS; Dutscher, France), 50 U/ml penicillin, 50 µg/ml streptomycin,
0.1 mM ß-mercaptoethanol and non essential amino acids (all reagents were
from Gibco BRL). This medium will be referred to as complete DMEM. The cells
were kept undifferentiated by the addition of recombinant leukemia inhibitory
factor (LIF) either purchased from Sigma or produced from COS cells
(Smith, 1991). ES cells
(5x106 cells) were transfected by electroporation with a gene
pulser (Eurogentec, Belgium) set at 500 V and 40 µF, in a total volume of
500 µl of PBS.
The established Chinese hamster lung fibroblast line CCL39 (American Type
Culture Collection) and PS120 MEK S222D
(Brunet et al., 1994) were
maintained in DMEM supplemented with 7.5% FCS, 50 U/ml penicillin and 50
µg/ml streptomycin. Bovine aortic endothelial cell (BAEC) were a generous
gift of H. Drexler (Max-Planck-Institut, Bad Nauheim, Germany). They were
maintained in low glucose DMEM supplemented with 5% FCS, 50 U/ml penicillin
and 50 µg/ml streptomycin. HEK 293 cells were maintained in DMEM
supplemented with 8% heat inactivated FCS, 50 U/ml penicillin and 50 µg/ml
streptomycin. Cells were maintained at 37°C in a humidified atmosphere of
5% CO2.
Transfection and luciferase assay
BAEC (1.5x105 cells/well), CCL39 (105
cells/well) and HEK 293 (2x105 cells/well) in 12-well dishes
were transiently transfected by CaPO4 precipitation with the
indicated plasmids (600 ng/well of the reporter plasmid and 300 ng/well of the
CMV ß-galactosidase as a control for transfection efficiency). Two days
later, cells were washed with cold PBS, and luciferase assays were performed
according to the Promega protocol and application guide. Briefly, cells were
lysed in lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1,
2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and
1% Triton X-100) for 15 minutes at room temperature, and the lysate was
cleared by centrifugation. Luciferase activity was quantified in a luminometer
(EG&G Wallac, Turku, Finland) in a buffer containing 20 mM Tricine, 1.07
mM (MgCO3)Mg(OH)2, 5H2O, 2.67 mM
MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 mM coenzyme A, 470 mM
luciferine, and 530 µM ATP. The protein concentrations were measured using
the Biorad protein assay (Hercules, CA).
In vitro differentiation and selection of ES-derived endothelial
cells
Two protocols were used, to induce growth and differentiation. In the first
one, freshly EDTA/trypsin (Biowhitaker, Walkersville, MD) dissociated ES cells
were aggregated into embryoid bodies (EBs) in hanging drops of complete DMEM
lacking LIF. 20 µl drops of cell suspension (4x104
cells/ml) were placed on the inside of lids of bacteriological Petri dishes.
The lids were then placed over PBS-filled Petri dishes and incubated at
37°C; this was designated day 0. After 3-4 days, the resulting EBs were
transferred to gelatin-coated 24-well tissue culture plates. In some cases,
the medium was supplemented with human rVEGF-165 either purchased from Sigma
or produced in our laboratory from Pichia pastoris after purification
on heparin binding affinity columns, using the pPICZA vector
(Scheidegger et al., 1999
)
kindly provided by K. Ballmer-Hofer (University of Zürich,
Switzerland).
In the second differentiation protocol, freshly dissociated ES cells were plated (3000 cells/cm2) in a 2D plane, on gelatin-coated dishes in complete DMEM lacking LIF. This method was used for selection of ES-derived endothelial cells. The concentration of puromycin to be applied on tie-1-puror expressing clones was determined using the non-resistant tie-1-EGFP cells as control cells. As tie-1-EGFP cells were killed by 0.25 µg/ml antibiotic, 1 µg/ml puromycin was added to the medium of differentiated tie-1-puror cells during 4 days. When indicated, selected cells were cultured on mouse laminin-1 (Sigma, 20 µg/ml) or in the presence of 5 ng/ml TGF-ß1 (TEBU) or 10 ng/ml PDGF-BB (TEBU).
