18286199.conll

BackgroundHuman O
embryonic I-CellType
stem I-CellType
cells I-CellType
(hESCs) O
offer O
a O
virtually O
unlimited O
source O
of O
neural B-Anatomy
cells I-CellType
for O
structural O
repair O
in O
neurological O
disorders, O
such O
as O
stroke. O
Neural B-Anatomy
cells I-CellType
can O
be O
derived O
from O
hESCs B-CellType
either O
by O
direct O
enrichment, O
or O
by O
isolating O
specific O
growth O
factor-responsive O
and O
expandable O
populations O
of O
human B-Species
neural I-CellType
stem I-CellType
cells I-CellType
(hNSCs). O
Studies O
have O
indicated O
that O
the O
direct O
enrichment O
method O
generates O
a O
heterogeneous O
population O
of O
cells O
that O
may O
contain O
residual O
undifferentiated O
stem B-CellType
cells I-CellType
that O
could O
lead O
to O
tumor O
formation O
in O
vivo.Methods/Principal O
FindingsWe O
isolated O
an O
expandable O
and O
homogenous O
population O
of O
hNSCs B-CellType
(named O
SD56) O
from O
hESCs B-CellType
using O
a O
defined O
media O
supplemented O
with O
epidermal B-GeneProtein
growth I-GeneProtein
factor I-GeneProtein
(EGF), O
basic B-GeneProtein
fibroblast I-GeneProtein
growth I-GeneProtein
factor I-GeneProtein
(bFGF) O
and O
leukemia B-GeneProtein
inhibitory I-GeneProtein
growth I-GeneProtein
factor I-GeneProtein
(LIF). O
These O
hNSCs B-CellType
grew O
as O
an O
adherent O
monolayer O
culture. O
They O
were O
fully O
neuralized O
and O
uniformly O
expressed O
molecular O
features O
of O
NSCs, O
including O
nestin, O
vimentin B-GeneProtein
and O
radial B-CellType
glial B-CellType
markers. O
These O
hNSCs B-CellType
did O
not O
express O
the O
pluripotency O
markers O
Oct4 B-GeneProtein
or O
Nanog, O
nor O
did O
they O
express O
markers O
for O
the O
mesoderm B-Anatomy
or O
endoderm B-Anatomy
lineages. O
The O
self-renewal O
property O
of O
the O
hNSCs B-CellType
was O
characterized O
by O
a O
predominant O
symmetrical O
mode O
of O
cell O
division. O
The O
SD56 B-CellLine
hNSCs I-CellType
differentiated O
into O
neurons, O
astrocytes B-CellType
and O
oligodendrocytes B-CellType
throughout O
multiple O
passages O
in O
vitro, O
as O
well O
as O
after O
transplantation. O
Together, O
these O
criteria O
confirm O
the O
definitive O
NSC O
identity O
of O
the O
SD56 B-CellLine
cell O
line. O
Importantly, O
they O
exhibited O
no O
chromosome O
abnormalities O
and O
did O
not O
form O
tumors O
after O
implantation O
into O
rat O
ischemic O
brains O
and O
into O
naïve O
nude O
rat O
brains B-Anatomy
and O
flanks. O
Furthermore, O
hNSCs B-CellType
isolated O
under O
these O
conditions O
migrated O
toward O
the O
ischemia-injured O
adult O
brain B-Anatomy
parenchyma I-Anatomy
and O
improved O
the O
independent O
use O
of O
the O
stroke-impaired O
forelimb O
two O
months O
post-transplantation.Conclusions/SignificanceThe O
SD56 B-CellLine
human I-CellType
neural I-CellType
stem I-CellType
cells I-CellType
derived O
under O
the O
reported O
conditions O
are O
stable, O
do O
not O
form O
tumors B-Anatomy
in O
vivo O
and O
enable O
functional O
recovery O
after O
stroke. O
These O
properties O
indicate O
that O
this O
hNSC O
line O
may O
offer O
a O
renewable, O
homogenous O
source O
of O
neural B-Anatomy
cells I-CellType
that O
will O
be O
valuable O
for O
basic O
and O
translational O
research. O
To O
date O
there O
have O
been O
no O
effective O
treatments O
for O
improving O
residual O
structural O
and O
functional O
deficits O
resulting O
from O
stroke. O
Current O
therapeutic O
approaches, O
such O
as O
the O
use O
of O
thrombolytics, O
benefit O
only O
1 O
to O
4% O
of O
patients O
[1]. O
Consequently, O
the O
majority O
of O
stroke O
patients O
experience O
progression O
of O
ischemia O
associated O
with O
debilitating O
neurological O
deficits. O
Recent O
evidence O
has O
suggested O
that O
the O
transplantation O
of O
cells O
derived O
from O
cord B-Anatomy
blood, O
bone B-Anatomy
marrow I-Anatomy
or O
brain O
tissue O
(fetal O
and O
adult) O
enhances O
sensorimotor O
function O
in O
experimental O
models O
of O
stroke O
[2], O
[3]. O
However, O
the O
normal O
human-derived B-CellType
somatic I-CellType
stem I-CellType
cells I-CellType
used O
in O
these O
studies O
have O
a O
limited O
capacity O
to O
differentiate O
into O
the O
diverse O
neural B-Anatomy
cell O
types O
optimal O
for O
structural O
and O
physiological O
tissue O
repair O
and O
are O
not O
amenable O
for O
large-scale O
cell O
production. O
Unlike O
other O
sources O
of O
stem O
cells, O
hESC O
lines O
possess O
a O
nearly O
unlimited O
self-renewal O
capacity O
and O
the O
developmental O
potential O
to O
differentiate O
into O
virtually O
any O
cell O
type O
of O
the O
organism. O
As O
such, O
they O
constitute O
an O
ideal O
source O
of O
cells O
for O
regenerative O
medicine. O
The O
successful O
derivation O
of O
hESC O
lines O
from O
the O
inner B-Anatomy
cell I-Anatomy
mass I-Anatomy
of O
preimplantation O
embryos B-Anatomy
and O
their O
long-term O
maintenance O
in O
vitro O
over O
multiple O
passages O
has O
been O
demonstrated O
[4] O
and O
standardized. O
Differentiation O
and O
enrichment O
processes O
that O
direct O
hESCs B-CellType
towards O
a O
neural B-Anatomy
lineage I-Anatomy
have O
also O
been O
achieved. O
To O
promote O
neuralization, O
ESCs B-CellType
were O
cultured O
in O
a O
defined O
media O
supplemented O
with O
morphogens O
or O
growth O
factors O
[5], O
[6], O
[7] O
or O
cultured O
under O
conditions O
that O
promote O
“rosettes”, O
structures O
morphologically O
similar O
to O
the O
developing O
neural B-Anatomy
tube I-Anatomy
[8], O
[9]. O
This O
neuralization O
process O
has O
proven O
invaluable O
in O
understanding O
the O
specification O
of O
hESC-derived O
neural B-Anatomy
tissue I-Anatomy
[10], O
[11], O
[12]. O
However, O
the O
enriched O
neural B-Anatomy
progeny I-CellType
derived O
displayed O
overgrowth O
and O
limited O
migration O
after O
grafting O
into O
normal O
newborn O
mice B-Species
[13] O
and O
lesioned O
adult O
rat B-Anatomy
striatum I-Anatomy
[12], O
[14], O
[15], O
[16]. O
The O
inner O
cores O
of O
these O
grafts O
contained O
tumorigenic B-CellType
precursor I-CellType
cells I-CellType
(reviewed O
in O
[17]). O
These O
findings O
suggest O
that O
neural B-Anatomy
cells I-CellType
generated O
by O
acute O
exposure O
to O
growth O
factors O
and/or O
morphogens O
may O
still O
be O
heterogeneous O
and O
potentially O
tumorigenic. O
We O
report O
an O
alternative O
method O
for O
the O
isolation O
and O
the O
perpetuation O
of O
a O
multipotent O
hNSC O
line O
from O
the O
hESCs B-CellType
with O
a O
primitive O
mode O
of O
self-renewal. O
We O
also O
demonstrate O
their O
long-term O
expansion, O
non-tumorigenic O
properties O
and O
functional O
engraftability O
in O
