Development of a new method for isolation and long-term
culture of organ-specific blood vascular and lymphatic
endothelial cells of the mouse
Takashi Yamaguchi, Taeko Ichise, Osamu Iwata, Akiko Hori, Tomomi Adachi, Masaru Nakamura,
Nobuaki Yoshida and Hirotake Ichise
Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Japan
As an indispensable component of the vascular system,
endothelial cells (ECs) have pivotal roles in develop-
ment and in health and disease [1]. Their properties
have been studied by a combination of in vitro analy-
ses of human primary ECs and in vivo analyses of
genetically modified mice exhibiting vascular pheno-
types. Human primary ECs are well-established
resources and are suitable for studying signal transduc-
tion and cellular physiology in vitro. However, it is still
difficult to control their gene expression strictly by
current overexpression and knockdown procedures. In
addition, they are not representative of all types of
ECs at various developmental stages and in vascular
beds [2]. On the other hand, the use of genetically
Keywords
Cre ⁄ loxP recombination; endothelial cell
culture; endothelial heterogeneity; SV40
tsA58 large T antigen; transgenic mouse
Correspondence
H. Ichise, Laboratory of Gene Expression
and Regulation, Center for Experimental
Medicine, Institute of Medical Science,
University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo 108-8639, Japan
Fax: +81 3 5449 5455

BEC, blood vascular endothelial cell; DiI, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; EC, endothelial cell; ESC,
embryonic stem cell; HRP, horseradish peroxidase; LDL, low-density lipoprotein; LEC, lymphatic endothelial cell; Lyve-1, lymphatic vessel
endothelial hyaluronan receptor-1; MACS, magnetic-activated cell separation; MAPK, mitogen-activated protein kinase; PFA,
paraformaldehyde; Prox-1, prospero-related homeobox-1; SV40T Ag, SV40 large T antigen; tsA58T Ag, large T antigen of SV40 mutant strain
tsA58; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.
1988 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS
modified mice has accelerated the understanding of
genetic mechanisms of endothelial development and
functions. However, further analyses of vascular phe-
notypes in vivo have been hampered by the compli-
cated relationship between ECs and non-ECs such as
mural, hematopoietic and mesenchymal fibroblast cells,
even though a conditional genetic modification such as
endothelium-specific knockouts can provide a partial
solution to this problem. Therefore, the isolation and
maintenance of murine endothelial cells from various
developmental stages and locations is important for
dissecting molecular and cellular mechanisms of endo-
thelial development and function.
Murine primary cells, including ECs, have a more
limited growth potential than human primary cells.
Thus, ‘immortalization’ techniques have been strongly
recommended for most analyses that require a large
quantity of transcripts, proteins or cells. For immortal-
ization of ECs, viral oncogenic proteins have been
used in previous studies. The polyoma middle T anti-
gen (PyMT Ag) allows selective proliferation of ECs in
mixed-cell populations [3–5], aiding in analyses of
genetically modified ECs in vitro [6–11]. However,
PyMT Ag causes endothelioma or hemangioma in vivo

formed 5-bromo-4-chloro-3-indolyl-b-d-galactopyrano-
side (X-gal) staining of embryoid bodies derived from
each clone and screened for the expression pattern of
b–geo in the embryoid bodies. Clone T26 had the most
favorable b–geo expression pattern among the G418-
resistant clones (data not shown). tsA58T Ag expres-
sion in ESCs after Cre-mediated excision was verified
by Western blotting (data not shown). The T26 trans-
genic mouse line was obtained through germline trans-
mission from chimeric mice. They grew normally, were
fertile, and did not display any defects.
Endothelium-specific expression of tsA58T Ag
in the transgenic mouse
We next crossed female T26 transgenic mice with
male Tie2–Cre transgenic mice [27], which removed a
loxP-flanked DNA fragment in endothelial cells and
T26 Tg
T26/Tie2-Cre Tg
Tie2-Cre Tg
tsA58T Ag-expressing
endothelial cell
pA
loxP loxP
pA
tsA58T
CAG
tsA58T
CAG
Enzymatic digestion of organs
Culture at 33 °C