For spheroid formation, purified endothelial cells were aggregated into hanging drops (20 µl drops, 105 cells/ml) for 5 days, in complete DMEM lacking LIF and in the presence of 10 ng/ml VEGF.
Genomic PCR for screening ES stable clone
PCR amplification on genomic DNA was carried out with the Taq PCR
master mix (Qiagen, France) containing Taq DNA polymerase, PCR buffer
(1.5 mM MgCl2) and dNTPs (200 µM for each dNTPs). After 35
cycles, the products were analyzed on ethidium-bromide-stained 1% agarose
gels.
For PCR detection of the tie-1-EGFP transgene, we used the following pair of primers: 5'-CCCAACCATCCCCAGATCTG-3' and 5'-TCCTCGCCCTTGCTCACCAT-3', that anneal to the 3' region of the tie-1 promoter and the 5' region of the EGFP cDNA, respectively. For PCR detection of the tie-1-puror transgene, we used the same tie-1 promoter primer and the primer 5'-GCGACCCACACCTTGCCG-ATG-3', which anneals to the 5' region of the puror cDNA. The annealing temperature was set at 57°C for both PCR reactions.
RT-PCR
Total RNAs from undifferentiated and from purified B9TP cells were isolated
using the RNA-now procedure (Biogentex, France). First-strand cDNAs were
generated using reverse transcriptase (Roche, France) and oligo dT (Gibco BRL)
using the manufacturer's instructions. For PCR amplification, cDNAs were
amplified using the Taq PCR master mix (Qiagen). The amplification parameters
were 95°C for 45 seconds, 55°C for 45 seconds and 72°C for 30
seconds, for 35 cycles. To ensure that the RT-PCR assay was semiquantitative,
the quantity of equivalent reverse-transcribed RNA chosen was in the linear
range of amplification. Hypoxanthine phosphoribosyl-transferase (HPRT) was
used as an internal standard. The sequences of the primers used are the
following:
Tumor formation in nude mice
The incorporation of ES-derived endothelial cells into sites of
neovascularization was analyzed by using a tumor transplantation model in
athymic nude mice (Harlan, France). To this end, 106 ES-derived
endothelial cells were co-injected subcutaneously with 106 PS120
MEK S222D cells into the left flank of male athymic nude mice (n=6).
For controls, 106 tumoral cells were injected into the right flank
of the same animal, and 106 ES-derived endothelial cells were
injected into the flank of five other mice.
Mice were euthanized after macroscopic tumor identification and frozen sections (4 µm) were processed for hematoxylin and immunofluorescence staining (see below).
Immunofluorescence staining
Differentiated ES cells, gelatin-bound embryoid bodies, spheroids and tumor
sections were fixed in 4% paraformaldehyde for 20 minutes at room temperature,
permeabilized with 0.2% Triton X-100 for 5 minutes and blocked in 2% BSA/PBS
for 2 hours. Cells were then incubated with the appropriate antibody for 1
hour at room temperature. After three washes in PBS, cells were incubated with
the appropriate fluorescent-conjugated secondary antibody or streptavidin
conjugates. Preparations were mounted in PBS:glycerol (1:9) and viewed under a
Leica microscope.
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Results |
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To this end, luciferase reporter vectors controlled by either the tie1 or the VE-cadherin promoter were transiently transfected in bovine aortic endothelial cells (BAEC) and in non-endothelial cells, namely the fibroblastic CCL39 and the epithelial HEK 293 cells. The constructs were co-transfected with a vector encoding the ß-galactosidase gene to monitor transfection efficiency. Relative luciferase activities were normalized to the values obtained with the empty pGL2-basic vector. Fig. 1 shows that the tie-1 promoter was active in BAEC and, to a lesser extent, in CCL39 cells. The low expression observed in CCL39 cells could reflect the lack of specificity reached in transient expression. In contrast, no activity could be detected in HEK 293 cells. A similar pattern was observed for the VE-cadherin promoter, but its relative activity was always lower than that of the tie-1 promoter. Transient expression of an EGFP reporter gene controlled by the same promoters gave similar results regarding their relative strength (data not shown). Based upon these results, we decided to test the cell-type specificity of the tie-1 promoter in the embryoid body model.
