Tài liệu Báo cáo khoa học: Development of a new method for isolation and long-term culture of organ-specific blood vascular and lymphatic endothelial cells of the mouse pdf

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

Tel: +81 3 5449 5754
E-mail:
(Received 27 November 2007, revised 13
February 2008, accepted 22 February 2008)
doi:10.1111/j.1742-4658.2008.06353.x
Endothelial cells are indispensable components of the vascular system, and
play pivotal roles during development and in health and disease. Their
properties have been studied extensively by in vivo analysis of genetically
modified mice. However, further analysis of the molecular and cellular phe-
notypes of endothelial cells and their heterogeneity at various developmen-
tal stages, in vascular beds and in various organs has often been hampered
by difficulties in culturing mouse endothelial cells. In order to overcome
these difficulties, we developed a new transgenic mouse line expressing the
SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary
expression system based on Cre ⁄ loxP recombination. tsA58T Ag-positive
endothelial cells in primary cultures of a variety of organs proliferate con-
tinuously at 33 °C without undergoing cell senescence. The resulting cell
population consists of blood vascular and lymphatic endothelial cells,
which could be separated by immunosorting. Even when cultured for two
months, the cells maintained endothelial cell properties, as assessed by
expression of endothelium-specific markers and intracellular signaling
through the vascular endothelial growth factor receptors VEGFR–2 and
VEGFR-3, as well as their physiological characteristics. In addition, lym-
phatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver
sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an
organ-specific endothelial characteristic was maintained. These results show
that our transgenic cell culture system is useful for culturing murine endo-
thelial cells, and will provide an accessible method and applications for
studying endothelial cell biology.
Abbreviations

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

[3] and mimics activated receptor tyrosine kinases [12],
which might obscure the analysis of endogenous recep-
tor-mediated signaling. Alternatively, tsA58T Ag, a
mutated SV40T Ag leading to temperature-dependent,
cell-type-independent cell proliferation [13,14], has
been used for ‘conditional immortalization’ of ECs of
wild-type and genetically modified mice [15–22].
Despite the fact that tsA58T Ag-directed immortaliza-
tion of ECs has been demonstrated, the method has
been under-utilized due to the specialized techniques
and expertise that are required for immunological iso-
lation of ECs [23,24] to prevent proliferation of
tsA58T Ag-expressing non-ECs.
Results and Discussion
Generation of a transgenic mouse line carrying
the CAG-bgeo-tsA58T Ag transgene
In order to circumvent the problems described above,
we developed a new transgenic mouse line expressing
tsA58T Ag under the control of a binary expression
system based on Cre ⁄ loxP recombination. To obtain a
transgenic mouse line with the potential to express
tsA58T Ag in a variety of tissues including ECs, we
exploited embryonic stem cell (ESC)-mediated trans-
genesis. Briefly, we constructed a transgene driven by
the CAG promoter [25] that expresses the b–geo gene
[26] in the absence of Cre recombinase, but expresses
the tsA58T Ag gene after Cre-mediated excision of
the lox P-flanked b–geo gene (Fig. 1). The plasmid
vector-free transgene was introduced into ESCs, and
G418-resistant clones were selected. We next per-

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

Serial passages every 2–3 days
at split ratio 1 : 3
day 20–30
day 0
βgeo
Fig. 1. An endothelial cell culture scheme based on endothelium-
specific expression of tsA58T antigen. pA, polyadenylation signal
sequence; Tg, transgenic mouse.
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 1989
hematopoietic cells (Fig. 1). The resulting T26 ⁄ Tie2–
Cre double-transgenic mice were born and grew
normally, but died suddenly within 6–12 weeks after
birth. To determine whether the expression of tsA58T
Ag was induced in ECs, we performed immunohisto-
chemistry using antibodies against the pan-EC mar-
ker, CD31, the lymphatic endothelial and liver
sinusoidal endothelial marker Lyve-1 (lymphatic vessel
endothelial hyaluronan receptor-1) [28–32] and SV40T
Ag. Immunostaining revealed that tsA58T Ag was
Brain
A
C
B
Uterus HeartLung Liver
SV40T CD31 SV40T Lyve-1
EmbryoYolk sac
SV40T CD31
ThymusCardiac valve
SV40T CD31