Fig. 2. Expression pattern of tsA58T Ag in T26 ⁄ Tie2–Cre double-transgenic mice. (A) tsA58T Ag (red) was expressed in CD31-positive ECs
(green) of an E9.5 T26 ⁄ Tie2–Cre double-transgenic embryo and its yolk sac. (B) tsA58T Ag (red) was expressed in CD31-positive ECs (green,
left panels) and Lyve-1-positive ECs (green, right panels) of 3)6-week-old T26 ⁄ Tie2–Cre double-transgenic mice. Lyve-1-positive ECs were
not detected in the brain (top right), which is known to be an LEC-free organ. (C) tsA58T Ag (red) was also expressed in non-endothelial cells
of the thymic medulla and interstitial cells of the cardiac valve. Arrowheads indicate CD31-positive ECs (green). All micrographs are shown
at the same magnification. Scale bar = 50 lm.
A new method for mouse endothelial cell culture T. Yamaguchi et al.
1990 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS
expressed in CD31-positive ECs of E9.5 embryos
proper and yolk sacs (Fig. 2A). Postnatally, tsA58T
Ag was not only expressed in CD31-positive ECs in
the brain, heart, lung, liver and uterus (Fig. 2B), but
was also expressed in Lyve-1-positive lymphatic endo-
thelial cells (LECs) in the heart, lung and uterus, and
sinusoidal ECs in the liver of 3–6-week-old double-
transgenic mice (Fig. 2B), indicating that endothe-
lium-specific expression of tsA58T Ag was achieved
as expected. Despite the mortality of the young dou-
ble-transgenic mice, no gross abnormalities, such as
endothelial hyperplasia, dysplasia or bleeding, could
be found in live or dead double-transgenic mice.
However, immunostaining revealed that tsA58T Ag
was expressed in non-ECs, including a subset of thy-
mocytes and cardiac valvular cells (Fig. 2C). These
observations are comparable to those of previous
studies using the same Tie2–Cre transgenic mouse
line, which showed that recombination occurred in
hematopoietic cells as well as ECs [27], and that
cardiac valvular cells were derived from endothelial
cells through an endothelial-to-mesenchymal transi-

shown), confirming that tsA58T Ag-directed prolifera-
tion was only achieved by Cre-mediated excision.
Characterization of tsA58T Ag-expressing
endothelial cell populations
In order to examine whether the tsA58T Ag-positive
cells maintained EC properties, we first performed
immunocytochemistry for EC markers and assessed the
uptake of acetylated low-density lipoproteins (LDLs).
The cell populations derived from the brain, lung, heart,
liver and uterus stained positive for CD31 (Fig. 3B for
the brain, liver and uterus; data not shown for the lung
and heart), strongly suggesting that the tsA58T Ag-posi-
tive cells originated from ECs. A subset of the cell popu-
lations from the lung and heart (data not shown) and a
Liver Uterus
Brain Liver Uterus
Brain
DAPI
A
B
C
D
tsA58T Ag Merge
Brain
Uterus
Brain
Fig. 3. Endothelial cell culture from organs of T26 ⁄ Tie2–Cre dou-
ble-transgenic mice. (A) Proliferating cells obtained from the brain
were immunostained for SV40T Ag. Proliferating cells without
undergoing senescence were tsA58T Ag-positive. DAPI, 4,6-diami-

diagnostic marker of liver cancer and cirrhosis [30], is
regulated in a cell-autonomous manner and is irrevers-
ible in the culture conditions used in this study. These
cultured ECs might allow us to investigate more prop-
erties of liver sinusoidal ECs in health and disease.
Isolation and characterization of BECs and LECs
We next isolated LECs from the mixed cell population
by magnetic immunosorting using an antibody against
Lyve-1 (Fig. 4A). We used uterine ECs for this purpose
because they contained large numbers of Lyve-1-posi-
tive cells as assessed by immunostaining (Fig. 3C) and
further confirmed by double staining for Lyve-1 and
another lymphatic endothelial marker, Prox-1 [34]
(Fig. 4A). As shown by the immunostaining of posi-
tively sorted or depleted cells (Fig. 4A), Lyve-1-positive
ECs were enriched as expected. Western blot analysis
revealed that Prox-1 and vascular endothelial growth
factor receptor 3 (VEGFR-3), which is expressed pre-
dominantly in LECs [35,36], were also expressed in
Lyve-1-positive ECs (Fig. 4B), confirming that LECs
were obtained from the mixed EC population.
tsA58T Ag-positive BECs and LECs transduced
signals of endothelial growth factors
We further examined whether isolated ECs constitu-
tively expressing tsA58T Ag could respond to
angiogenic and lymphangiogenic growth factors.
Serum-depleted LECs were treated with vascular endo-
thelial growth factors A or C (VEGF-A or VEGF-C)
(Fig. 4C). Phosphorylation of VEGFR-2 and mitogen-
activated protein kinases (MAPKs), but not of

LECs for at least 5 days after transfection under drug-selection pressure. Bars = 200 lm. All cells were cultured at 33 °C, and day 40–50 ECs
were used for experiments shown in B–E.
A new method for mouse endothelial cell culture T. Yamaguchi et al.
1992 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS
250 kDa
150
kDa
250
kDa
250 kDa
250 kDa
150
kDa
50 kDa
37
kDa
50 kDa
37
kDa
IP: VEGFR-3
IB: P-Y
IP: VEGFR-3
IB: VEGFR-3
IB: P-VEGFR-2
IB: P-MAPK
IB: MAPK
IB: VEGFR-2
No treatment
VEGF-C
VEGF-A