|
Endothelial specificity of the tie-1 promoter in
differentiating ES cells
When differentiated into embryoid bodies (EBs), ES cells reproduce the
stages of embryonic development, including vasculogenesis and angiogenesis
(Risau et al., 1988). We first
characterized vascular-like structures in wild-type EBs treated or not with 10
ng/ml VEGF, 5 and 8 days after leukemia inhibitory factor (LIF) removal to
induce differentiation. Three different endothelial markers were used: the
adhesion molecule CD31, VEGFR-2 and VE-cadherin.
Five days after LIF removal, control EBs contained numerous CD31-positive
(CD31+) cell clusters (Fig.
2A), which also stained for VEGFR-2 but not for VE-cadherin (not
shown). These cell clusters, in which CD31 staining was concentrated at
cell-cell contacts, probably represent colonies of endothelial progenitors.
After 8 days of differentiation, stained cells started to reorganize into
primitive cord-like structures, and CD31 redistributed from cell-cell contacts
to the entire cell membrane. The addition of VEGF accelerated the formation of
the vascular network, already apparent at day 5 and fully developed after 8
days of differentiation (Fig.
2A). Cells forming these networks also expressed VEGFR-2 and
VE-cadherin (Fig. 2B),
indicating that VEGF promoted endothelial cell maturation. The time course of
marker expression, in which VEGFR-2 and CD31 are expressed before VE-cadherin,
is in agreement with previous studies
(Hirashima et al., 1999;
Vittet et al., 1996
).
|
To test the cell-type specificity of the tie-1 promoter in the EB
model, undifferentiated ES cells were transfected with the tie-1-EGFP
construct. G418-resistant clones were screened by PCR to verify transgene
integration. Two independent clones, designated A1TG and E3TG, were
differentiated into EBs in the presence of VEGF. EGFP protein expression was
undetectable until day 6 or 7 of differentiation, whereas CD31 and VEGFR-2
appeared earlier, at day 4 (not shown). Again, this was in agreement with the
work of Vittet et al., who showed that expression of CD31 and VEGFR-2
transcripts preceded that of tie-1
(Vittet et al., 1996). As
shown in Fig. 3A, extensive
vascular-like structures were visualized by CD31 staining in 10-day-old EBs
derived from the A1TG clone (Fig.
3A, top). Cells forming these structures also expressed EGFP. All
EGFP-positive (EGFP+) cells were found in the vascular network,
thus demonstrating the endothelial specificity of the tie-1 promoter
in the EB model. CD31-positive (CD31+) cell clusters that were
negative for EGFP could be detected in some areas. As CD31 is an earlier
marker than Tie-1 in the process of endothelial cell maturation, these
clusters are likely to represent endothelial progenitors. In contrast, in EBs
derived from a selected clone that did not carry the tie-1-EGFP
transgene, vascular-like structures expressed only CD31 and not EGFP. The
punctate green staining apparent in these control EBs represent
autofluorescent dead cells (Fig.
3A, bottom).
|
To further check the tissue specificity of the tie-1 promoter, cells from embryoid bodies were enzymatically dissociated and replated on coverslips for 1 day. All EGFP+ cells also expressed CD31 as shown in Fig. 3B and all autofluorescent dead cells were lost. Changes in the cell morphology that correlate with EGFP expression are most obvious in this figure. Whereas CD31+EGFP- cells were rounded and tightly bound to each other, CD31+EGFP+ cells were more elongated and CD31 was found on the entire cell membrane, instead of being concentrated at cell-cell contacts.