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-

tion during early development [33]. The presence of
these cells may cause a dysfunctional cardiac flow
and cause the sudden death of the transgenic mice,
although it remains to be determined whether T anti-
gen-expressing cardiac valves are functionally affected.
Endothelial cell culture from organs of
T26/Tie2–Cre double-transgenic mice
Following the demonstration of endothelium-specific
expression of tsA58T Ag in vivo, we performed primary
cell culturing (Fig. 1). Several organs (the brain, heart,
lung, liver and uterus) were obtained from 3-week-old
T26 single- or T26 ⁄ Tie2–Cre double-transgenic mice,
dissected, and dissociated by enzymatic digestion. Dis-
persed cell suspensions were plated onto gelatin-coated
plastic dishes and cultured at 33 °C (day 0 in Fig. 1).
For the initial 2 weeks, primary cells, including both
tsA58T Ag-negative cells (primarily fibroblasts) and
tsA58T Ag-positive cells, proliferated, and ECs could
barely be morphologically distinguished. However,
tsA58T Ag-negative cells gradually stopped proliferat-
ing and underwent senescence at about 2 weeks, as
assessed by morphology (data not shown). In contrast,
the remaining cells continued to proliferate over the
2 weeks and formed colonies that were distinguishable
under light-field microscopy (data not shown). tsA58T
Ag-negative senescent cells were progressively excluded
by serial passages. At day 30, the dishes consisted
almost exclusively of viable tsA58T Ag-positive cells
(Figs 1 and 3A). Cells obtained from T26 single-trans-
genic mice did not grow beyond 2–3 weeks (data not

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-

dino-2-phenylindole. (B,C) Immunostaining revealed that tsA58T
Ag-positive proliferating cells obtained from each organ maintained
expression of the endothelial-specific markers CD31 (B) and Lyve-1
(C). (D) 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine per-
chlorate (DiI)-labeled acetylated LDLs are taken up by these cells.
Bar = 50 lm (D, right panel) or 200 lm (all other panels).
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 1991
substantial proportion of the cell population from the
uterus (Fig. 3C) also stained positive for Lyve-1, indi-
cating that cell populations obtained from these tissues
were a mixture of blood vascular ECs (BECs) and
LECs. DiI-labeled acetylated LDLs were taken up by
all types of cell populations (Fig. 3D for brain and uter-
ine ECs; data not shown for others), but not by non-
endothelial NIH3T3 cells (data not shown), indicating
that the cells maintained the physiological characteristic
of acetylated-LDL uptake.
Lyve-1-positive liver sinusoidal endothelial cells
in vitro
Intriguingly, almost all of the cell population from the
liver were also positive for Lyve-1 (Fig. 3C), and Wes-
tern blot analysis revealed that they were Lyve-1-posi-
tive, prospero-related homeobox-1 (Prox-1)-negative
[34] ECs (Fig. 4B), suggesting that the population rep-
resented Lyve-1-positive liver sinusoidal ECs [30–32]
and maintained the property of Lyve-1 expression
in vitro. These results also suggest that Lyve-1 expres-
sion in liver sinusoidal ECs, reported as a marker of
differentiated organ-specific ECs [32] and a potential

