16316465.conll

BackgroundUsing O
antibodies O
to O
specific O
protein O
antigens O
is O
the O
method O
of O
choice O
to O
assign O
and O
identify O
cell O
lineage O
through O
simultaneous O
analysis O
of O
surface O
molecules O
and O
intracellular O
markers. O
Embryonic O
stem O
cell O
research O
can O
be O
benefited O
from O
using O
antibodies O
specific O
to O
transcriptional O
factors/markers O
that O
contribute O
to O
the O
"stemness" O
phenotype O
or O
critical O
for O
cell O
lineage.ResultsIn O
this O
report, O
we O
have O
developed O
and O
validated O
antibodies O
(either O
monoclonal O
or O
polyclonal) O
specific O
to O
human B-Species
embryonic I-CellType
stem I-CellType
cell I-CellType
antigens O
and O
early O
differentiation O
transcriptional O
factors/markers O
that O
are O
critical O
for O
cell O
differentiation O
into O
definite O
lineage.ConclusionThese O
antibodies O
enable O
stem O
cell O
biologists O
to O
conveniently O
identify O
stem O
cell O
characteristics O
and O
to O
quantitatively O
assess O
differentiation. O
Although O
the O
stem O
cell O
concept O
was O
introduced O
decades O
ago, O
to O
date, O
stem B-CellType
cells I-CellType
can O
only O
be O
defined O
functionally, O
not O
morphologically O
or O
phenotypically. O
Two O
functions O
define O
stem B-CellType
cells. O
Firstly, O
they O
are O
self-renewing, O
thus O
able O
to O
propagate O
to O
generate O
additional O
stem B-CellType
cells. O
Secondly O
they O
can O
differentiate O
into O
various O
progenitor B-CellType
cells, O
which O
commit O
to O
further O
maturation O
along O
a O
specific O
lineage. O
While O
stem B-CellType
cells I-CellType
can O
be O
best O
defined O
functionally, O
a O
good O
number O
of O
molecular O
markers O
have O
been O
used O
to O
prospectively O
identify O
various O
stem O
cell O
populations. O
Although O
the O
functional O
importance O
of O
many O
of O
these O
antigens O
remains O
unknown, O
their O
unique O
expression O
pattern O
and O
timing O
of O
expression O
provide O
a O
useful O
tool O
for O
scientists O
to O
identify O
as O
well O
as O
isolate O
stem B-CellType
cells. O
Embryonic B-Anatomy
stem I-CellType
cells I-CellType
(ESC), O
derived O
from O
the O
inner B-Anatomy
cell I-Anatomy
mass I-Anatomy
of O
pre-implantation O
embryos, O
have O
been O
recognized O
as O
the O
earliest O
stem O
cell O
population O
[1,2]. O
This O
pluripotent B-Anatomy
population I-Anatomy
can O
differentiate O
into O
all O
somatic B-Anatomy
tissue I-Anatomy
including O
germ B-CellType
cells. O
In O
the O
case O
of O
human B-Species
ESC, O
they O
can O
differentiate O
into O
some O
extra-embryonic B-CellType
derivatives I-CellType
as O
well. O
Like O
mouse B-Species
ESC, O
human B-Species
ES I-CellType
cells I-CellType
can O
be O
maintained O
and O
propagated O
on O
mouse B-Species
fibroblast I-CellType
feeders O
for O
extended O
periods O
in O
media O
containing O
basic B-GeneProtein
fibroblast I-GeneProtein
growth I-GeneProtein
factor I-GeneProtein
(bFGF) O
[3]. O
Gene O
expression O
of O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
has O
been O
investigated O