As an alternative, we also tested another ES differentiation system that
was initiated by plating cells in 2D culture without LIF and in the presence
of VEGF. This model was previously shown to support endothelial cell
differentiation (Hirashima et al.,
1999; Nishikawa et al.,
1998
). Thus, A1TG cells were analyzed for the expression of CD31,
CD34 (another marker of the endothelial lineage) and EGFP after 8 days of
differentiation in the presence of VEGF. A population of cells expressing
CD31, EGFP (Fig. 3C) and CD34
(not shown) was identified. DAPI analysis shows the heterogeneity of the
differentiated population, which includes cells that express neither CD31,
EGFP (Fig. 3C) nor CD34 (not
shown). Since all EGFP+ cells also expressed CD31, this experiment
confirmed the endothelial specificity provided by the tie-1 promoter
in the 2D differentiation system.
Puromycin selection of ES-cell-derived endothelial cells
Stable clones carrying both tie-1-EGFP and
tie-1-puror transgenes were obtained by co-transfecting
the A1TG clone described above with the tie-1-puror
transgene and a hygromycin resistance gene. Hygromycin-resistant clones were
screened by PCR for transgene integration
(Fig. 4A). Note that positive
clones also carry the tie-1-EGFP transgene.
|
Two independent clones, B9TP and C4TP, were induced to differentiate in EBs and in 2D culture in the presence of 10 ng/ml VEGF. We found that puromycin addition to EBs was not appropriate for proper endothelial cell selection. First, the antibiotic could not efficiently penetrate into the whole 3D structures and, second, endothelial cells became surrounded by dying cells, which affected their growth capacity. Prior enzymatic dissociation of EBs was not efficient, since it damaged the cells and gave rise to only small numbers of selected cells. For these reasons, 2D cultures were preferentially used for endothelial cell selection.
Fig. 4B shows the efficiency of selection on differentiated B9TP cells after a 4-day puromycin treatment (1 µg/ml puromycin), started at day 7 of differentiation after LIF removal. Endothelial cells were visualized by both CD31 staining and EGFP expression. Nuclei were stained with DAPI to detect cells present in the field, including CD31-EGFP-cells. On the left panel, corresponding to 10-day-old differentiated B9TP cells without puromycin treatment, EGFP+CD31+ cells are encompassed in a monolayer of cells negative for both markers. After a 4-day puromycin-selection followed by 1 day without the antibiotic to allow cells to recover, 96-98% of cells were positive for CD31 and EGFP. Upon selection, EGFP+CD31+ cell spreading was less efficient and cells were more tightly associated (Fig. 4B). EGFP protein expression also decreased in some cells when selection was released, but all selected cells retained the CD31 marker, which was then found concentrated at cell-cell contacts. It is possible that environmental changes caused by the removal of all other cell types results in a partial dedifferentiation of endothelial cells towards an endothelial progenitor phenotype.
The C4TP clone gave results similar to B9TP, which clearly demonstrates the efficiency of ES-cell-derived endothelial cell purification through genetic selection using the tie-1 promoter. Subsequent analyses were performed on the B9TP clone.
Characterization of selected cells
Purified cells were further characterized for the expression of various
endothelial markers. We did not visualize VE-cadherin and VEGFR-2 proteins in
2D cultures with conventional immufluorescence techniques. Reasoning that the
detection of these antigens could be easier in pseudo-vascular structures than
in selected colonies, we aggregated purified CD31+EGFP+
cells into spheroids. After 3 days, spheroids were plated on gelatin, and
stained for endothelial markers 2 days later.
Fig. 5A shows that although
most cells were CD31+, EGFP expression was restricted to certain
cells organized into vascular networks. Such structures were also found to
express VE-cadherin, VEGFR-2 and CD34. However, VEGFR-2 and CD34 were also
expressed in cell clusters negative for EGFP. These clusters probably
represent endothelial progenitors.
|
Expression of three other endothelial markers, VEGFR-1, Tie-1 and ICAM-2, was monitored by a semi-quantitative RT-PCR approach. Fig. 5B shows that VEGFR-2 and VEGFR-1 were expressed at basal levels in undifferentiated cells. However, their expression was strongly increased in purified endothelial cells. In contrast, Tie-1 and ICAM-2 mRNA could be detected only in selected cells.