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

VEGFR-3, was induced by VEGF-A, whereas phos-
phorylation of VEGFR-2, VEGFR-3 and MAPKs was
induced by VEGF-C, as reported in a previous study
using human primary LECs [36]. These results suggest
that growth factor signals were transduced properly
via endothelium-specific receptors in these cells. Mes-
enteric BECs and LECs (Fig. 5A,B) were also obtained
by the same strategy as illustrated in Figs 1 and 4, and
were treated with VEGF-A or VEGF-C (Fig. 5C).
MAPK and Akt phosphorylation were induced in both
BECs and LECs by stimulation with VEGF-A or
VEGF-C, indicating that the cultured ECs responded
to the endothelial growth factors.
Implications for tube formation-based assays and
transfection assays of tsA58T Ag-expressing ECs
We also examined whether the cells formed tube-like
structures on collagen gel. Both uterine BECs and
LECs could form tube-like structures (Fig. 4D). In
addition, an SV40-ori-containing plasmid carrying a
GFP expression cassette could be introduced by lipo-
fection and maintained for at least 5 days after trans-
fection as assessed by GFP expression (Fig. 4E). These
Fig. 4. Isolation and characterization of uterine BECs and LECs expressing tsA58T Ag. (A) Scheme for sorting of LECs from the uterine EC
population (days 30–40). A substantial proportion of uterine ECs were positive for Lyve-1 and Prox-1 (red and green on the top panel, respec-
tively), indicating that the uterine EC population was a mixed cell population of BECs and LECs. Lyve-1-positive LECs were isolated from
mixed ECs by magnet immunosorting using anti-Lyve-1 antibody. Scale bars ¼ 200 nm. (B) Western blotting revealed that Lyve-1-positive
uterine ECs maintained expression of Lyve-1, Prox-1 and VEGFR-3, indicating that they represent LECs. In contrast, liver ECs were positive
for Lyve-1 but not for Prox-1, indicating that they represent liver sinusoidal ECs. (C) The uterine LECs transduced growth-factor signals via
VEGFR-2 and VEGFR-3. IP, immunoprecipitation; IB, immunoblot; P-Y, phosphotyrosine. (D) The uterine BECs and LECs formed tube-like
structures. Bars = 200 lm. (E) SV40-ori-positive plasmids bearing GFP and drug-resistance genes were maintained in the uterine BECs and

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

LECs
LECs
BECs
Brain ECs
Heart ECs
Liver ECs
Lung ECs
Uterine ECs (Lyve-1+)
Uterine ECs (Lyve-1–)
Uterine ECs (unsorted)
50 kDa
75 kDa
250
kDa
150
kDa
100
kDa
100 kDa
75 kDa
50
kDa
100 kDa
75 kDa
Lyve-1
Prox-1
VEGFR-3
tsA58 T Ag
/ tublin
Lyve-1 DaAPI

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.

Thus, our system provides an accessible method to
examine the endothelial cell biology of the mouse, and
will accelerate the molecular and cellular analysis of
ECs and their heterogeneity in various vascular beds.
The early mortality of the T26 ⁄ Tie2–Cre double-
transgenic mice is a disadvantage for the generation of
T26 ⁄ Tie2–Cre double-transgenic mice with a mutation
in a gene-of-interest. In addition, non-ECs expressing
tsA58T Ag derived from Tie2-expressing hematopoietic
cells and embryonic endothelial cells may be contami-
nants in the present culture system. In order to overcome
these disadvantages, the use of tamoxifen-inducible
Cre-expressing mice, such as the VE–cadherin–CreER
T2
mice [37], may be preferable. Homozygous T26 trans-
genic mice may also facilitate production of these
animals, although it remains to be determined whether
the homozygous mice are viable and fertile.
Experimental procedures
Mice
C57BL ⁄ 6J mice and MCH:ICR mice were purchased from
CLEA Japan (Tokyo, Japan). Tie2–Cre transgenic mice
(B6.Cg-Tg(Tek-cre)12Flv ⁄ J, #004128) [27] were purchased
from the Jackson Laboratory (Bar Harbor, ME, USA). All
mice were housed under pathogen-free conditions. All of
the work with mice conformed to guidelines approved by
the Institutional Animal Care and Use Committee of the
University of Tokyo.
Construction of the transgene
Total RNA was extracted using TRIzol (Invitrogen, Carls-