among O
several O
ES O
cell O
lines O
by O
a O
variety O
of O
techniques. O
They O
include O
comparison O
with O
databases, O
reverse O
transcriptase-polymerase O
chain O
reaction, O
focused O
cDNA O
microarrays, O
and O
immunocytochemistry. O
A O
list O
of O
molecules O
comprised O
of O
known O
ES-specific O
or O
-highly O
expressed O
genes O
and O
candidates O
that O
can O
serve O
as O
markers O
for O
human B-Species
ESCs I-CellType
and O
may O
also O
contribute O
to O
the O
"stemness" O
phenotype O
has O
been O
established O
[3-11]. O
For O
example, O
pluripotent B-CellType
ESC I-CellType
can O
be O
characterized O
by O
high O
level O
expression O
of O
Oct3/4 B-GeneProtein
(POU O
domain, I-GeneProtein
class I-GeneProtein
5, I-GeneProtein
transcription I-GeneProtein
factor I-GeneProtein
1, O
Pou5f1) O
and O
Nanog, O
which O
are O
a O
member O
of O
POU O
domain O
and O
homeobox O
transcription O
factors O
respectively. O
A O
critical O
amount O
of O
Oct3/4 B-GeneProtein
and O
Nanog B-GeneProtein
expression O
is O
required O
to O
sustain O
stem-cell O
pluripotency O
and O
both O
of O
these O
markers O
are O
downregulated O
as O
cells O
differentiate O
in O
vitro O
and O
in O
vivo O
[4-9]. O
Antibodies O
to O
Oct3/4 B-GeneProtein
which O
cross O
react O
with O
human B-Species
Oct I-GeneProtein
3/4 I-GeneProtein
have O
been O
widely O
used O
to O
monitor O
the O
presence O
of O
undifferentiated O
ESC. O
No O
single O
marker O
however O
is O
sufficient O
or O
unique O
for O
identifying O
ESCs. O
Oct3/4 B-GeneProtein
for O
example O
is O
expressed O
by O
germ B-CellType
cells I-CellType
and O
may O
be O
expressed O
by O
specific O
populations O
later O
in O
development. O
Likewise, O
Nanog B-GeneProtein
has O
been O
shown O
to O
express O
in O
other O
tissues. O
We O
and O
other O
have O
noted O
however, O
that O
while O
no O
single O
marker O
is O
sufficient O
a O
constellation O
of O
positive O
and O
negative O
markers O
can O
in O
concert O
unambiguously O
allow O
one O
to O
define O
the O
state O
of O
ESC O
cultures O
and O
that O
surface O
markers O
in O
combination O
can O
be O
used O
to O
prospectively O
sort O
for O
ESC. O
Based O
on O
published O
data O
at O
the O
level O
of O
gene O
expression, O
we O
have O
cloned O
a O
number O
of O
candidate O
marker O
genes. O
We O
have O
also O
expressed O
the O
recombinant O
protein O
and O
generated O
a O
panel O
of O
monoclonal O
or O
polyclonal O
antibodies O
to O
these O
proteins. O
Using O
these O
antibodies O
we O
have O
confirmed O
the O
specificity O
and O
selectivity O
of O
these O
antibodies O
on O
several O
ESC O
lines O
and O
established O
their O
utility O
as O
stem O
cells O
markers. O
Our O
results O
confirm O
the O
expression O
pattern O
and O
timing O
of O
these O
cell O
markers O
at O
the O
protein O
level, O
whereas O
previous O
data O
reported O
at O
the O
level O
of O
gene O
expression. O
Characterization O
of O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
and O
differentiated O
EBs B-Anatomy
by O
antibodiesAll O
monoclonal O
antibodies O
were O
initially O
selected O
for O