Interestingly, selected cells contained a small percentage (2-4%) of
CD31-/EGFP- cells when analyzed 24 hours after puromycin
removal. These cells were highly spread and expressed -smooth muscle
actin (
-SMA; Fig. 6).
Their proportion was strongly enhanced at high cell density and in spheroids.
In the light of recent results (Yamashita
et al., 2000
), we formulated the hypothesis that
-SMA+ cells may be derived from purified endothelial cells
themselves or their progenitors. Thus, we tested the effects of TGF-ß and
laminin-1, two known inducers of SMC differentiation
(Arciniegas et al., 1992
;
Hayashi et al., 1998
;
Hirschi et al., 1998
;
Morla and Mogford, 2000
) on
the purified population. Fig.
6A shows that providing 5 ng/ml of TGF-ß or plating cells
onto laminin-1 dramatically increased the number of
-SMA+
cells after 2 days. Laminin-1 had the strongest effect, even when cells were
cultured in the presence of 10 ng/ml of VEGF. PDGF, a potent growth factor for
SMC, was less efficient than TGF-ß or laminin-1, suggesting that the
occurrence of
-SMA+ cells in the purified endothelial cell
population was due to the differentiation of endothelial cells or their
progenitors towards an SMC phenotype rather than to their proliferation.
Finding cells that expressed both CD31 and
-SMA+
(Fig. 6B) further supported
this hypothesis.
|
Interestingly, and unlike previous results
(Yamashita et al., 2000), we
did not obtain a pure population of
-SMA+ cells, even after
a treatment with either TGF-ß1, laminin-1 or PDGF for several days. This
might be due to the different stages of cell differentiation at the time of
purification. Whereas Yamashita et al. selected VEGFR-2+
progenitors by flow cytometry at day 4 of differentiation, we waited until day
7 before adding puromycin for 4 additional days. It is possible that our
selection protocol allows endothelial cells or their progenitors to reach a
more advanced stage of differentiation that could prevent some of them from
entering the smooth muscle cell lineage.
Incorporation of selected endothelial cells into neovascularization
sites in vivo
To investigate the capacity of purified endothelial cells to participate in
the process of neovascularization in vivo, we used a tumor transplantation
model in athymic nude mice. For that purpose, we chose a clone of PS120 cells
expressing an active mutated form of MEK-1 (MEK S222D) conferring a weak
tumoral potential in nude mice (Brunet et
al., 1994). Six mice were injected subcutaneously with a mixture
of 106 selected endothelial cells and 106 PS120/MEK
S222D cells on the left flank, and 106 PS120/MEK S222D cells alone
on the right flank. Other controls were provided by five other mice injected
only with 106 endothelial cells. Mice injected with 106
endothelial cells alone never developed tumors, even after 5 months. PS120
MEK/S222D cells produced small tumors, if any, visible 3-4 weeks after
inoculation; their weight ranged from 0.3 to 70 mg, confirming their
previously described low tumorigenic potential. In contrast, co-injection of
selected endothelial cells and PS120 MEK S222D resulted in the formation of
tumors visible 2 weeks after injection. Three weeks after injection, their
weight was almost 10 times higher than control tumors (ranged from 75 to 610
mg). This experiment was performed three times and always gave similar
results.
When stained with hematoxylin and eosin, sections of 3-week-old co-injected and control tumors were homogenous and revealed no overt difference (not shown). These tumors were next analyzed for the presence of EGFP+ endothelial cells in the neovasculature (Fig. 7A). Immunofluorescence staining for CD31 showed that the extent of neovasculazisation was similar in both types of tumors. EGFP+ cells were found in microvessels at the periphery of tumors resulting from the co-injection of the two cell types. Surprisingly, there was no EGFP staining in the center of the tumors. Whether this means that injected cells are simply absent in this location or that the tie-1 promoter is no longer active in these particular cells is not known and will be discussed below.
|
We also did not detect any EGFP+ cells in tumors resulting from the injection of PS120/MEK S222D alone in the same mice, indicating that injected endothelial cells did not migrate to the other flank of the animals through the circulation.