an O
experimental O
model O
of O
stroke. O
1. O
In O
vitro O
isolation, O
growth O
and O
differentiation O
of O
hESC-derived O
hNSCsThe O
hESCs B-CellType
were O
maintained O
and O
expanded O
on O
mouse B-Species
feeder O
layer O
in O
media O
supplemented O
with O
bFGF B-GeneProtein
(Figure O
1A). O
After O
cell O
dissociation, O
a O
portion O
of O
the O
hESCs B-CellType
was O
cultured O
in O
serum O
free O
medium O
containing O
EGF, O
bFGF B-GeneProtein
and O
LIF. O
These O
factors O
are O
known O
to O
stimulate O
the O
proliferation O
of O
human B-Species
fetal-derived I-CellType
NSCs I-CellType
[18], O
[19]. O
After O
3 O
days O
in O
vitro O
(DIV), O
there O
was O
selective O
survival O
and O
growth O
of O
cells O
that O
aggregated O
in O
clusters O
or O
spheres O
(Figure O
1B). O
These O
primary O
spheres O
were O
harvested O
and O
replated O
in O
fresh O
media. O
During O
the O
following O
week, O
the O
spheres O
attached O
to O
the O
flask O
and O
a O
fibroblast-like O
cell O
population O
began O
to O
migrate O
out O
(Figure O
1C). O
Secondary O
spheres O
(2° O
spheres) O
were O
generated O
from O
these O
cultures O
and O
lifted O
off O
by O
the O
end O
of O
the O
week O
leaving O
a O
hollow O
in O
the O
middle O
of O
the O
attached O
cells O
(Figure O
1D). O
The O
floating O
2° O
spheres O
were O
collected O
and O
replated O
in O
fresh O
growth O
medium O
for O
2 O
weeks. O
The O
cultures O
were O
then O
passaged O
by O
collagenase B-GeneProtein
cell O
dissociation O
every O
7 O
DIV O
for O
an O
additional O
4 O
passages O
(Figure O
S1). O
At O
the O
5th O
and O
6th O
passages, O
spheres O
were O
dissociated O
into O
a O
single-cell O
suspension O
using O
trypsin-EDTA. O
At O
this O
stage O
there O
was O
a O
change O
in O
the O
hNSCs' O
adherent O
properties, O
and O
the O
cells O
began O
to O
grow O
as O
a O
monolayer O
with O
multiple O
foci O
of O
cells O
throughout O
the O
culture O
(Figure O
1F). O
The O
adherent O
hNSC O
culture O
stained O
uniformly O
for O
nestin B-GeneProtein
(Figure O
1K), O
vimentin B-GeneProtein
(Figure O
1L) O
and O
with O
the O
radial B-CellType
glial I-CellType
marker O
3CB2 B-GeneProtein
(Figure O
1M) O
indicating O
their O
homogeneity O
and O
NSC O
identity. O
Under O
these O
culture O
conditions, O
it O
is O
noteworthy O
that O
we O
did O
not O
observe O
the O
formation O
of O
rosettes B-Anatomy
which O
has O
been O
previously O
reported O
to O
occur O
under O
certain O
conditions O
during O
neuralization O
of O
hESCs B-CellType
[8], O
[20], O
[21]. O
RT-PCR O
analysis O
confirmed O
that O
these O
hNSCs B-CellType
did O
not O
express O
the O
pluripotency O
transcripts O
Oct-4 B-GeneProtein
and O
Nanog B-GeneProtein
(Figure O
1I). O
Furthermore, O
the O
hNSCs B-CellType
did O
not O
express O
transcripts O
for O
brachyury B-GeneProtein
and O
foxa2, O
marker O
genes O
for O
mesoderm B-Anatomy
and O
endoderm B-Anatomy
respectively O
(negative O
result, O
data O
not O
shown).10.1371/journal.pone.0001644.g001Figure O
1Isolation O
and O
purification O
of O
hNSCs B-CellType
from O
the O
hESCs.The O
hESCs B-CellType
were O
grown O
on O
a O
mouse B-Species
feeder O
layer O
(A). O
Primary O
neurospheres B-Anatomy
(B) O
were O
isolated O
and O
replated O
to O
eliminate O
other O
non-neural O
cells. O
The O
selectively O
harvested O
secondary O
neurospheres B-Anatomy
(arrow O
in O
C), O
left O
behind O
hollow O
cores O
in O
the O
surface O
area O
(star O
in O
D) O
where O
they O
attached O
earlier. O
They O
were O
perpetuated O
for O
an O
additional O
5 O
passages O
(E). O
These O
2° O
spheres O
were O
then O
passaged O
using O
a O
single O
cell O
dissociation O
protocol O
(F). O
Arrow O
in O
F O
shows O
an O
example O
of O
a O
focus O
of O
proliferating O
cells. O
(G, O
H) O
The O
hNSCs B-CellType
were O
passaged O
every O
5–7 O
days, O
as O
described O
in O
the O
Methods O
section. O
Starting O
from O
an O
initial O
population O
of O
1 O
million O
cells, O
the O
cumulative O
cell O
number O
was O
calculated O
at O
each O
passage O
as O
the O
fold O
of O
increase×the O
total O
cell O
number O
and O
plotted O
as O
logarithm O
with O
base O
2 O
in O
function O
of O
time O
(G). O
The O
cell O
perpetuation O
(G) O
and O
population O
doubling O
(H) O
analysis O
demonstrated O
the O
continuous O
and O
stable O
growth O
of O
the O
hNSCs. O
(I) O
RT-PCR O
analysis O
showing O
the O
down O
regulation O
of O
the O
pluripotency O
transcripts O
Oct4 B-GeneProtein
and O
Nanog B-GeneProtein
in O
secondary O
neurospheres B-Anatomy
and O
in O
expanded O
hNSCs B-CellType
at O
passage O
8 O
(P8). O
(J) O
Cytogenetic O
evaluation O
of O
the O
SD56 B-CellLine
hNSCs B-CellType
line O
at O
passage O
12 O
by O
standard O
G-banding O
was O
performed. O
Twenty O
metaphase O
cells O
were O
analyzed O
and O
showed O
a O
normal O
female B-Anatomy
chromosome O
complement O
(46,XX). O
Isolated O
and O
expanded O
hNSCs B-CellType
expressed O
the O
neural B-Anatomy
precursor I-CellType
cell I-CellType
markers O
nestin B-GeneProtein
(K), O
Vimentin B-GeneProtein
(L) O
and O
the O
radial B-CellType
glial I-CellType
cell I-CellType
marker O
3CB2 B-GeneProtein
(M) O
in O
virtually O
all O
the O
progeny. O
(N-P) O
Clonal O
self-renewal O
ability O
of O
the O
isolated O
hNSCs O
through O
symmetrical O
cell O
division. O
Single O
(N), O
two-cell O
stage O
(O) O
and O
four-cell O
stage O
(P) O
of O
a O
hNSC O
proliferating O
over O
a O
3-day O
culture O
period. O
Note O
the O
symmetrical O
segregation O
of O
BrdU O
and O
nestin B-GeneProtein
in O
the O
progeny. O
Bars: O
(A, O
B, O
C, O
D) O
200 O
µm; O
(E, O
F) O
100 O
µm; O
(K–M) O
20 O
µm; O
(N–P) O
10 O
µm.To O
ascertain O
self-renewal O
ability O
under O
clonal O
conditions, O
a O
single O
cell O
suspension O
was O
plated O
at O
clonal O
density O
(1–2 O
cell/10 O
µl). O
To O
determine O
if O
the O
hNSCs B-CellType
divide O
symmetrically, O
we O
pulsed O
cultures O
with O
the O
thymidine O
analog, O
bromodeoxyuridine O
(BrdU), O
after O
plating O
and O
looked O
for O
the O
expression O
of O
nestin B-GeneProtein
in O
the O
progeny. O
Cultures O
were O
fixed O
after O
1, O
2 O
or O
3 O
DIV O
(Figure O
1N–P). O
After O
2 O
days, O
plated O
single O
cells O
first O
underwent O
a O
symmetric O
cell O
division O
and O
gave O
rise O
to O
daughter O
cells O
that O
were O
both O
positive O
for O
BrdU O
and O
nestin. O
The O
clone O
of O
cells O
continued O
to O
grow O
over O
the O
3 O
DIV O
and O
all O
the O
progeny B-CellType
expressed O
nestin. O
BrdU O
labeling O
demonstrated O
that O
it O
was O
rare O
for O
only O
one O
daughter O
cell O
to O
inherit O
the O
BrdU O
and O
thus O
had O
undergone O
asymmetric O
segregation O
of O
the O
chromatids O
(Figure O
S2). O
G-band O
karyotyping O
of O
these O
hNSCs B-CellType
confirmed O