DaAPI Lyve-1 Prox-1
CDa31 DaAPI
Magnetic
A
B
C
D
E
immunosorting
using anti-Lyve-1 Ab
Uterine ECs
Positive Negative
24

h 5 days 24

h 5 days
BECs
T. Yamaguchi et al. A new method for mouse endothelial cell culture
FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1993
results suggest that these cells can not only be used in
functional analyses based on tube-like formation, but
also used in gain-of-function, knockdown or rescue
analyses using expression vectors.
Taken together, these results demonstrate that
tsA58T Ag-positive BECs and LECs can be isolated
by a simple method using our transgenic system and
maintained at 33 °C without overt alterations in endo-
thelial properties, including specific gene expression,
physiological functions and intracellular signaling.

bad, CA, USA) from COS-7 cells harboring the wild-type
SV40T Ag gene (purchased from Health Science Research
Resources Bank, Osaka, Japan). The RNA was reverse-tran-
scribed using SuperScript II (Invitrogen) and used for clon-
ing the SV40T Ag cDNA. The cDNA encoding the wild-type
SV40T Ag and the 3¢ portion of the tsA58T Ag cDNA carry-
ing the A438V mutation were PCR-amplified from COS-7
cDNAs using the following primers: LTA-1F, 5¢-CTC
GAGATGGATAAAGTTTTAAACAGAG-3¢ and LTA-
1R, 5¢-TGAAGGCAAATCTCTGGAC-3¢ for the former,
and LTA–M2F, 5¢-CAGCTGTTTTGCTTGAATTATG-3¢
and LTA–2R, 5¢-GAATTCATTATGTTTCAGGTTCA
GGGG-3¢ for the latter. The PCR products were cloned into
the EcoRV site of pZErO-2 (Invitrogen). A XhoI–PvuII-
digested fragment of the wild-type SV40T Ag cDNA and a
PvuII–EcoRI-digested fragment of the ts58T Ag cDNA were
re-ligated and subcloned into XhoI–EcoRI-digested pZErO-2
and sequence-verified. The pCGX vector was constructed by
replacing the EcoRI–HindIII fragment of pCAGGS [25]
(kindly provided by J I. Miyazaki, Osaka University, Japan)
with the following fragments: b–geo cDNA with the poly-
adenylation signal sequence of the bovine growth hormone
gene derived from pSA–bgeo [26] (kindly provided by
H. Niwa, RIKEN Center for Developmental Biology,
VEGF-C
VEGF-A
No treatment
No treatment
VEGF-A
VEGF-C

A new method for mouse endothelial cell culture T. Yamaguchi et al.
1994 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS
Kobe, Japan), two synthetic lox P sequences and cloning
sites, a polyadenylation signal sequence of the mouse Pgk
gene derived from pGT-N28 (NEB, Ipswich, MA, USA),
and a portion of the multiple cloning site derived from
pMCS5 (MoBiTec, Goettingen, Germany). Briefly, the lox
P-flanked b–geo cassette was cloned under the control of
the CAG promoter, followed by several cloning sites
including a SwaI site, polyadenylation signal sequence of
the Pgk gene, and a portion of the multiple cloning site
of pMCS5. XhoI–EcoRI-digested tsA58T Ag cDNA was
blunted and cloned into the SwaI site of pCGX, and
the direction was verified by enzymatic digestion and
sequencing.
Generation of a transgenic mouse line carrying
the CAG–b–geo–tsA58T Ag transgene
SalI-digested pCGX harboring the tsA58T Ag cDNA was
resolved by electrophoresis, and a plasmid vector-free
fragment was electro-eluted, phenol-extracted, ethanol-pre-
cipitated, and dissolved in NaCl ⁄ P
i
.A10lg aliquot of
the transgene was introduced into E14.1 ESCs by electro-
poration. ESCs expressing the transgene were selected by
incubation for 7 days in medium containing a concentra-
tion of G418 (Invitrogen) of 400 lgÆmL
)1
. G418-resistant
colonies were picked and expanded for PCR genotyping

dishes.
Immunohistochemistry and
immunocytochemistry
Embryos and tissues were collected, fixed in 4% parafor-
maldehyde (PFA) overnight at 4 °C, processed in NaCl ⁄ P
i
containing 20% sucrose, and embedded in OCT (optimum
cutting temperature) compound (Sakura Finetec, Tokyo,
Japan). Sections (10–15 lm) of several tissues were cut
using a cryotome (Sakura Finetech). The sections were
mounted onto Matsunami adhesive silane-coated slides
(Matsunami, Osaka, Japan) and dried overnight at room
temperature. The dried specimens were rehydrated in
NaCl ⁄ P
i
and then antigen-retrieved for the detection of
SV40T Ag by incubation in NaCl ⁄ P
i
containing 0.1–
0.25% trypsin and 0.5 mm EDTA at 37 °C or room tem-
perature for 10–25 min. Prior to incubation with primary
antibodies, all sections were incubated in NaCl ⁄ P
i
or
methanol containing 3% H
2
O
2
at room temperature for
10–15 min. The primary antibodies used in this study were