As puromycin-selected cells could also give rise to -SMA+
cells in vitro, tumor sections were co-stained for
-SMA and CD31
(Fig. 7B). First, we detected
few, if any,
-SMA+ blood vessels in control tumors,
suggesting that these vessels were not mature. In contrast, most blood vessels
in co-injected tumors were lined by
-SMA+ cells. It is
unclear whether these
-SMA+ cells were of host or ES cell
origin. In addition, we detected, in the co-injected tumors only, areas rich
in
-SMA+ cells, probably derived from the injected
endothelial cells. The size difference between co-injected and control tumors
might thus be due to the growth of these
-SMA+ cells. This
hypothesis and alternatives will be discussed below.
Subcutaneously injected ES cells are known to give rise to teratomas, which
are tumors containing various types of differentiated tissues. Even if we
found that the expression of oct-4, a marker for undifferentiated ES cells
(Yeom et al., 1996), was
downregulated in purified cells (data not shown), we thought it was important
to check that selected endothelial cells do not give rise to teratomas in
vivo. Although we did not detect any tumor formation in mice injected with
106 endothelial cells alone, we repeated the experiment using
107 endothelial cells instead. This protocol did not result in
teratoma formation but hemangiomas did develop 3 weeks after injection (not
shown). Hemangiomas consisted of both blood-filled cavities and tumor masses,
with cavities surrounded by several layers of endothelial cells positive for
EGFP, CD31 and CD34, while the rest of the tumor mass contained mostly
CD34+ cells, with occasional CD31 staining.
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Discussion |
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A novel experimental model for easily monitoring angiogenesis and the
regulation of tie-1 promoter activity in embryoid bodies
Both tie-1 and VE-cadherin promoters were previously
found to drive endothelial-specific expression in vivo
(Gory et al., 1999;
Korhonen et al., 1995
). In the
present study, we compared the activity of these two promoters in various cell
lines. In agreement with previous reports
(Gory et al., 1999
;
Iljin et al., 1999
), both
promoters displayed functional activity in endothelial cell lines, but they
also exhibited a weaker activity in fibroblast cells in transient transfection
experiments. Whereas previous studies have not directly compared their
respective activity, our experiments demonstrate that the tie-1
promoter allows a more potent induction of reporter genes in endothelial
cells. Therefore, we chose the tie-1 promoter for the rest of the
study.
In spite of its weak activity in fibroblast cell lines in transient
experiments, the tie-1 promoter retained its cellular specificity
when expressed in differentiating ES cells. Reporter EGFP expression could be
detected from day 5 of differentiation in VEGF-treated EBs and from day 6 in
2D cultures. This occurred only 1 day later than the expression of CD31, an
early marker for endothelial precursors and endothelial cells
(Nishikawa et al., 1998;
Vittet et al., 1996
). In
embryoid bodies, EGFP expression was restricted to cells organized into
cord-like structures also expressing CD31. VEGF enhanced the number of both
EGFP+ cells and vascular-like structures. However, EGFP was absent
from clusters of CD31+VEGFR-2+ cells, which probably
represent endothelial progenitors. This confirms previous reports showing that
the VEGF receptor R2 is an early marker for endothelial cell precursors
(Hirashima et al., 1999
;
Vittet et al., 1996
;
Yamaguchi et al., 1993
),
whereas VE-cadherin and tie-1 transcript expression start at
a later stage of differentiation
(Hirashima et al., 1999
;
Vittet et al., 1997
;
Vittet et al., 1996
).