the O
normal, O
non-transformed O
nature O
of O
cells O
after O
12 O
passages O
(Figure O
1J). O
We O
named O
the O
derived O
hNSCs B-CellType
SD56 B-CellLine
(intermittently O
referred O
to O
as O
SD56 B-CellLine
hNSCs B-CellType
or O
hNSCs).Under O
these O
defined O
growth O
conditions, O
the O
hNSCs B-CellType
showed O
stable O
growth O
with O
a O
2.7±0.2 O
fold O
increase O
every O
5 O
to O
7 O
days O
(Figure O
1G). O
The O
population O
doubling O
at O
each O
passage O
averaged O
at O
1.4±0.1 O
(Figure O
1H). O
The O
viability O
of O
hNSCs B-CellType
at O
each O
passage O
was O
consistent O
at O
the O
approximate O
value O
of O
98%. O
The O
projected O
cumulative O
cell O
numbers O
demonstrated O
that O
trillions O
of O
cells O
could O
be O
generated O
over O
a O
period O
of O
5 O
months O
(Figure O
1G). O
We O
expanded O
the O
isolated O
hNSCs B-CellType
lines O
for O
over O
20 O
passages O
with O
a O
stable O
phenotype. O
An O
initial O
cell O
bank O
of O
75 O
vials O
containing O
2 O
to O
5 O
million O
cells O
each O
was O
generated O
and O
cryopreserved.Upon O
removal O
of O
the O
mitogenic O
factors O
and O
plating O
on O
a O
coverslip O
pre-coated O
with O
poly-L-ornithine O
(PLO) O
substrate, O
the O
hNSCs B-CellType
spontaneously O
differentiated O
into O
neurons, O
astrocytes B-CellType
and O
oligodendrocytes, O
a O
property O
that O
is O
consistent O
with O
normal O
multipotent O
hNSCs B-CellType
(Figure O
2). O
After O
2 O
DIV, O
hNSCs B-CellType
expressed O
transcripts O
for O
the O
neural-specific O
genes O
nestin, O
Notch1 B-GeneProtein
and O
neural B-GeneProtein
cell I-GeneProtein
adhesion I-GeneProtein
molecule I-GeneProtein
(N-CAM) O
(Figure O
2A) O
and O
for O
the O
lineage O
specific O
markers O
β-tubulin B-GeneProtein
class I-GeneProtein
III, O
medium-size B-GeneProtein
neurofilament I-GeneProtein
(NF-M) O
and O
microtubule-associated B-GeneProtein
protein I-GeneProtein
2 I-GeneProtein
(MAP-2) O
for O
neurons, O
GFAP B-GeneProtein
for O
astrocytes B-CellType
and O
myelin B-GeneProtein
basic I-GeneProtein
protein I-GeneProtein
(MBP) O
for O
oligodendrocytes B-CellType
(Figure O
2A). O
Transcripts O
for O
the O
GABAergic B-CellType
cell I-CellType
marker O
glutamic B-GeneProtein
acid I-GeneProtein
decarboxylase I-GeneProtein
(GAD) O
were O
expressed, O
but O
transcripts O
for O
the O
tyrosine B-GeneProtein
hydroxylase I-GeneProtein
(TH), O
a O
marker O
for O
dopaminergic B-CellType
neurons, O
were O
undetectable. O
Immunocytochemical O
analysis O
(Figure O
2B–F) O
of O
10 O
day-old O
cultures O
demonstrated O
that O
the O
proportion O
of O
nestin+ B-CellType
cells O
was O
36.6±2.7%, O
62.5±2.8% O
expressed O
the O
neuronal B-CellType
marker O
TuJ1, O
1.9±0.3% O
expressed O
the O
astrocytic B-CellType
marker O
GFAP B-GeneProtein
and O
7.1±0.4% O
were O
oligodendrocytes B-CellType
and O
expressed O
galactocerebrocide B-GeneProtein
(GC) O
(Figure O
2F). O
A O
subset O
(9.8±1.6%) O
of O
the O
GFAP+ O
astrocytes B-CellType
co-expressed O
nestin.10.1371/journal.pone.0001644.g002Figure O
2hESC-derived O
hNSCs B-CellType
spontaneously O
differentiated O
into O
the O
3 O
principal O
central B-Anatomy
nervous I-Anatomy
system I-Anatomy
cell I-CellType
types.Dissociated O
hNSCs B-CellType
were O
washed O
free O
of O
growth O
factors O
and O
plated O
on O
poly-L-onithine O
coated O
glass O
coverslips. O
Differentiated O
cultures O
were O
either O
harvested O
after O
2 O
DIV O
for O
total O
RNA O
extraction O
and O
RT-PCR O
analysis O
or O
fixed O
after O
10 O
DIV O
and O
processed O
for O
indirect O
immunocytochemistry. O
(A) O
Differentiated O
hNSCs B-CellType
expressed O
the O
neural-specific O
transcripts O
nestin B-GeneProtein
and O
Notch1 B-GeneProtein
as O
well O
as O
transcripts: O
for O
neurons B-CellType
(β-tubulin O
class I-GeneProtein
III, O
MAP-2, O
NCAM B-GeneProtein
and O
medium-size B-GeneProtein
neurofilament, O
NF-M), O
for O
astrocytes B-CellType
(GFAP) O
and O
for O
oligodendrocytes B-CellType
(MBP). O
The O
hNSCs B-CellType
expressed O
transcripts O
for O
GAD, O
but O
not O
for O
TH. O
B, O
C O
& O
D, O
morphology O
of O
NSC-derived B-CellType
progeny I-CellType
differentiated O
into O
GFAP+ O
astrocytes B-CellType
(B), O
GC-expressing O
oligodendrocytes B-CellType
(C) O
and O
TuJ1+ O
neurons B-CellType
(D), O
DAPI O
(blue) O
show O
life O
cell O
nuclei. O
(E) O
Photo O
showing O
cultures O
double-immunostained O
for O
TuJ1 B-GeneProtein
(green) O
and O
nestin B-GeneProtein
(red) O
(DAPI, O
blue). O
(F) O
Quantitative O
analysis O
of O
immunostained O
10 O
day-old O
cultures O
for O
the O
3 O
neural B-Anatomy
cell O
types. O
Results O
are O
mean±s.e.m. O
of O
three O
independent O
experiments, O
each O
performed O
in O
duplicate. O
Bars: O
(c) O
20 O
µm; O
(d, O
e) O
10 O
µm. O
2. O
The O
isolated O
hNSCs B-CellType
are O
normal O
and O
do O
not O
form O
tumors B-Anatomy
in O
normal O
nude O
ratsThe O
self-renewal O
and O
pluripotent O
abilities O
of O
the O
hESCs B-CellType
are O
also O
associated O
with O
tumorigenic O
properties. O
Therefore, O
the O
first O
critical O
step O
toward O
developing O
therapeutic O
hNSCs B-CellType
is O
to O
verify O
that O
they O
are O
non-tumorigenic. O
The O
monolayer O
culture O
of O
SD56 B-CellLine
hNSCs I-CellType
clearly O
demonstrated O
contact O
inhibition O
of O
growth, O
a O
normal O
karyotype O
and O
did O
not O
express O
the O
pluripotency O
transcripts O
Oct-4 B-GeneProtein
and O
Nanog. O
Removal O
of O
mitogenic O
factors O
and O
continued O
culture O
on O
plastic O
resulted O
in O
cell O
senescence O
that O
is O
characteristic O
of O
non-transformed O
cells. O
To O
determine O
whether O
SD56 B-CellLine
hNSCs I-CellType
form O
tumors B-Anatomy
in O
vivo, O
we O
transplanted O
them O
at O
high O
density O
(see O
Methods) O
into O
the O
forebrain B-Anatomy
and O
subcutaneously B-Anatomy
into O
the O
flank O
of O
nude O
rats. O
The O
animals O
were O
kept O
for O
a O
two-month O
post-transplant O
survival O
period. O
To O
label O
mitotically O
active O
cells O
in O
vivo O
during O
S-phase, O
the O
rats B-Species
were O
injected O
IP O
with O
BrdU O
(50 O
mg/kg) O
every O
8 O
hours O
during O
the O
last O
24 O
hours O
before O
euthanasia. O
The O
transit B-CellType
amplifying I-CellType
endogenous I-CellType
precursors I-CellType
located O
in O
the O
subventricular B-Anatomy
zone I-Anatomy
(SVZ) O
were O
labeled O
(Figure O
S3); O
however, O
we O
were O
unable O
to O
detect O
grafted O
SD56 B-CellLine
hNSCs I-CellType
co-localizing O
the O
human-specific O
nuclear B-CellComponent
marker O
hNuc B-GeneProtein
and O
BrdU O
(Figure O
S3). O
No O
surviving O
SD56 B-CellLine
hNSCs I-CellType
were O
detected O
in O
the O
flank O
of O
the O
transplanted O
animals O
suggesting O
that O
the O