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

p-MAPK
MAPK
p-Akt
Akt
BECs
LECs
BECsAB
C
LECs
CD31
SV40T
Lyve-1
SV40T
BECs
LECs
Prox-1
VEGFR-3
Lyve-1
Fig. 5. Isolation and characterization of mesenteric BECs and LECs
expressing tsA58T Ag. Mesenteric ECs were obtained from an
8-week-old T26 ⁄ Tie2–Cre double-transgenic mouse as illustrated in
Fig. 1. (A) CD31-positive, Lyve-1-negative BECs and CD31-positive,
Lyve-1-positive LECs expressing tsA58T Ag were separated by
magnetic immunosorting using anti-Lyve-1 antibody by the method
illustrated in Fig. 4A. Red, Lyve–1 and CD31; green, SV40T Ag.
Bar = 200 lm. (B) Prox–1 and VEGFR–3 were also expressed in
Lyve–1-positive ECs, confirming that they maintained lymphatic
endothelial properties. (C) VEGF–A and VEGF–C induced MAPK and
Akt activation in both populations of mesenteric ECs. Day 30–40
endothelial cells were cultured at 33 °C and used in (B) and (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

and the formation of embryoid bodies. One ESC clone
(T26) out of 48 G418-resistant clones was further exam-
ined for the presence of the tsA58T cDNA expression unit
and for widespread expression of b–geo in embryoid
bodies. To validate binary expression of the transgene, a
plasmid vector harboring CAG–Cre (kindly provided by
I. Saito, Institute of Medical Science, University of
Tokyo, Japan) was introduced into T26 ESCs by electro-
poration. The resulting b–geo-free subclones (T26d) were
selected and propagated for Western blot analysis of the
SV40T Ag.
For production of transgenic mice, T26 ESCs were
injected into B6 blastocysts. The resulting blastocysts were
transplanted into the uterus of pseudo-pregnant MCH:ICR
female mice. Chimeric male mice were then crossed with B6
female mice. T26 transgenic mice were back-crossed five
times or more with B6 mice and used for analysis. For
genotyping, PCR and ⁄ or X-gal staining of tail tips were
performed.
ESCs were maintained on a layer of irradiated, G418-
resistant mouse primary embryonic fibroblasts in high-
glucose (4.5 gÆL
)1
) Dulbecco’s modified Eagle’s medium
supplemented with 15% fetal bovine serum, 0.1 mm 2-mer-
captoethanol and a culture supernatant of leukemia inhibi-
tory factor (LIF)-producing BMT10 cells (kindly provided
by J. I. Miyazaki, Osaka University, Japan). Embryoid
bodies were obtained by culturing ESCs in the medium not
supplemented with LIF-conditioned medium on non-coated

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

as follows: anti-PECAM-1 (BD Pharmingen, Franklin
Lakes, NJ, USA), anti-Lyve-1 (R&D, Minneapolis, MN,
USA), anti-Prox-1 (Acris Antibodies, Hiddenhausen,
Germany), and anti-SV40 T Ag (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA). The corresponding secondary
antibodies labeled with horseradish peroxidase (HRP)
(Biosource, Invitrogen), AlexaFluor 488 or AlexaFluor 546
(Molecular Probes, Invitrogen) were used. Alternatively,
Histofine (Nichirei Biosciences, Tokyo, Japan) was used.
For double immunostaining using HRP-conjugated anti-
bodies for both of the secondary antibodies, sections
stained with the first primary and secondary antibody
were incubated in NaCl ⁄ P
i
containing 3% H
2
O
2
at room
temperature for 15 min prior to incubation with the
second primary antibody. The signal-enhancing TSA Plus
fluorescence system (Perkin-Elmer, Waltham, MA, USA)
for HRP-conjugated secondary antibodies was also used
for visualization. 4,6-diamidino-2-phenylindole (Molecular
Probes) was used for nuclear staining. Fluorescent micro-
scopic photographs were acquired using an Olympus IX70
microscope with DP70 imaging system (Olympus, Tokyo,
Japan).
For immunocytochemistry, cells were fixed on ice with
4% PFA in NaCl ⁄ P