Interestingly, the onset of tie-1 promoter activity correlated with changes in cell morphology. Whereas CD31+EGFP- cells were round and formed clumps, CD31+EGFP+ cells displayed elongated processes and reorganized into pseudo-vascular structures. It is not clear whether these morphological changes are induced by Tie-1 expression or whether they are simply a part of a more general maturation program, but it would be interesting to analyze the effects of Tie-1 expression on endothelial cell migration and cytoskeleton organization.
The availability of ES cell clones carrying an integrated
tie-1-EGFP transgene is of great interest for several reasons. First,
we have confirmed the endothelial specificity of the tie-1 promoter
in the EB model. This finding is in agreement with recently published work
(Gustafsson et al., 2001).
Second, this model can be used to trace the activity of the tie-1
promoter in single cells within EBs whereas, in most studies published so far,
tie-1 expression was assayed only by RT-PCR in a mix of different
cell types. Third, tie-1-EGFP clones constitute a good model for
easily monitoring the effects of potential pro- or anti-angiogenic factors.
Two examples are given by our work, in which VEGF (this study) and bFGF (S.M.,
unpublished) strongly promoted the formation of EGFP+ vascular-like
structures. Finally, these cells can be used to generate mice expressing EGFP
in their vasculature. This may help to understand the molecular mechanisms
regulating tie-1 promoter activity, in particular, physiological and
pathological conditions.
A novel approach for selecting endothelial cells from differentiating
embryonic stem cells
We took advantage of the cellular specificity of the tie-1
promoter to genetically select endothelial cells from differentiating ES
cells, using puromycin as a selection marker. Such an approach has been
previously used for selecting neuronal and cardiac cells from a population of
differentiating ES cells (Klug et al.,
1996; Li et al.,
1998
). Starting from a few undifferentiated ES cells, large
numbers of cells expressing the endothelial markers CD31, CD34, VEGFR-1,
VEGFR-2, Tie-1, VE-cadherin and ICAM-2 can be obtained using this method.
While this work was under way, an alternative method was proposed to
isolate vascular progenitors by flow cytometry cell sorting based on VEGFR-2
expression (Yamashita et al.,
2000). VEGFR-2+ cells were isolated at day 4 of
differentiation and counter-selected for E-cadherin expression, which is a
marker for undifferentiated ES cells. VEGFR-2 is an early marker for
endothelial progenitors (Hirashima et al.,
1999
; Vittet et al.,
1996
; Yamaguchi et al.,
1993
) and we detected its expression by RT-PCR in undifferentiated
ES cells. This was in agreement with the weak vegfr-2 promoter
activity previously observed in undifferentiated ES cells by the same
experimental method, using lacZ as a reporter gene
(Hidaka et al., 1999
).
Although LIF removal is theoretically sufficient to induce the differentiation
of most ES cells over a few days, we thought it might be advantageous to wait
longer than 4 days to avoid contamination by undifferentiated ES cells. For
this reason, we chose a promoter regulating the expression of a late marker of
endothelial cell maturation, [i.e. tie-1
(Hirashima et al., 1999
;
Vittet et al., 1996
)]. Because
EGFP expression driven by the tie-1 promoter was not detectable
before day 5 or 6 in differentiating ES cells, we waited until day 7 to start
selection with puromycin. After 4 days selection, almost all cells (96-98%)
were CD31+EGFP+. The other cells expressed
-SMA,
a marker for smooth muscle cells. When maintained in culture,
-SMA+ cells later expressed transcripts for two other smooth
muscle markers, SM22 and calponin (S.M. and C.G., unpublished). Two known
inducers of SMC differentiation, TGF-ß1 and laminin-1, strongly increased
the proportion of
-SMA+ in the cell population. The fact
that some cells express both CD31 and
-SMA suggested the existence of
either a common progenitor for endothelial and SMC or a transdifferentiation
mechanism from endothelial towards SMC. This finding is reminiscent of
previous works by other groups (Arciniegas
et al., 1992
; DeRuiter et al.,
1997
; Hirschi et al.,
1998
; Yamashita et al.,
2000
). A few days after puromycin removal, CD31+ cells
selected with our method adopted a morphology more characteristic of
endothelial progenitors than more mature endothelial cells
(Bautch et al., 2000
). We
interpreted this phenomenon as being a possible dedifferentiation of
endothelial cells towards a more immature phenotype. Whether a
dedifferentiation process is required for further differentiation into smooth
muscle cells or whether transdifferentiation may occur between both lineages
needs to be explored in more depth. If the common origin for both lineages is
confirmed, our differentiation system might serve as a new model for studying
the effects of TGF-ß1 and laminin-1 on SMC differentiation.