their O
abilities O
to O
recognize O
recombinant O
proteins O
in O
direct O
ELISAs. O
A O
subset O
were O
also O
tested O
by O
Western O
Blot O
analysis O
using O
recombinant O
proteins O
and O
cell O
lysate O
to O
confirm O
binding O
to O
a O
single O
epitope. O
The O
best O
clone O
was O
later O
screened O
for O
its O
applications O
for O
immunocytochemistry O
and O
flow O
cytometry O
using O
various O
cell O
lines. O
Human B-Species
peripheral B-Anatomy
blood I-Anatomy
platelets B-CellComponent
were O
used O
for O
screening O
mouse B-Species
anti-human O
CD9 B-GeneProtein
antibody. O
MCF-7 B-CellLine
cells O
were O
used O
for O
screening O
mouse B-Species
anti-human O
E-Cadherin B-GeneProtein
and O
PODXL B-GeneProtein
(podocalyxin-like) O
antibodies. O
MG-63 B-CellLine
cells O
were O
used O
for O
screening O
mouse B-Species
anti-human O
GATA1 B-GeneProtein
(GATA O
binding I-GeneProtein
protein I-GeneProtein
1) O
antibody. O
Beta-TC6 B-CellLine
cells O
were O
used O
for O
screening O
for O
mouse B-Species
anti-human/mouse O
PDX-1 B-GeneProtein
(pancreatic O
duodenal I-GeneProtein
homeobox-1) O
antibody. O
NTERA-2 B-CellLine
cells O
were O
used O
for O
screening O
mouse B-Species
anti-human O
Oct3/4 B-GeneProtein
and O
SOX2 B-GeneProtein
(sex-determining O
region I-GeneProtein
Y-box I-GeneProtein
2) O
antibodies. O
All O
polyclonal O
antibodies O
were O
affinity-purified O
using O
recombinant O
proteins O
and O
validated O
by O
direct O
ELISAs O
and O
Western. O
Caco-2 B-CellLine
cells O
were O
used O
for O
validation O
of O
goat O
anti-human O
GATA6 B-GeneProtein
antibody O
and O
NTERA-2 B-CellLine
cells O
were O
used O
for O
validation O
of O
goat O
anti-human O
Nanog B-GeneProtein
and O
anti-human O
Oct3/4 B-GeneProtein
antibodies O
(Summarized O
in O
Table O
1).Table O
1Summary O
list O
of O
antibody O
verification O
by O
western O
blot.AntibodySample O
used O
for O
analysisMol. O
Wt. O
(KD)Gt O
× O
hBrachyurymouse O
ES-derived O
EB O
lysate48Ms O
× O
hDPPA5N/AN/AGt O
× O
hGATA6Caco2 O
cell I-CellLine
lysate65Gt O
× O
hNanogNTERA-2 O
cell I-CellType
lysate33Gt O
× O
hOct O
3/4NTERA-2 O
cell I-CellType
lysate39Gt O
× O
hPDX1beta-TC O
6 I-CellLine
cell I-CellType
lysate32Gt O
× O
hSOX17mouse O
ES-derived O
EB O
lysate45Ms O
× O
hCD9PBMC25Rt O
× O
hGATA-1N/AN/AMs O
× O
hE-CadherinMCF-7 O
cell I-CellLine
lysate97Ms O
× O
hPODXLMCF-7 O
cell I-CellLine
lysate57Ms O
× O
hSOX2NTERA-2 O
cell I-CellType
lysate36N/A: O
1. O
DPPA5 B-GeneProtein
is O
still O
being O
subcloned. O
Only O
Elisa O
verification O
is O
available.2. O
The O
clone O
for O
GATA-1 B-GeneProtein
(MAB1779) O
does O
not O
work O
for O
Western O
blot O
application O
but O
is O
useful O
for O
IHC, O
The O
clone O
picked O
for O
Western O
blot O
analysis O
does O
not O
work O
for O
IHC O
(MAB17791, O
see O
data O
in O
).After O
antibodies O
were O
validated O
in O
direct O
ELISAs, O
Western O
blot O
or O
cell O
lines O
(Fig. O
1 O
and O
data O
not O
shown), O
they O
were O
used O
to O
examine O
the O
expression O
of O
individual O
molecules O
in O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