i
for 10 min, incubated in methanol at
)20 °C for 20 min, and rehydrated in NaCl ⁄ P
i
. For detec-
tion of Prox-1, cells were further bleached and the TSA
Plus fluorescence system was used. For the detection of
other proteins, AlexaFluor-conjugated secondary antibodies
were used for visualization.
Western blot analysis
Cell lysates (40 lg, or 20 lg for lysates from cells shown
in Fig. 5) were loaded, resolved by SDS–PAGE, and
wet- or semi-dry-blotted onto poly(vinylidene difluoride)
T. Yamaguchi et al. A new method for mouse endothelial cell culture
FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS 1995
membranes (Bio-Rad, Hercules, CA,USA). Western blot anal-
ysis was performed using the following primary antibodies:
goat anti-Lyve-1 polyclonal IgG (1 : 500; Santa Cruz), rab-
bit anti-Prox-1 polyclonal IgG (1 : 500; Upstate ⁄ Millipore,
Billerica, MA, USA), rat anti-VEGFR-3 monoclonal IgG
(AFL4, 1 : 500; eBioscience, San Diego, CA, USA), rat
anti-VEGFR-2 monoclonal IgG (Avas2a, 1 : 500; eBio-
science), rabbit phospho-VEGFR-2 monoclonal IgG
(1 : 1000; Cell Signaling Technology, Danvers, MA, USA),
rabbit anti-SV40 large T antigen polyclonal IgG (1 : 1000;
Santa Cruz), rabbit anti-a ⁄ b-tubulin polyclonal IgG
(1 : 1000; Cell Signaling Technology), rabbit anti-phospho-
p42 ⁄ 44 MAPK polyclonal IgG (1 : 1000, Cell Signaling
Technology), rabbit anti-p42 ⁄ 44 MAPK polyclonal IgG
(1 : 1000, Cell Signaling Technology), rabbit anti-phospho–

by agitation in Hanks’ balanced salt solution containing
0.2% type IV collagenase or NaCl ⁄ P
i
containing 0.1%
trypsin for 30–60 min at 37 °C. After pipetting the solution
containing digested tissues several times, the enzyme-con-
taining buffer was thoroughly removed by centrifugation
and washing several times with NaCl ⁄ P
i
. Dissociated cells
were filtered through a 100 lm nylon mesh to remove
undissociated tissues, and cultured in microvascular
endothelial cell medium 2 (EGM-2MV) (Lonza, Basel,
Switzerland) at 33 °C, the permissive temperature for
tsA58T Ag. The initial cells attached to dishes were
passaged when sub-confluent to remove dead cells and
small pieces of tissues. Confluent cells were usually pas-
saged every 2–3 days at a split ratio of 1 : 3, but several
passages around day 20 were performed without splitting
because tsA58T-negative cells had undergone senescence,
decreasing the cell number.
Isolation of Lyve-1-positive endothelial cells was per-
formed using magnetic immunosorting. Magnetic-activated
cell separation (MACS) columns and MACS goat anti-rat
IgG microbeads (Miltenyi Biotec, Bergisch Galdbach,
Germany) were used according to the manufacturer’s pro-
tocol. Attached cells were trypsinized, collected and
counted. Cells (1 · 10
7
) were resuspended with 50 lLof

cells on collagen gel
Matrigel (9.5 mgÆmL
)1
; BD Pharmingen) was placed into
24-well dishes, and 2–4 · 10
4
endothelial cells were seeded
on the Matrigel and cultured for 24 h at 33 °C.
Uptake of DiI-labeled acetylated LDL into
tsA58T-expressing endothelial cells
DiI-labeled acetylated LDL was added to the culture med-
ium at a final concentration of 10 lgÆmL
)1
, and the cells
were incubated at 37 °C overnight. Dishes were washed
with NaCl ⁄ P
i
and observed by fluorescent microscopy.
A new method for mouse endothelial cell culture T. Yamaguchi et al.
1996 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS
NIH3T3 cells (Health Science Research Resources Bank,
Osaka, Japan) were used as a negative control.
Transfection of plasmids harboring a
GFP-expressing cassette into
tsA58T-expressing endothelial cells
Transfection was performed using Lipofectamine LTX and
pcDNAÔ6.2-GW ⁄ miR-neg control plasmid (Invitrogen)
according to the manufacturer’s protocol. GFP signals were
used to assess transfection and expression of the plasmid.
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