Puromycin-selected endothelial cells are incorporated into
neo-vessels in vivo
Yamashita et al. showed that FACS-selected VEGFR-2+ cells
participated in the formation of the vascular network in the developing chick
embryo, and that they differentiated into both endothelial and vascular smooth
muscle cells in vivo (Yamashita et al.,
2000). In the present study, we showed that puromycin-resistant
cells could be incorporated into microvessels at sites of neovascularisation
in a tumor transplantation model in athymic nude mice, as revealed by EGFP
fluorescence. Surprisingly, there were no EGFP+ endothelial cells
in the vessels found in the core of the tumors, rather they were incorporated
into peripheric vessels. The simplest explanation for this phenomenon would be
that EGFP+ cells are not integrated at this particular location for
unknown reasons. Alternatively, the activity of the tie-1 promoter
might be downregulated in microvessels found in the tumor center. Another
member of the Tie family, Tie-2, was shown to be preferentially expressed in
vessels at the periphery of certain tumors
(Asahara et al., 1999a
;
Peters et al., 1998
). Because
Tie-1, like Tie-2, is involved in angiogenesis and blood vessel maturation, it
is tempting to speculate that the two promoters might be similarly
regulated.
We consistently found that the size of the PS120/MEK S222D tumors was
larger when tumor cells were co-injected with purified endothelial cells.
Providing exogenous endothelial cells may have promoted the vascularization
and subsequent growth of the tumors, as suggested by the presence of
EGFP+ cells in newly formed blood vessels. In this respect, it
should be noted that, although vascular densities were comparable in control
and co-injected tumors, overall angiogenesis was increased as the co-injected
tumors were larger. However, there might be other causes for this difference
in tumor size. First, we found numerous -SMA+ cells in
co-injected tumors. Although we do not have a marker for these cells (the
tie-1 promoter is not active in
-SMA+ cells), it is
possible that they are derived from the injected endothelial population, as
they were absent in the control tumors.
-SMA+ cells were
found both located around blood vessels and organized in cell aggregates. On
the one hand, blood vessel lining by
-SMA+ cells, observed
only in the co-injected tumors, may have an additional impact on angiogenesis
and subsequent tumor growth. On the other hand, the size of SMC-containing
aggregates could be increased by tumor-secreted cytokines, such as members of
the TGF family. Alternatively, injected endothelial cells may themselves
secrete growth factors, such as PDGF, triggering the survival and
proliferation of PS120 MEK S222D cells at the initial stage following
injection. Although it is likely that the tumor size difference results from a
combination of all these effects, the fact remains that puromycin-selected
endothelial cells can be incorporated in tumors at sites of
neovascularization. Importantly, experiments performed in mice indicated that
endothelial cells selected by our protocol did not produce teratomas over a
period of several months when subcutaneously injected in large amounts,
suggesting that the purified cells did not contain undifferentiated cells.
Further studies will be necessary to assess the ability of endothelial cells selected by our method to promote revascularization of wounded or ischemic tissues. Their potential ability to give rise to both endothelial and smooth muscle cells in vivo could be particularly advantageous if mature vessels are to be formed. If they prove to be competent, ES-cell-derived endothelial cells could provide an appealing alternative to adult stem cells in the context of pro-angiogenic therapy. As we have shown here, a large number of endothelial cells or progenitors can be obtained from few undifferentiated ES cells and they can also be easily genetically modified. Hence, a combination of stem cell and gene therapy could be of great benefit for treating degenerative diseases in which endothelial cells are involved.
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