and O
differentiated O
EBs. O
When O
examined O
by O
immunohistochemistry, O
high O
level O
of O
expressions O
of O
Oct3/4, O
SOX2, O
E-Cadherin, O
PODXL B-GeneProtein
and O
Nanog B-GeneProtein
were O
observed O
in O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
(Fig. O
2A, O
2B O
and O
2C). O
DPPA5 B-GeneProtein
(developmental O
pluripotency I-GeneProtein
associated I-GeneProtein
5) O
expression O
was O
also O
observed O
in O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
(data O
not O
shown). O
We O
noted O
that O
a O
subset O
of O
the O
proteins O
used O
were O
membrane B-CellComponent
bound O
proteins. O
To O
test O
if O
any O
of O
the O
antibodies O
generated O
could O
recognize O
an O
extracellular B-CellComponent
epitope I-CellComponent
and O
thus O
be O
used O
for O
live O
cell O
sorting, O
we O
repeated O
staining O
of O
live O
cells O
as O
previously O
described. O
The O
CD9, O
E-Cadherin B-GeneProtein
and O
PODXL B-GeneProtein
antibodies O
recognized O
an O
extracellular B-CellComponent
epitope I-CellComponent
and O
their O
ability O
to O
select O
cells O
by O
FACS O
was O
confirmed O
(Fig. O
3). O
Minimal O
or O
no O
expressions O
of O
Oct3/4, O
E-Cadherin, O
PODXL B-GeneProtein
and O
Nanog B-GeneProtein
were O
detected O
in O
the O
differentiated O
EBs B-Anatomy
(Fig. O
2D, O
2E O
and O
2F). O
However, O
SOX2 B-GeneProtein
expression, O
which O
is O
observed O
in O
neural B-Anatomy
progenitor I-CellType
cells, O
is O
persistent O
in O
subsets O
of O
EBs.Figure O
1Western O
blot O
analysis O
for O
Gt O
× O
hOct3/4 O
(A), O
Gt O
× O
hNanog O
(B) O
and O
Ms O
× O
hSOX2 O
(C) O
in O
NTERA-2 B-CellLine
cell I-CellType
lysate, O
Ms O
× O
hE-Cadherin O
(D) O
in O
MCF-7 B-CellLine
cell I-CellType
lysate, O
Ms O
× O
hCD9 B-GeneProtein
(E) O
in O
PBMC B-CellType
lysate O
and O
Ms O
× O
hPDX-1(F) O
in O
β-TC-6 B-CellLine
cell I-CellType
lysate. O
Numbers O
indicate O
the O
positions O
of O
molecular O
weight O
markers.Figure O
2Undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
(A, O
B, O
and O
C) O
and O
differentiated O
EBs B-Anatomy
(D, O
E O
and O
F) O
were O
analyzed O
using O
antibodies O
to O
indicated O
molecular O
markers. O
Immunostaining O
with O
goat O
anti-human O
Oct3/4 B-GeneProtein
(Red O
in O
A O
and O
D), O
mouse B-Species
anti-human O
SOX2 B-GeneProtein
(Green O
in O
A O
and O
D), O
goat O
anti-human O
E-Cadherin B-GeneProtein
(Red O
in O
B O
and O
E), O
mouse B-Species
anti-human O
PODXL B-GeneProtein
(Green O
in O
B O
and O
E), O
and O
goat O
anti-human O
Nanog B-GeneProtein
(Red O
in O
C O
and O
F), O
are O
contrasted O
with O
DAPI O
nuclear B-CellComponent
staining O
(Blue O
in O
C-F). O
Note O
the O
dramatic O
downregulation O
of O
ESC O
specific O
markers O
(Oct3/4, O
E-Cadherin, O
PODXL, O
and O
Nanog) O
in O
EBs. O
However, O
SOX2 B-GeneProtein
expression O
is O
persistent O
in O
subsets O
of O
EB B-CellType
cells. O
Scale O
bars O
= O
100 O
μm.Figure O
3Human O
embryonic I-CellType
stem I-CellType
cells I-CellType
stained O
with O
anti-CD9 O
(A), O
anti-E-Cadherin O
(B), O
and O
anti-PODXL O
(C) O
and O
antigen O