grafted O
cells O
are O
not O
tumorigenic. O
3. O
Transplanted O
cells O
survived, O
migrated O
toward O
and O
engrafted O
into O
the O
stroke-damaged O
host O
tissueTo O
investigate O
the O
survival O
and O
functional O
engraftment O
in O
an O
injury O
environment, O
hNSCs B-CellType
(4×105) O
were O
transplanted O
into O
the O
ischemic O
boundary O
zone O
in O
the O
rat O
striatum B-Anatomy
one O
week O
after O
the O
middle B-Anatomy
cerebral I-Anatomy
artery I-Anatomy
occlusion O
(MCAO) O
was O
performed. O
Animals O
were O
euthanized O
two O
months O
later O
and O
the O
brains B-Anatomy
processed O
for O
histo-pathology O
and O
immunocytochemistry. O
Grafted O
SD56 B-CellLine
hNSCs, O
identified O
with O
hNuc, O
demonstrated O
a O
37.0±15.8% O
survival O
rate O
and O
a O
remarkable O
dispersion O
toward O
the O
stroke-damaged O
tissue O
with O
no O
sign O
of O
overgrowth O
or O
tumorigenesis. O
The O
majority O
of O
grafted O
cells O
(61.2±4.7%) O
migrated O
at O
least O
200 O
µm O
away O
from O
the O
injection O
site O
and O
penetrated O
an O
average O
distance O
of O
806.4±49.3 O
µm O
into O
the O
stroke-damaged O
tissue O
(Figure O
3A–C). O
Immunostaining O
with O
the O
blood B-Anatomy
vessel O
marker, O
GluT1, O
revealed O
dilated O
vessels O
in O
the O
infarcted O
striatum B-Anatomy
and O
a O
close O
association O
between O
vessels B-Anatomy
and O
the O
grafted O
hNSCs B-CellType
(Figure O
3B, O
3C). O
The O
grafted O
cells O
rarely O
expressed O
the O
proliferation O
marker O
Ki67 B-GeneProtein
(Figure O
3D), O
29.8±4.4% O
expressed O
nestin B-GeneProtein
(Figure O
3E), O
6.5±0.9% O
expressed O
doublecortin B-GeneProtein
(DCX) O
and O
60.8±8.1% O
were O
TuJ1+ O
(Figure O
3F, O
G). O
Grafted O
cells O
rarely O
co-expressed O
the O
astroglial B-CellType
marker O
GFAP B-GeneProtein
(Figure O
3H) O
or O
differentiated O
into O
CNPase-expressing O
oligodendrocytes B-CellType
(Figure O
3I). O
Immunostaining O
for O
GAD B-GeneProtein
demonstrated O
that O
25.1±2.3% O
of O
grafted O
hNSCs B-CellType
differentiated O
into O
GABAergic B-CellType
neurons I-CellType
while O
less O
than O
2% O
were O
positive O
for O
glutamate B-GeneProtein
(Figure O
3J, O
K).10.1371/journal.pone.0001644.g003Figure O
3Dispersion, O
engraftment O
and O
differentiation O
of O
the O
hNSCs B-CellType
in O
stroke-lesioned O
animals.(A) O
Schematic O
drawing O
of O
a O
frontal O
section O
through O
the O
striatum B-Anatomy
illustrating O
the O
dispersion O
of O
grafted O
hNSCs B-CellType
in O
the O
focal O
ischemia-lesioned O
parenchyma B-Anatomy
(shaded O
area). O
(B, O
C) O
Photos O
show O
frontal O
sections O
through O
the O
graft O
in O
the O
striatum B-Anatomy
immunostained O
with O
the O
human B-Species
specific O
antibodies: O
anti-hNuc O
(green O
in O
B O
& O
C) O
and O
anti-GluT1 O
(red, O
B O
& O
C) O
showing O
blood B-Anatomy
vessels I-Anatomy
and O
dispersed O
hNSCs B-CellType
in O
the O
graft O
zone. O
C: O
higher O
magnification O
of O
the O
inset O
in O
B. O
(D–I) O
Photos O
taken O
from O
frontal O
sections O
through O
the O
graft O
in O
the O
striatum B-Anatomy
double O
immunoprocessed O
for O
cell O
proliferation O
and O
neural B-Anatomy
lineage O
markers. O
(D) O
Note O
the O
endogenous O
Ki67+ O
cells O
(red O
cells, O
arrow) O
in O
the O
stroke O
damaged O
area O
and O
the O
hNuc+ O
(green)/Ki67- O
grafted O
hNSCs B-CellType
(arrowheads). O
(E) O
Examples O
of O
grafted O
SD56 B-CellLine
hNSCs I-CellType
showing O
co-expression O
of O
hNuc B-GeneProtein
(green) O
and O
nestin B-GeneProtein
(red). O
(F) O
Confocal O
3D O
reconstructed O
orthogonal O
images O
of O
the O
hNuc+(green)/DCX+(red) O
NSCs B-CellType
(arrowheads) O
viewed O
in O
the O
x-z O
plan O
on O
the O
top O
and O
y-z O
plan O
on O
the O
right. O
(G) O
Examples O
show O
the O
majority O
of O
grafted O
NSC O
progeny O
co-expressing O
hNuc B-GeneProtein
(red) O
and O
the O
neuronal B-Anatomy
marker O
TuJ1 B-GeneProtein
(green). O
Grafted O
NSCs B-CellType
rarely O
differentiate O
into O
GFAP+ O
astrocytes B-CellType
(H). O
In O
I, O
rare O
example O
of O
grafted O
NSC O
progeny O
becoming O
an O
oligodendrocyte O
identified O
by O
the O
expression O
of O
CNPase B-GeneProtein
(green). O
Grafted O
NSCs B-CellType
expressed O
the O
GABAergic B-CellType
marker O
GAD65/67 B-GeneProtein
(J) O
and O
rarely O
expressed O
glutamate B-GeneProtein
(K). O
(Abbreviations: O
Cx: O
cortex, O
Str: O
striatum). O
Bars: O
(B, O
C) O
100 O
µm; O
(D, O
F) O
20 O
µm; O
(E, O
G–K) O
10 O
µm. O
4. O
Transplanted O
cells O
improve O
sensorimotor O
function O
of O
the O
stroke-disabled O
forelimbWe O
asked O
whether O
transplanted O
SD56 B-CellLine
hNSCs I-CellType
could O
enhance O
the O
recovery O
of O
sensorimotor O
function O
that O
is O
compromised O
in O
the O
stroke-injured O
rats. O
We O
used O
the O
cylinder O
test O
to O
measure O
the O
sensorimotor O
asymmetry O
in O
forelimb O
use O
during O
spontaneous O
exploration O
[22]. O
To O
establish O
the O
baseline O
of O
the O
stroke-induced O
sensorimotor O
deficit, O
spontaneous O
behavior O
of O
rats B-Species
in O
a O
transparent O
cylinder O
was O
videotaped O
one O
week O
after O
stroke O
(pre-transplant, O
Figure O
4). O
Tests O
were O
then O
conducted O
4 O
and O
8 O
weeks O
after O
vehicle O
and O
SD56 B-CellLine
hNSCs I-CellType
transplantation. O
Stable O
asymmetry O
in O
forelimb O
use O
was O
observed O
7 O
days O
post-stroke O
(pre-transplant, O
Figure O
4). O
Ischemic O
rats B-Species
used O
their O
impaired O
forelimbs B-Anatomy
(contralateral O
to O
lesion) O
during O
lateral O
exploration O
less O
than O
they O
did O
before O
stroke. O
Transplantation O
of O
SD56 B-CellLine
hNSCs I-CellType
significantly O
enhanced O
the O
independent O
use O
of O
the O
impaired O
contralateral B-Anatomy
forelimb O
4 O
weeks O
post O
transplantation O
(P<0.05 O
vs O
pre-transplant). O
Eight O
weeks O
after O
transplantation O
the O
improvement O
in O
the O
use O
of O
the O
impaired O
forelimb O
was O
stable O
and O
significant O
when O
compared O
to O
the O
pre-transplant O
group O
and O
significantly O
improved O
in O
comparison O
to O
vehicle O
treated O
group O
at O
8 O
weeks O
(Figure O
4). O
In O
the O
vehicle O
treated O
group, O
the O
independent O
use O
of O
the O
contralateral B-Anatomy
forelimb O
remained O
impaired O
4 O
and O
8 O
weeks O
post-injection. O
In O
an O
independent O
study O
and O
using O
the O
same O
MCAO O
rat O
animal O
model, O
we O
found O
that O
transplantation O
of O
dermal B-Anatomy
fibroblasts I-Anatomy
did O
not O
improve O
the O
stroke-induced O
motor O
deficits O
(unpublished O
data).10.1371/journal.pone.0001644.g004Figure O
4Transplantation O
of O
NSCs B-CellType
improves O
sensorimotor O
function O
of O
the O
stroke-disabled O