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–

Akt polyclonal IgG (1 : 1000, Cell Signaling Technology)
and anti-Akt polyclonal IgG (1 : 1000, Cell Signaling
Technology). The secondary antibodies were swine anti-
goat IgG (HRP) (1 : 1000; Biosource), goat anti-rabbit IgG
(HRP) (1 : 1000; Cell Signaling Technology or GE Health-
care, Piscataway, NJ, USA; 1 : 2000 used for the detection
of rabbit primary antibodies purchased from Cell Signal-
ing Technology), and goat anti-rat IgG (HRP) (1:1000;
Biosource). Skim milk (5%) in Tris-buffered saline con-
taining 0.05-0.1% Tween 20 was used for blocking non-
specific antibody binding. Antibody-labeled bands were
visualized using an enhanced chemiluminescence kit (GE
Healthcare) and X-ray film (Fujifilm, Tokyo, Japan).
To detect phosphorylated tyrosine residues in VEGFR-
3, rabbit anti-VEGFR-3 polyclonal IgG (Santa Cruz) and
protein G (Calbiochem ⁄ Merck, Darmstadt, Germany)
were used for immunoprecipitation. Blocking One-P
(Nakarai-tesque, Kyoto, Japan) was used for blocking.
The membranes were incubated with mouse anti-phos-
photyrosine monoclonal IgG (4G10, 1 : 2000; Upstate) at
4 °C overnight, followed by incubation with sheep
anti-mouse IgG (HRP) (1 : 4000; GE Healthcare)
at room temperature for 1 h. ‘Can Get Signal’ solution
(Toyobo, Osaka, Japan) was used for dilution of both
antibodies.
Cell culture and isolation of lymphatic endothelial
cells from an endothelial-cell population
Tissues were collected, washed in NaCl ⁄ P
i
, and dissociated

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

MACS buffer containing 50 lgÆmL
)1
antibody against
Lyve-1 (MAB2125, R&D) and incubated on ice for
5–10 min. The primary antibody-labeled cells were washed
twice with 500–1000 lL MACS buffer, resuspended in
100 lL MACS buffer containing microbeads, and incu-
bated on ice for 15 min. Following a rinse with MACS buf-
fer, the cells were suspended with 500 lL MACS buffer
and applied onto MACS columns. Magnetically selected
positive cells and depleted cells were cultured independently
as lymphatic endothelial cells and blood vascular endothe-
lial cells, respectively.
Prior to activation with growth factors, cells were cul-
tured in endothelial cell basal medium 2 (EBM-2) basal
medium (Cambrex) without either serum or supplemental
growth factors for 16–18 h. Recombinant human VEGF-A
(Peprotech EC, London, UK) or rat VEGF-C (R&D) was
added to EBM medium at a final concentration of
100 ngÆmL
)1
. Cells were then incubated at 33 °C for 20 min
and harvested for analysis.
For the experiment shown in Fig. 5, mesenteric endo-
thelial cells were isolated by the method described above
with slight modification. Prior to treatment with growth
factors, cells were cultured in EBM-2 basal medium with
0.5% serum for 24 h. Cells were incubated with growth
factors at 33 ° C for 10 min and harvested for analysis.
Tube formation of tsA58T-expressing endothelial

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.
Acknowledgements

We thank Dr Jun-ichi Miyazaki (Osaka University,
Japan) for providing pCAGGS, pCAG-LIF and
BMT10 cells; Dr Hitoshi Niwa (RIKEN Center for
Developmental Biology, Kobe, Japan) for providing
pSA-bgeo; Dr Izumo Saito (Institute of Medical
Science, University of Tokyo, Japan) for providing
pCAG-Cre; Dr Ikuo Yana (Institute of Medical
Science, University of Tokyo, Japan) for help in per-
forming endothelial tube formation experiments. This
work was supported by grants from the Japan Society
for the Promotion of Science (to T. I.) and the Minis-
try of Education, Culture, Sports, Science and Tech-
nology, Japan (to T. Y., T. I., N. Y. and H. I.).
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