expression O
detected O
by O
a O
flow O
cytometer. O
The O
specific O
staining O
is O
indicated O
by O
green O
histogram O
and O
corresponding O
isotype O
control O
is O
indicated O
by O
black O
histogram.Suspension O
culture O
with O
FGF B-GeneProtein
withdrawal O
is O
known O
to O
induce O
differentiation O
of O
ES B-CellType
cells I-CellType
to O
all O
three O
germ B-CellType
layer I-CellType
precursors I-CellType
[12]. O
The O
differentiation O
status O
of O
the O
EB O
used O
here O
was O
detected O
to O
contain O
all O
germ O
cell O
markers O
by O
RT-PCR O
(Fig. O
4). O
In O
order O
to O
examine O
how O
more O
antibodies O
can O
be O
used O
for O
characterization O
of O
early O
differentiation O
events O
from O
human B-Species
ES I-CellType
cells, O
we O
examined O
the O
expressions O
of O
endodermal B-Anatomy
markers, O
SOX17, O
GATA6 B-GeneProtein
and O
PDX-1, O
and O
mesodermal B-Anatomy
markers, O
Brachyury B-GeneProtein
and O
GATA1, O
in O
the O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
and O
differentiated O
EBs. O
Expressions O
of O
SOX17, O
GATA6, O
PDX-1, O
Brachyury B-GeneProtein
and O
GATA1 B-GeneProtein
were O
not O
detected O
in O
undifferentiated O
human B-Species
ES I-CellType
cells I-CellType
(data O
not O
shown). O
In O
contrast O
to O
the O
undifferentiated O
ES B-CellType
cells, O
subpopulations O
of O
SOX17-, O
GATA6-, O
Brachyury- O
and O
GATA1-positive O
cells O
were O
observed O
(Fig O
4). O
These O
results O
suggest O
that O
both O
endodermal B-Anatomy
and O
mesodermal B-Anatomy
precursors I-CellType
exist O
in O
EBs B-Anatomy
with O
FGF B-GeneProtein
withdrawal O
for O
8 O
days. O
However, O
no O
PDX-1-positive O
cells O
were O
seen O
in O
EBs B-Anatomy
differentiated O
with O
the O
same O
treatment O
(data O
not O
shown).Figure O
4Differentiated O
EBs B-Anatomy
were O
analyzed O
by O
either O
immunocytochemistry O
or O
RT-PCR O
to O
the O
indicated O
molecular O
markers. O
(A) O
Immunostaining O
with O
goat O
anti-human O
SOX17 B-GeneProtein
(Red), O
is O
contrasted O
with O
Fluoro O
Nissl O
nuclear B-CellComponent
staining O
(Green). O
(B) O
Immunostaining O
with O
goat O
anti-human O
GATA6 B-GeneProtein
(Red), O
is O
contrasted O
with O
DAPI O
nuclear B-CellComponent
staining O
(Blue). O
(C) O
Immunostaining O
with O
goat O
anti-human O
brachyury B-GeneProtein
(Red), O
is O
contrasted O
with O
DAPI O
nuclear B-CellComponent
staining O
(Blue). O
(D) O
Immunostaining O
with O
mouse B-Species
anti-human O
GATA1 B-GeneProtein
(Red). O
Note O
that O
each O
antibody O
recognizes O
subsets O
of O
EB B-CellType
cells. O
Scale O
bars O
= O
100 O
μm. O
(E) O
The O
differentiation O
status O
of O
EB O
is O
detected O
by O
RT-PCR O
using O
different O
germ B-Anatomy
layer I-Anatomy
cell I-CellType
markers. O
Selected O
endoderm B-Anatomy
markers O
AFP, O
FoxA2; O
mesoderm B-Anatomy
markers O
Hand1, O
MSX1 B-GeneProtein
and O
ectoderm B-Anatomy
marker O
Msl1 B-GeneProtein
were O
all O
highly O
expressed O
in O
the O
EB O
samples O
while O
their O
expression O
was O
either O
undetectable O
or O
at O
low O
level O
in O
the O