forelimb.Forelimb O
use O
during O
spontaneous O
lateral O
exploration O
was O
measured O
in O
the O
cylinder O
test O
(see O
Method O
and O
Results O
sections O
for O
details). O
Groups O
of O
vehicle O
injected O
(n O
= O
7) O
and O
hNSCs B-CellType
(n O
= O
10) O
transplanted O
are O
represented. O
The O
animals O
were O
tested O
as O
described O
in O
Method O
section. O
Note O
the O
significant O
increase O
in O
the O
independent O
use O
of O
the O
impaired O
contralateral B-Anatomy
forelimb O
at O
4 O
and O
8 O
weeks O
post O
transplantation O
(P<0.05 O
vs O
pre-transplant O
group). O
The O
contralateral B-Anatomy
forelimb O
remained O
impaired O
in O
the O
vehicle O
treated O
group O
at O
4 O
and O
8 O
weeks O
post-injection. O
Bars O
represent O
percentages±s.e.m. O
of O
steps O
taken O
by O
the O
ipsilateral, O
contralateral B-Anatomy
and O
both O
forelimbs B-Anatomy
simultaneously. O
*P<0.05 O
vs O
pre-transplant O
group; O
#P<0.05 O
vs O
vehicle O
groups. O
Our O
results O
indicate O
that O
a O
self-renewable O
and O
homogenous O
population O
of O
hNSCs, O
SD56, O
was O
derived O
from O
hESCs B-CellType
using O
defined O
media O
supplemented O
with O
a O
specific O
combination O
of O
growth O
factors. O
The O
SD56 B-CellLine
hNSCs I-CellType
grew O
as O
an O
adherent O
monolayer O
culture, O
uniformly O
expressed O
molecular O
features O
of O
hNSCs B-CellType
including O
nestin, O
vimentin B-GeneProtein
and O
the O
radial B-CellType
glial I-CellType
marker O
3CB2, O
and O
did O
not O
express O
the O
pluripotency O
markers O
Oct4 B-GeneProtein
or O
Nanog. O
The O
self-renewal O
property O
of O
the O
hNSCs B-CellType
was O
characterized O
by O
a O
predominant O
symmetrical O
mode O
of O
cell O
division. O
They O
exhibited O
no O
chromosomal O
abnormalities O
and O
demonstrated O
non-tumorigenic O
properties O
after O
implantation O
into O
ischemic O
brains B-Species
and O
into O
naïve O
nude O
rat O
brains B-Anatomy
and O
flanks. O
Furthermore, O
the O
transplanted O
SD56 B-CellLine
hNSCs I-CellType
migrated O
toward O
the O
stroke-damaged O
adult O
brain B-Anatomy
parenchyma, O
engrafted O
and O
improved O
the O
independent O
use O
of O
the O
stroke-impaired O
forelimb. O
Maintenance O
of O
stem O
cells O
requires O
symmetrical O
and O
asymmetrical O
cell O
divisions O
to O
both O
expand O
and O
to O
give O
rise O
to O
specialized O
progeny B-CellType
of O
a O
specific O
tissue O
(reviewed O
in O
[23]). O
In O
vivo, O
a O
complex O
cellular O
micro-environment O
or O
niche O
ensures O
the O
self-maintenance O
property O
of O
NSCs B-CellType
[24], O
[25], O
[26], O
[27]. O
In O
vitro, O
defined O
growth O
factors O
and O
extracellular B-CellComponent
matrices I-CellComponent
support O
stem O
cell O
self-renewal O
[28], O
[29]. O
The O
embryonic B-Anatomy
stem I-CellType
cells I-CellType
can O
propagate O
in O
a O
predominantly O
proliferative O
symmetrical O
mode, O
leading O
to O
homogeneous O
cell O
cultures O
growing O
relatively O
quickly O
with O
minimal O
cell O
differentiation O
[30], O
[31], O
[32], O
[33], O
[34]. O
Tissue O
specific O
stem B-CellType
cells, O
however, O
self-renew O
in O
a O
predominant O
asymmetric O
mode O
to O
maintain O
them O
selves O
and O
compensate O
for O
the O
loss O
of O
differentiated O
cells O
due O
to O
disease O
or O
injury. O
Thus, O
NSCs B-CellType
isolated O
from O
developing O
or O
adult O
brain O
grow O
as O
a O
mixture O
of O
undifferentiated O
and O
differentiated O
cells O
due O
the O
predominant O
asymmetrical O
mode O
of O
cell O
division O
[35], O
[36], O
[37], O
[38], O
[39], O
[40], O
[41]. O
A O
recent O
study O
has O
reported O
that O
a O
murine B-Species
ESC-derived O
NSC O
line O
(LC1) O
is O
propagated O
as O
an O
adherent O
homogenous O
culture O
with O
a O
dominant O
mode O
of O
symmetrical O
self-renewal O
[21]. O
A O
combination O
of O
EGF B-GeneProtein
and O
FGF2 B-GeneProtein
was O
sufficient O
to O
propagate O
these O
NSCs B-CellType
as O
an O
adherent O
monolayer. O
However, O
the O
SD56 B-CellLine
hNSC O
line O
described O
here O
required O
the O
combination O
of O
EGF, O
bFGF B-GeneProtein
and O
LIF B-GeneProtein
for O
self-maintenance. O
Although O
there O
are O
morphological O
and O
molecular O
similarities O
between O
our O
hNSCs B-CellType
and O
the O
NSCs B-CellType
previously O
described O
[21], O
the O
methods O
of O
isolation O
and O
growth O
are O
different. O
In O
addition O
to O
the O
different O
combination O
of O
growth O
factors O
used, O
the O
hNSC O
line O
we O
have O
isolated O
did O
not O
go O
through O
the O
rosette-structure O
stage. O
The O
in O
vitro O
analysis O
of O
BrdU O
incorporation O
and O
nestin O
expression O
indicated O
that O
our O
hNSCs B-CellType
divide O
predominantly O
symmetrically. O
This O
type O
of O
growth O
pattern O
is O
characteristic O
of O
primitive O
normal O
stem O
cells O
undergoing O
mostly O
symmetric O
cell O
division O
to O
increase O
the O
stem O
cell O
pool O
at O
the O
early O
stage O
of O
development O
or O
during O
tissue O
regeneration O
after O
injury O
[23]. O
RT-PCR O
and O
immunocytochemistry O
analysis O
demonstrated O
that O
these O
undifferentiated O
SD56 B-CellLine
cells I-CellType
did O
not O
express O
any O
pluripotency, O
endodermal B-Anatomy
or O
mesodermal B-Anatomy
markers. O
Furthermore, O
the O
SD56 B-CellLine
hNSCs I-CellType
described O
here O
exhibited O
the O
multipotential O
characteristic O
to O
differentiate O
into O
neurons, O
astrocytes B-CellType
and O
oligodendrocytes B-CellType
both O
in O
vitro O
and O
upon O
transplantation. O
Together O
these O
findings O
suggest O
that O
the O
hNSC O
line O
we O
isolated O
are O
appropriately O
programmed O
and O
share O
similar O
characteristics O
with O
the O
definitive O
NSCs B-CellType
of O
the O
developing O
brain. O
The O
SD56 B-CellLine
hNSCs I-CellType
demonstrated O
a O
remarkable O
ability O
to O
migrate O
toward O
the O
stroke-damaged O
parenchyma B-Anatomy
of O
the O
adult O
rat O
brain. O
This O
directed O
migration O
by O
the O
majority O
of O
the O
grafted O
cells O
could O
be O
due O
to O
their O
uniform O
cellular O
composition, O
which O
results O
in O
an O
equal O
response O
to O
the O
host O
microenvironment. O
In O
previous O
studies, O
subpopulations O
of O
transplanted O
hESCs B-CellType
that O
were O
enriched O
in O
neural B-Anatomy
cells I-CellType
migrated O
in O
host O
microenvironments O
conducive O
to O
cell O
migration, O
such O
as O
the O
developing O
brain O
or O
in O
structures O
such O
as O
the O
rostral B-Anatomy
migratory I-Anatomy
stream I-Anatomy
[13], O
[20]. O
In O
the O
adult O
lesioned O
brain, O
the O
grafted O
hESC-derived O
neural B-Anatomy
cells I-CellType
proliferated O
and O
formed O
rosettes B-Anatomy
[14], O