ES B-CellType
samples. O
G3PDH B-GeneProtein
was O
a O
positive O
control O
showing O
similar O
amount O
of O
RNA O
samples O
were O
used O
for O
analysis. O
Examination O
of O
cross-reactivity O
of O
antibodies O
on O
mouse B-Species
ES I-CellType
and O
differentiated O
cellsWe O
have O
also O
examined O
the O
cross-reactivities O
of O
these O
antibodies O
to O
mouse B-Species
ES I-CellType
cells I-CellType
using O
mouse B-Species
D3 I-CellType
ES I-CellType
cell I-CellType
line O
and O
mouse B-Species
fetal B-Anatomy
endodermal B-Anatomy
tissue. O
Cross-reactivity O
to O
mouse B-Species
of O
goat O
anti-Oct3/4, O
goat O
anti-PDX-1, O
goat O
anti-SOX17 O
and O
mouse B-Species
anti-SOX2 O
was O
detected. O
Minimal O
cross-reactivity O
to O
mouse, O
measured O
by O
10% O
intensity O
to O
human B-Species
by O
higher O
than O
control O
cells, O
was O
observed O
in O
mouse B-Species
anti-CD9 O
and O
mouse B-Species
anti-E-cadherin O
antibodies. O
Goat B-Species
anti-Nanog O
and O
mouse B-Species
anti-PODXL O
antibodies O
appear O
to O
be O
human-specific O
as O
well O
(data O
not O
shown). O
The O
subtypes O
of O
monoclonal O
antibodies O
were O
also O
identified O
in O
the O
best O
clones. O
These O
results O
are O
summarized O
in O
Table O
2.Table O
2Summary O
of O
antibodies O
detection O
in O
ES B-CellType
and O
EB O
samples.AntibodyESEBReactivity O
to O
mouseIsotype O
of O
monoclonal O
antibody O
(Clone O
No.)Gt O
× O
hBrachyuryNoYesNT*Ms O
× O
hDPPA5YesNT*NT*ND*Gt O
× O
hGATA6NoYesNT*Gt O
× O
hNanogYesDownNoGt O
× O
hOct O
3/4YesDownYesGt O
× O
hPDX-1NoNoYesGt O
× O
hSOX17NoYesYesMs O
× O
hCD9YesNoMinimalMouse O
IgG2B I-GeneProtein
(clone O
209306)Ms O
× O
hE-cadherinYesNoMinimalMouse O
IgG2B I-GeneProtein
(clone O
180224)Ms O
× O
hGATA1NoYesNT*Rat O
IgG2B I-GeneProtein
(clone O
234732)Ms O
× O
hPODXLYesNoNoMouse O
IgG2A I-GeneProtein
(clone O
222328)Ms O
× O
hSOX2YesYesYesMouse O
IgG2A I-GeneProtein
(clone O
245610)*NT, O
Not O
tested; O
ND, O
Not O
determined. O
The O
expression O
patterns O
detected O
using O
antibodies O
developed O
in O
our O
facility O
are O
consistent O
with O
data O
reported O
using O
reverse O
transcriptase-polymerase O
chain O
reaction O
or O
cDNA O
microarrays. O
Moreover O
several O
of O
the O
monoclonal O
antibodies O
have O
differing O
heavy O
chain O
subunits O
allowing O
double O
labeling O
using O
subtype O
specific O
markers O
to O
be O
performed. O
In O
summary, O
we O
have O
developed O
a O
useful O
collection O
of O
antibodies O
that O
would O
be O
useful O
for O
identification O
of O
stem O
cell O
characteristics O
and O
assessment O
of O
differentiation. O
Several O
additional O
antibodies O
to O
the O
molecules O
that O
have O
been O
identified O
as O
potential O
cell O
lineage O
markers O
[13] O
are O
currently O
under O
development O
using O
the O
same O
approach. O
Cloning O
and O
expression O
of O
Brachyury, O
DPPA5, O
CD9, O
E-Cadherin, O
GATA1, O
GATA6, O
Nanog, O
Oct3/4, O
PDX-1, O
PODXL, O
SOX2 B-GeneProtein
and O
SOX17Brachyury O
(aa. O
1–202), O
DPPA5 B-GeneProtein
(a.a. O
1–116), O
GATA1 B-GeneProtein
(a.a. O
1–413), O
GATA6 B-GeneProtein
(aa. O
1–449), O
Nanog B-GeneProtein
(aa. O
153–305), O
Oct3/4 B-GeneProtein
(aa. O
1–265), O
PDX-1 B-GeneProtein
(aa. O
1–283), O
SOX2 B-GeneProtein
(aa. O
135–317) O
and O
SOX17 B-GeneProtein
(aa. O
177–414) O
were O
expressed O
in O
E. O
Coli O
and O
extracellular O
domains O
of O
CD9, O
E-Cadherin, O
PODXL B-GeneProtein
were O
expressed O
in O
mouse B-Species
NSO I-CellType
cells. O
All O
proteins O
were O
purified O
and O
sequenced O
before O
they O
were O
used O
as O
antigens O
for O
immunizations O
and O
as O
substrate O
for O
antibody O
screening O
and O
subcloning. O
Production O
and O
purification O
of O
antibodiesAll O
monoclonal O
antibodies O
were O
derived O
from O
fusions O
of O
mouse B-Species
myeloma I-Anatomy
with O
B B-CellType
cells I-CellType
obtained O
from O
BALB/c B-Species
mice I-Species
which O
had O
been O
immunized O
with O
purified O
antigen. O
The O
IgG B-GeneProtein
fraction O
of O
the O
culture O
supernatant O
was O
purified O
by O
Protein B-GeneProtein
G I-GeneProtein
affinity O
chromatography O
(Sigma). O
Each O
panel O
of O
antibodies O
was O
screened O
and O
selected O
for O
their O
abilities O
to O
detect O
purified O
recombinant O
antigen O
in O
direct O
ELISA O
and O
Western O
blot. O
All O
polyclonal O
antibodies O
were O
derived O
from O
sera O
of O
goats B-Species
which O
had O
been O
immunized O
and O
boost O
it O
with O
purified O
antigen. O
Antibody O
was O
purified O
from O
the O
sera O
by O
an O
antigen-affinity O
chromatography. O
Cells O
and O
cell O
cultureHuman O
Caco-2, O
MG-63, O
MCF-7, O
NTERA-2 B-CellLine
and O
mouse B-Species
D3 I-CellType
cells I-CellType
were O
purchased O
from O
American O
Type O
Culture O
Collection O
(ATCC). O
Cells O
were O
cultured O
according O
to O
the O
ATCC O
instructions. O
Information O
regarding O
human B-Species
ES B-CellType
cell O
line O
HSF-6 B-CellLine
(NIH O
code O
UC06) O
can O
be O
obtained O
at O
the O
website O
[14]. O
Undifferentiated O
human B-Species
ES B-CellType
cells I-CellType
were O
cultured O
according O
to O
the O
protocol O
provided O
by O
the O
University O
of O
California, O
San O
Francisco O
in O
human B-Species
ES B-CellType
culture O
medium O
[DMEM O
supplemented O
with O
20% O
KnockOut O
Serum O
Replacement O
(Invitrogen) O
and O
5 O
ng/mL O
of O
bFGF B-GeneProtein
(R&D O
Systems)]. O
To O
induce O
formation O
of O
embryoid B-Anatomy
bodies I-Anatomy
(EBs), O
ES B-CellType
colonies B-Anatomy
were O
harvested, O
separated O
from O
the O
MEF B-CellType
feeder B-CellType
cells I-CellType
by O
gravity, O
gently O
resuspended O
in O
ES B-CellType
culture B-Anatomy
medium O
and O
transferred O
to O
non-adherent O
suspension O
culture B-Anatomy
dishes O
(Corning). O
Unless O
otherwise O
noted, O
EBs B-Anatomy
derived O
from O
human B-Species
ES O
cell O
aggregates B-Anatomy
were O
cultured O
for O
8 O
days O
in O
ES B-CellType
culture O
medium O
deprived O
of O
bFGF B-GeneProtein
and O
used O
for O
analysis O
by O
immunohistochemistry O
as O
described. O
Western O
blotCells O
are O
solubilized O
in O
hot O
2× O
SDS O