teratomas B-Anatomy
[12], O
[15] O
or O
a O
cellular O
mass O
that O
induced O
a O
gliotic O
host O
response O
whereby O
local O
astrocytes B-CellType
demarcated O
the O
grafts O
[16]. O
Enriched O
neural B-Anatomy
cultures O
derived O
from O
mouse B-Species
[42] O
and O
monkey B-Species
ESCs B-CellType
[43] O
have O
produced O
behavioral O
improvements O
when O
transplanted O
into O
animal O
models O
of O
stroke O
and O
brain O
injury. O
However, O
in O
these O
cases, O
the O
transplanted O
non-human B-Species
ESCs B-CellType
also O
formed O
a O
mass O
with O
signs O
of O
overgrowth O
in O
the O
core, O
as O
well O
as O
deformations O
[44], O
[45], O
[46]. O
ESCs B-CellType
plated O
at O
low O
density O
acquire O
neural O
identity O
within O
few O
hours O
after O
plating O
[47]. O
Interestingly, O
nearly O
all O
viable O
cells O
expressed O
nestin, O
the O
early O
neural O
fate O
marker O
Sox1, O
and O
the O
pluripotency O
marker O
Oct4. O
Together, O
these O
studies O
are O
seminal O
and O
suggest O
that O
complete O
neuralization O
may O
not O
be O
achieved O
through O
certain O
enrichment O
processes, O
consequently O
the O
neural B-Anatomy
cells I-CellType
could O
revert O
to O
a O
pluripotent O
stage O
[17]. O
The O
dispersion O
of O
the O
grafted O
hNSCs B-CellType
within O
host O
parenchyma B-Anatomy
may O
allow O
for O
more O
graft-host O
interactions O
that O
could O
stabilize O
differentiation, O
inhibit O
growth O
and O
prevent O
gliotic O
host O
response. O
In O
the O
present O
study, O
SD56 B-CellLine
hNSC-transplanted O
animals O
demonstrated O
a O
stable O
improvement O
in O
the O
sensorimotor O
function O
when O
evaluated O
for O
spontaneous O
exploratory O
activity O
in O
the O
cylinder O
test O
that O
detects O
long-lasting O
deficits O
in O
forelimb O
use O
in O
the O
experimental O
models O
of O
stroke O
[22]. O
The O
transplantation O
of O
hNSCs B-CellType
significantly O
enhanced O
the O
independent O
use O
of O
the O
impaired O
contralateral O
forelimb O
8 O
weeks O
post O
transplantation. O
This O
is O
the O
first O
report O
demonstrating O
that O
the O
transplantation O
of O
hNSCs B-CellType
derived O
from O
hESCs B-CellType
can O
improve O
neurologic O
behavior O
after O
experimental O
stroke. O
Together, O
these O
findings O
are O
encouraging O
and O
suggest O
that O
these O
cells O
are O
promising O
for O
future O
development. O
In O
addition O
to O
their O
therapeutic O
application, O
the O
hNSCs B-CellType
isolated O
under O
the O
reported O
conditions O
offer O
a O
means O
to O
interrogate O
host O
environments O
and O
unravel O
mechanistic O
features O
of O
self-renewal, O
non-tumorigenicity O
and O
functional O
engraftability O
in O
animal O
models O
of O
neurological O
disorders. O
hESC O
and O
NSC O
CulturesThe O
hESC O
line O
H9 B-CellLine
(WiCell O
Research O
Institute) O
was O
propagated O
every O
5 O
to O
7 O
days O
on O
irradiated O
mouse B-Species
embryonic B-Anatomy
fibroblasts. O
The O
cell O
culture O
media O
consisted O
of O
a O
1∶1 O
mixture O
of O
Dulbecco's O
modified O
Eagle's O
medium O
(DMEM) O
and O
F12 O
nutrient, O
20% O
serum O
replacement O
(Invitrogen), O
0.1 O
mM O
β-mercaptoethanol, O
2 O
µg/ml O
heparin B-GeneProtein
and O
4 O
ng/ml O
bFGF B-GeneProtein
(R&D O
Systems). O
To O
generate O
the O
NSCs, O
dissociated O
hESCs B-CellType
were O
cultured O
in O
a O
chemically O
defined O
medium O
composed O
of O
DMEM/F12 O
(1∶1) O
including O
glucose O
(0.6%), O
glutamine O
(2 O
mM), O
sodium O
bicarbonate O
(3 O
mM), O
and O
HEPES O
buffer O
(5 O
mM) O
[all O
from O
Sigma O
except O
glutamine O
(Invitrogen)]. O
A O
defined O
hormone O
mix O
and O
salt O
mixture O
(Sigma), O
including O
insulin B-GeneProtein
(25 O
mg/ml), O
transferrin B-GeneProtein
(100 O
mg/ml), O
progesterone O
(20 O
nM), O
putrescine O
(60 O
mM), O
and O
selenium O
chloride O
(30 O
nM) O
was O
used O
in O
place O
of O
serum. O
The O
medium O
was O
supplemented O
with O
EGF B-GeneProtein
(20 O
ng/ml), O
bFGF B-GeneProtein
(10 O
ng/ml) O
and O
LIF B-GeneProtein
(10 O
ng/ml). O
Dissociated O
hNSCs B-CellType
were O
plated O
at O
a O
density O
of O
100,000 O
cell/ml O
in O
Corning O
T75 O
(Invitrogen) O
culture O
flasks O
in O
the O
defined O
media O
together O
with O
the O
growth O
factors. O
After O
5–7 O
DIV, O
the O
adherent O
culture O
was O
incubated O
in O
0.025%trypsin/0.01% O
EDTA O
(w/v) O
for O
1 O
min O
followed O
by O
the O
addition O
of O
trypsin B-GeneProtein
inhibitor O
(Invitrogen) O
then O
gently O
triturated O
to O
achieve O
single O
cell O
suspension. O
The O
cells O
were O
then O
washed O
twice O
with O
fresh O
medium O
and O
reseeded O
in O
fresh O
growth O
factor-containing O
media O
at O
100,000 O
cells/ml. O
This O
procedure O
was O
performed O
for O
21 O
passages O
and O
the O
fold O
of O
increase O
and O
population O
doubling O
were O
calculated O
at O
each O
passage. O
For O
clonal O
analysis, O
single O
spheres O
or O
confluent O
hNSC O
cultures O
were O
single O
cell O
dissociated O
and O
serially O
diluted O
to O
yield O
1–2 O
cell/10 O
µl. O
A O
10-µl-cell O
suspension O
was O
then O
added O
to O
each O
of O
96 O
or O
24 O
well O
plates O
containing O
200–300 O
µl O
of O
growth O
media. O
Wells O
containing O
one O
viable O
cell O
were O
marked O
the O
next O
day O
and O
re-scored O
5 O
to O
7 O
days O
later O
for O
cell O
proliferation. O
The O
differentiation O
of O
the O
hNSCs B-CellType
was O
performed O
as O
previously O
described O
[48]. O
Dissociated O
hNSCs B-CellType
were O
plated O
at O
a O
density O
of O
106 O
cells/ml O
in O
control O
(media/hormone O
mix) O
medium O
devoid O
of O
any O
growth O
factors O
and O
supplemented O
with O
1% O
fetal B-Anatomy
bovine B-Species
serum O
(FBS) O
on O
poly-L-ornithine-coated O
(15 O
mg/ml; O
Sigma) O
glass O
coverslips O
in O
24-well O
Nunclon O
culture O
dishes O
with O
0.5 O
ml/well. O
After O
2, O
7–15 O
DIV O
cultures O
were O
fixed O
and O
processed O
for O
immunocytochemistry O
or O
used O
for O
RT-PCR O
analysis. O
Karyotype O
analysisLong-term O
cultures O
of O
hNSCs B-CellType
were O
incubated O
at O
37°C O
and O
harvested O
for O
metaphase O
chromosomes O
when O
the O
cultures O
were O
75% O
confluent. O
Metaphase O
chromosomes B-CellComponent
were O
obtained O
by O
standard O
chromosome O
harvest O
methods O
by O
exposure O
to O
Colcemid O
at O
0.1 O
µg/ml O
for O
1 O
hour O
at O
37°C, O
a O
2-minute O
exposure O
to O
trypsin/EDTA, O
hypotonized O
with O
0.057 O
M O
KCl O
and O
fixed O
with O
3∶1 O
methanol:acetic O
acid. O
Slide O
preparations O
were O
made O
by O
dropping O
the O
fixed O
cell O
pellet O
onto O
cold, O
wet O
slides O
and O
air-dried. O
After O
incubating O
the O
slides O
at O
90°C O
for O
30 O
minutes, O
chromosomes B-CellComponent