gel O
sample O
buffer O
(20 O
mM O
dithiothreitol, O
6% O
SDS, O
0.25 O
M O
Tris, O
pH O
6.8, O
10% O
glycerol, O
10 O
mM O
NaF O
and O
bromophenyl O
blue) O
at O
2 O
× O
106 O
per O
mL. O
The O
extracts O
are O
heated O
in O
a O
boiling O
water O
bath O
for O
5 O
minutes O
and O
sonicated O
with O
a O
probe O
sonicator O
with O
3–4 O
bursts O
of O
5–10 O
seconds O
each. O
Samples O
are O
diluted O
with O
1× O
SDS O
sample O
buffer O
to O
the O
desired O
loading O
of O
1–5 O
× O
103 O
per O
lane. O
Lysates O
were O
resolved O
by O
SDS-PAGE, O
transferred O
to O
Immobilon-P O
membrane, O
and O
immunoblotted O
with O
0.5 O
μg/mL O
primary O
Abs O
as O
described O
in O
R&D O
Systems O
Website O
[15]. O
ImmunohistochemistryAntibodies O
were O
used O
with O
the O
appropriate O
secondary O
reagents O
at O
a O
concentration O
of O
5 O
to O
10 O
μg/ml. O
Cells O
or O
sections O
of O
EBs B-Anatomy
were O
fixed O
with O
4% O
paraformaldehyde O
in O
PBS O
at O
room O
temperature O
for O
20 O
min, O
then O
blocked O
and O
permeabilized O
with O
0.1% O
Triton O
X-100, O
1% O
BSA, O
10% O
normal O
donkey B-Species
serum O
in O
PBS O
at O
room O
temperature O
for O
45 O
min. O
After O
blocking, O
cells O
were O
incubated O
with O
diluted O
primary O
antibody O
overnight O
at O
4°C O
followed O
by O
coupled O
anti-mouse O
or O
anti-goat O
IgG B-GeneProtein
(Molecular O
Probes) O
at O
room O
temperature O
in O
the O
dark O
for O
an O
hour. O
Between O
each O
step O
cells O
were O
washed O
with O
PBS O
with O
0.1% O
BSA. O
RT-PCRTotal O
RNA O
was O
extracted O
from O
EBs B-Anatomy
using O
Trizol O
LS O
(Invitrogen). O
cDNA O
was O
synthesized O
by O
using O
Superscript O
II O
reverse O
transcriptase O
(Invitrogen) O
according O
to O
the O
manufacturer's O
recommendations. O
The O
PCR O
primers O
are O
available O
upon O
request. O
Flow O
cytometryAntibodies O
were O
prepared O
at O
the O
concentration O
of O
0.1 O
mg/mL. O
10 O
μL O
of O
the O
stock O
solution O
was O
added O
to O
1 O
– O
2.5 O
× O
105 O
cells O
in O
a O
total O
reaction O
volume O
not O
exceeding O
200 O
μL. O
The O
sample O
was O
then O
incubated O
for O
20 O
min O
at O
2–8 O
°C. O
Following O
incubation, O
excess O
antibody O
was O
removed O
by O
washing O
cells O
twice O
with O
FACS O
buffer O
(2% O
FCS O
and O
0.1% O
sodium O
azide O
in O
Hank's O
buffer). O
After O
wash, O
cells O
were O
resuspend O
in O
200 O
μL O
of O
FACS O
buffer O
and O
the O
binding O
of O
unlabeled O
monoclonal O
antibodies O
was O
visualized O
by O
adding O
10 O
μL O
of O
a O
25 O
μg/mL O
stock O
solution O
of O
a O
secondary O
developing O
reagent O
such O
as O
goat O
anti-mouse O
IgG B-GeneProtein
conjugated O
to O
a O
fluorochrome O
for O
20 O
min O
at O
2–8°C. O
Following O
incubation, O
cells O
were O
washed O
once O
with O
FACS O
buffer, O
once O
with O
PBS. O
After O
wash, O
cells O
were O
resuspend O
in O
400 O
μL O
of O
PBS O
and O
analyzed O
on O
a O
FACScant O
flow O
cytometer O
(Becton-Dickinson, O
Mountain O
View, O
CA). O
Five O
thousand O
events O
were O
collected O
and O
analyzed O
using O
CELL O
Quest O
software. O