were O
trypsin B-GeneProtein
banded O
and O
then O
Wright/Giemsa O
stained O
for O
G-banding O
analysis. O
Twenty O
metaphase O
cells O
were O
completely O
analyzed O
and O
a O
normal O
female B-Anatomy
chromosome O
complement O
was O
found O
(46,XX). O
Tumorigenicity O
in O
nude O
ratsAll O
animal O
experiments O
were O
conducted O
according O
to O
the O
National O
Institute O
of O
Health O
(NIH) O
guidelines O
and O
approved O
by O
the O
IACUC. O
Normal O
adult O
NIH-Nude O
rats B-Species
(n O
= O
5, O
8 O
week-old, O
Taconic, O
Germantown, O
New O
York, O
United O
States) O
were O
used O
to O
test O
the O
tumorigenic O
potential O
of O
the O
SD56 B-CellLine
hNSCs. O
Undifferentiated O
hNSCs B-CellType
from O
passage O
9 O
were O
single O
cell O
dissociated O
using O
trypsin-EDTA O
and O
suspended O
at O
the O
concentration O
of O
125,000 O
cell/µl O
in O
preparation O
for O
cell O
transplantation. O
Two O
µl O
of O
the O
cell O
suspension O
were O
stereotaxically O
transplanted O
into O
4 O
sites O
within O
the O
striatum O
at O
the O
following O
coordinates: O
AP: O
+1.0 O
mm, O
ML: O
+3.2 O
mm, O
DV: O
−5.0; O
AP: O
+0.5 O
mm, O
ML: O
+3.0 O
mm, O
DV: O
−5.0; O
AP: O
−0.5 O
mm, O
ML: O
+3.0 O
mm, O
DV: O
−5.0; O
AP: O
−1.0 O
mm, O
ML: O
+3.5 O
mm, O
DV: O
−5.0 O
mm O
with O
the O
incisor O
bar O
set O
at O
3.4 O
mm. O
The O
injection O
rate O
was O
1 O
µl/min, O
and O
the O
cannula O
was O
left O
in O
place O
for O
an O
additional O
5 O
min O
before O
retraction. O
For O
the O
flank O
tumor O
assay, O
2×106 O
cells O
(125,000 O
cell/µl) O
were O
injected O
subcutaneously B-Anatomy
to O
the O
side O
of O
the O
adult O
nude O
rats. O
To O
label O
mitotically O
active O
cells O
in O
vivo O
during O
S-phase, O
the O
rats B-Species
were O
injected O
IP O
with O
the O
BrdU O
(50 O
mg/kg, O
Sigma) O
every O
8 O
hours O
during O
the O
last O
24 O
hours O
before O
euthanasia. O
After O
2-month O
survival O
time, O
rats B-Species
were O
euthanized O
and O
a O
postmortem O
examination O
for O
tumor O
formation O
was O
performed. O
Induction O
of O
Focal O
Ischemia O
and O
Cell O
TransplantationAll O
animal O
experimentations O
were O
conducted O
according O
to O
the O
National O
Institute O
of O
Health O
(NIH) O
guidelines O
and O
approved O
by O
the O
IACUC. O
Sprague B-Species
Dawley I-Species
adult I-Species
male I-Species
rats I-Species
(n O
= O
17, O
275g–310g, O
Charles O
River O
Laboratories, O
Wilmington, O
Massachusetts, O
United O
States) O
were O
subjected O
to O
one O
and O
a O
half O
hour O
suture O
occlusion O
of O
the O
middle B-Anatomy
cerebral I-Anatomy
artery I-Anatomy
(MCAO), O
as O
previously O
described O
[49] O
and O
immunosuppressed O
2 O
days O
before O
cell O
transplantation O
and O
daily O
thereafter O
for O
one O
week O
with O
i.p. O
injections O
of O
cyclosporine O
A O
(20 O
mg/ml, O
Sandimmune, O
Novartis O
Pharmaceuticals). O
Thereafter O
oral O
cyclosporine O
was O
used O
at O
210 O
µg/ml O
in O
drinking O
water O
until O
euthanasia. O
Undifferentiated O
SD56 B-CellLine
hNSCs B-CellType
from O
passages O
between O
P9 O
and O
P13 O
were O
single O
cell O
dissociated O
using O
trypsin-EDTA O
in O
preparation O
for O
cell O
transplantation. O
One O
week O
after O
the O
stroke O
lesion, O
2 O
µl O
of O
the O
hNSCs, O
at O
a O
concentration O
of O
50,000 O
cell/µl, O
were O
stereotaxically O
transplanted O
into O
4 O
sites O
within O
the O
lesioned O
striatum O
(n O
= O
10) O
at O
the O
following O
coordinates: O
AP: O
+1.0 O
mm, O
ML: O
+3.2 O
mm, O
DV: O
−5.0; O
AP: O
+0.5 O
mm, O
ML: O
+3.0 O
mm, O
DV: O
−5.0; O
AP: O
−0.5 O
mm, O
ML: O
+3.0 O
mm, O
DV: O
−5.0; O
AP: O
−1.0 O
mm, O
ML: O
+3.5 O
mm, O
DV: O
−5.0 O
mm O
with O
the O
incisor O
bar O
set O
at O
3.4 O
mm. O
The O
injection O
rate O
was O
1 O
µl/min, O
and O
the O
cannula O
was O
left O
in O
place O
for O
an O
additional O
5 O
min O
before O
retraction. O
As O
a O
control O
group, O
we O
used O
rats B-Species
subjected O
to O
ischemia O
and O
injected O
with O
the O
vehicle O
(n O
= O
7). O
All O
animals O
underwent O
baseline O
motor O
behavioral O
assessment O
before O
and O
after O
the O
ischemic O
lesion, O
and O
4 O
& O
8 O
weeks O
after O
cell O
transplantation. O
The O
animals O
were O
killed O
after O
2-month O
survival O
time O
by O
transcardial O
perfusion O
with O
phosphate O
buffered O
saline O
(PBS) O
followed O
by O
4% O
paraformaldehyde. O
The O
brains O
were O
cryoprotected O
in O
an O
increasing O
gradient O
of O
10, O
20 O
and O
30% O
sucrose O
solution O
and O
cryostat O
sectioned O
at O
40 O
µm O
and O
processed O
for O
immunocytochemistry. O
ImmunocytochemistryCultures O
were O
fixed O
with O
4% O
paraformaldehyde O
for O
15 O
min. O
Both O
cells O
and O
brain O
sections O
were O
rinsed O
in O
PBS O
for O
3×5 O
min O
then O
incubated O
for O
2 O
hrs O
(cultures) O
or O
overnight O
(brain O
sections) O
with O
the O
appropriate O
primary O
antibodies O
for O
multiple O
labeling. O
Secondary O
antibodies O
raised O
in O
the O
appropriate O
hosts O
and O
conjugated O
to O
FITC, O
RITC, O
AMCA, O
CY3 O
or O
CY5 O
chromogenes O
(Jackson O
ImmunoResearch) O
were O
used. O
Cells O
and O
sections O
were O
counterstained O
with O
the O
nuclear O
marker O
4′,6-diamidine-2′-phenylindole O
dihydrochloride O
(DAPI). O
Positive O
and O
negative O
controls O
were O
included O
in O
each O
run. O
Immunostained O
sections O
were O
coverslipped O
using O
fluorsave O
(Calbiochem) O
as O
the O
mounting O
medium. O
The O
following O
antibodies O
were O
used: O
Anti-human B-GeneProtein
Nuclei I-GeneProtein
(hNuc, O
monoclonal O
1∶100, O
Chemicon), O
Anti-TuJ1 B-GeneProtein
(monoclonal O
1∶100, O
Covance; O
Polyclonal O
1∶200, O
Aves O
Labs); O
anti-GAD65/67 B-GeneProtein
(polyclonal O
1∶1000, O
Chemicon); O
Anti-glial B-GeneProtein
fibrillary I-GeneProtein
acidic I-GeneProtein
protein I-GeneProtein
(GFAP, O
monoclonal O
1∶1000, O
Chemicon; O
polyclonal O
1∶200, O
Aves O
Labs); O
Anti-galactocerebrocide B-GeneProtein
(GC, O
monoclonal O
1∶250, O
Chemicon); O
Anti-CNPase B-GeneProtein
(polyclonal O
1∶200, O
Aves O
Labs); O
Anti-Glucose B-GeneProtein
Transporter I-GeneProtein
type I-GeneProtein
1 I-GeneProtein
(Glut-1 O
polyclonal, O
1∶500, O
Chemicon); O
Anti-Nestin B-GeneProtein
(polyclonal O
1∶1000, O
Chemicon); O
Anti-vimentin B-GeneProtein
(monoclonal O
1∶500, O
Calbiochem); O
Anti-3CB2 B-GeneProtein
(monoclonal O
1∶500, O
Developmental O
Studies O
Hybridoma O
Bank); O
Anti-doublecortin B-GeneProtein
(DCX, O
polyclonal O
1∶250, O
SantaCruz O
Biotechnology); O
Anti-Ki67 B-GeneProtein
(polyclonal O
1∶250, O
SantaCruz O
Biotechnology). O
Fluorescence O
was O
detected, O
analyzed O
and O
photographed O
with O
a O
Zeiss O
LSM550 O
laser O
scanning O
confocal O
photomicroscope. O
For O
each O
animal, O
quantitative O
estimates O
of O
the O
total O
number O
of O
grafted O
cells O
were O
stereologically O
determined O
using O
the O
optical O
fractionator O
procedure O
[50]. O
A O
computer-assisted O
image O
analysis O
system O
was O
performed O
using O
Stereo O
Investigator O
software O
(MicroBrightField, O
Inc.). O
The O
rostral B-Anatomy
and O
caudal B-Anatomy
limits O
of O
the O
reference O
volume O
were O
determined O
by O
first O
and O
last O
frontal O
sections O
containing O
grafted O
cells. O
The O
striatum B-Anatomy
and O
cortex B-Anatomy
were O
accurately O
outlined O
at O
low O
magnification O
(2.5× O
objective). O
The O
optical O
fractionator O
probe O
was O
selected O
to O
perform O
systematic O
sampling O
of O
the O
immunoreactive O
cell O
population O
distributed O
within O
the O
serial O
sections O
to O
estimate O
the O
population O
number O
in O
the O
volume O
of O
tissue. O
The O
counting O
frame O
of O
the O
optical O
fractionator O
was O
defined O
at O
50×50 O
µm O
squares O
and O
the O
systematic O
sampling O
was O
performed O
by O
translating O
a O
grid O
with O
200×200 O
µm O
squares O
onto O
the O
sections O
of O
interest O
using O
the O
Stereo O
Investigator O
software. O
The O
sample O
sites O
were O
systematically O
and O
automatically O
generated O
by O
the O
computer O
and O
examined O
using O
a O
60× O
objective O
of O
a O
Nikon O
Eclipse O
TE O
300 O
microscope. O
The O
counting O
frame O
displayed O
inclusion O
and O
exclusion O
lines O
and O
only O
immunoreactive O
cell O
bodies O
falling O
within O
the O
counting O
frame O
with O
no O
contact O
with O
the O
exclusion O
lines O
were O
counted. O
The O
cell O
dispersion O
was O
measured O
by O
counting O
the O
number O
of O
cells O
within O
200 O
µm O
distance O
from O
the O
graft O
site. O
The O
number O
and O
distance O
in O
µm O
of O
cells O
dispersed O
beyond O
200 O
µm O
was O
also O
measured. O
An O
average O
of O
2,000 O
cells O
was O
counted O
per O
animal. O
Double O
labeling O
was O
determined O
using O
the O
confocal O
laser O
scanning O
microscope O
by O
random O
sampling O
of O
100 O
or O
more O
cells O
per O
marker O
for O
each O
animal, O
scoring O
first O
for O
hNuc+, O
followed O
by O
DAPI+ O
nuclei B-CellComponent
and O
then O
the O
marker O
of O
choice. O
The O
double O
labeling O
was O
always O
confirmed O
in O
x-z O
and O
y-z O
cross-sections O
produced O
by O
the O
orthogonal O
projections O
of O
z-series. O
Reverse O
Transcription-Polymerase O
Chain O
Reaction O
(RT-PCR) O
analysisTotal O
RNA O
was O
extracted O
from O
cultured O
cells O
using O
RNAeasy O
kit O
(Quiagen). O
Aliquots O
(1 O
µg) O
of O
total O
RNA O
from O
the O
cells O
were O
reverse O
transcribed O
in O
the O
presence O
of O
50 O
mM O
Tris-HCl, O
pH O
8.3, O
75 O
mM O
KCl, O
3 O
mM O
MgCl2, O
10 O
mM O
DTT, O
0.5 O
µM O
dNTPs, O
and O
0.5 O
µg O
oligo-dT(12–18) O
with O
200 O
U O
Superscript O
RNase B-GeneProtein
H-Reverse O
Transcriptase B-GeneProtein
(Invitrogen). O
PCR O
amplification O
was O
performed O
using O
standard O
procedure O
with O
Taq O
Polymerase. O
Aliquots O
of O
cDNA O
equivalent O
to O
50 O
ng O
of O
total O
RNA O
were O
amplified O
in O
25 O
µl O
reactions O
containing O
10 O
mM O
Tris-HCl, O
pH O
8.3, O
50 O
mM O
KCl, O
1.5 O
mM O
MgCl2 O
, O
50 O
pmol O
of O
each O
primer, O
400 O
µM O
dNTPs, O
and O
0.5 O
U O
AmpliTaq O
DNA O
polymerase B-GeneProtein
(Perkin-Elmer). O
PCR O
was O
performed O
using O
the O
following O
thermal O
profile: O
4 O
min O
at O
94°C; O
1 O
min O
at O
94°C, O
1 O
min O
at O
60°C, O
1.5 O
min O
at O
72°C, O
for O
30–40 O
cycles; O
7 O
min O
at O
72°C, O
and O
finally O
a O
soak O
at O
4°C O
overnight. O
The O
following O
day, O
10 O
µl O
aliquots O
of O
the O
amplified O
products O
were O
run O
on O
a O
2% O
agarose O
Tris–acetate O
gel O
containing O
0.5 O
mg/ml O
ethidium O
bromide. O
The O
products O
were O
visualized O
through O
a O
UV O
transilluminator, O
captured O
in O
a O
digital O
format O
using O
Quantify O
One O
Gel O
Analysis O
software O
(Bio-Rad O
Laboratories) O
on O
a O
Macintosh O
G4 O
computer.The O
PCR O
primers O
specific O
to O
each O
transcript O
were O
as O
follows: O
GFAP, O
forward O
(F), O
5′-TCATCGCTCAGGAGGTCCTT–3′ O
Reverse O
(R), O
5′-CTG O
TTGCCAGAGATGGAGGTT–3′; O
MAP2 B-GeneProtein
(F) O
5′-GAAGACTCGCATCCGAATGG–3′, O
(R) O
5′-CGCAGGATAGGAGGAAGAGACT–3′; O
MBP B-GeneProtein
(F) O
5′-TTAGCTGAATTC O
GCGTGTGG–3′, O
(R) O
5′-GAGGAAGTGAATGAGCCGGTTA-3′ O
were O
deigned O
using O
the O
Primer O
Designer O
software, O
Version O
2.0 O
(Scientific O
and O
Educational O
Software) O
[48]. O
18S, O
β-tubulin B-GeneProtein
class I-GeneProtein
III, O
N-CAM, O
Nestin, O
NF-M, O
Notch-1 B-GeneProtein
primers O
[51]. O
Oct4, O
Nanog B-GeneProtein
primers O
[11]. O
FOXa2 B-GeneProtein
(HNF3B), O
Brachyury B-GeneProtein
primers O
[52]. O
Behavioral O
testsThe O
cylinder O
test O
was O
used O
to O
assess O
the O
spontaneous O
forelimb O
use O
during O
lateral O
exploration O
movement O
[22]. O
Rats B-Species
were O
placed O
in O
a O
transparent O
acrylic O
cylinder O
(20 O
cm O
diameter) O
for O
5 O
minutes. O
The O
cylinder O
encourages O
use O
of O
the O
forelimbs B-Anatomy
for O
vertical O
exploration. O
A O
mirror O
was O
placed O
behind O
the O
cylinder O
so O
that O
the O
forelimbs B-Anatomy
could O
be O
viewed O
at O
all O
times. O
Testing O
sessions O
were O
videotaped O
and O
forelimb O
use O
was O
scored O
by O
a O
blinded O
operator. O
Movements O
scored O
were O
the O
independent O
use O
of O
the O
left O
or O
right O
forelimb O
or O
simultaneous O
use O
of O
both O
the O
left O
and O
right O
forelimb O
to O
contact O
the O
wall O
of O
the O
cylinder O
during O
a O
full O
rear, O
to O
initiate O
a O
weight-shifting O
movement, O
or O
to O
regain O
center O
of O
gravity O
while O
moving O
laterally O
in O
a O
vertical O
posture O
along O
the O
wall. O
Animals O
were O
tested O
for O
their O
baselines O
after O
stroke O
and O
4 O
and O
8 O
weeks O
after O
cell O
transplantation. O
Statistical O
analysisOutcome O
measurement O
for O
each O
experiment O
was O
reported O
as O
mean±SEM. O
All O
data O
were O
analyzed O
using O
SPSS O
11 O
for O
Mac O
OS O
X O
(SPSS O
Inc.). O
Significance O
of O
inter-group O
differences O
was O
performed O
by O
applying O
Student's O
t-test O
where O
appropriate. O
The O
One-Way O
ANOVA O
analysis O
was O
used O
to O
compare O
group O
differences O
for O
the O
forelimb O
use O
as O
the O
dependant O
variable O
and O
groups O
as O
the O
single O
independent O
factor O
variable. O
Differences O
between O
the O
groups O
were O
determined O
using O
Bonferroni's O
post O
hoc O
test. O
A O
P-value O
of O
less O
than O
0.05 O
was O
considered O
to O
be O
statistically O
significant. O