16623949.conll

BackgroundMany O
novel O
studies O
and O
therapies O
are O
possible O
with O
the O
use O
of O
human B-Species
embryonic I-CellType
stem I-CellType
cells I-CellType
(hES O
cells) O
and O
their O
differentiated O
cell B-CellType
progeny. O
The O
hES B-CellType
cell O
derived O
CD34 B-GeneProtein
hematopoietic I-CellType
stem I-CellType
cells I-CellType
can O
be O
potentially O
used O
for O
many O
gene O
therapy O
applications. O
Here O
we O
evaluated O
the O
capacity O
of O
hES O
cell O
derived O
CD34 B-GeneProtein
cells I-CellType
to O
give O
rise O
to O
normal O
macrophages B-CellType
as O
a O
first O
step O
towards O
using O
these O
cells O
in O
viral O
infection O
studies O
and O
in O
developing O
novel O
stem O
cell O
based O
gene O
therapy O
strategies O
for O
AIDS.ResultsUndifferentiated O
normal O
and O
lentiviral O
vector O
transduced O
hES B-CellType
cells I-CellType
were O
cultured O
on O
S17 B-CellLine
mouse B-Species
bone B-Anatomy
marrow I-Anatomy
stromal B-Anatomy
cell O
layers B-Anatomy
to O
derive O
CD34 B-GeneProtein
hematopoietic I-CellType
progenitor I-CellType
cells. O
The O
differentiated O
CD34 B-GeneProtein
cells I-CellType
isolated O
from O
cystic B-Anatomy
bodies I-Anatomy
were O
further O
cultured O
in O
cytokine O
media O
to O
derive O
macrophages. O
Phenotypic O
and O
functional O
analyses O
were O
carried O
out O
to O
compare O
these O
with O
that O
of O
fetal B-Anatomy
liver B-Anatomy
CD34 B-GeneProtein
cell O
derived O
macrophages. O
As O
assessed O
by O
FACS O
analysis, O
the O
hES-CD34 O
cell O
derived O
macrophages B-CellType
displayed O
characteristic O
cell O
surface O
markers O
CD14, O
CD4, O
CCR5, O
CXCR4, O
and O
HLA-DR B-GeneProtein
suggesting O
a O
normal O
phenotype. O
Tests O
evaluating O
phagocytosis, O
upregulation O
of O
the O
costimulatory O
molecule O
B7.1, O
and O
cytokine O
secretion O
in O
response O
to O
LPS O
stimulation O
showed O
that O
these O
macrophages B-CellType
are O
also O
functionally O
normal. O
When O
infected O
with O
HIV-1, O
the O
differentiated O
macrophages B-CellType
supported O
productive O
viral O
infection. O
Lentiviral B-Anatomy
vector B-Anatomy
transduced O
hES B-CellType
cells I-CellType
expressing O
the O
transgene O
GFP B-GeneProtein
were O
evaluated O
similarly O
like O
above. O
The O
transgenic O
hES B-CellType
cells I-CellType
also O
gave O
rise O
to O
macrophages B-CellType
with O
normal O
phenotypic O
and O
functional O
characteristics O
indicating O
no O
vector O
mediated O
adverse O
effects O
during O
differentiation.ConclusionPhenotypically O
normal O
and O
functionally O
competent O
macrophages B-CellType
could O
be O
derived O
from O
hES-CD34 O
cells. O
Since O
these O
cells B-Anatomy
are O
susceptible O
to O
HIV-1 B-Anatomy
infection, O
they O
provide O
a O
uniform O
source O
of O
macrophages B-CellType
for O
viral B-Anatomy
infection O
studies. O
Based O
on O
these O
results, O
it O
is O
also O
now O
feasible O
to O
transduce O
hES-CD34 O
cells I-CellType
with O
anti-HIV B-Anatomy
genes O
such O
as O
inhibitory O
siRNAs B-GeneProtein
and O
test O
their O
antiviral B-Anatomy
efficacy O
in O
down O
stream O
differentiated O
cells O
such O
as O
macrophages B-CellType
which O
are O
among O
the O
primary B-CellType
cells I-CellType
that O
need O
to O
be O
protected O
against O
HIV-1 B-Anatomy
infection. O
Thus, O
the O
potential O
utility O
of O
hES B-CellType
derived O
CD34 B-GeneProtein
hematopoietic I-CellType
cells I-CellType
for O
HIV-1 B-Anatomy
gene O
therapy O
can O
be O
evaluated. O
Human B-Species
embryonic I-CellType
stem I-CellType
cells I-CellType
(hES O
cells) O
show O
great O
promise O
for O
many O
novel O
cellular O
therapies O
due O
to O
their O
pluripotent O
nature O
[1]. O
These O
cells O
have O
the O
capacity O
to O
give O
rise O
to O
mature O
cells O
and O
tissues O
that O
arise O
from O
all O
three O
germ B-Anatomy
layers I-Anatomy
during O
embryonic B-Anatomy
development O
[2-4]. O
Several O
pluripotent O
hES O
cell O
lines O
have O
so O
far O
been O
derived O
from O
the O
inner B-Anatomy
cell I-Anatomy
mass I-Anatomy
of O
human B-Species
blastocysts B-Anatomy
and O
can O
be O
cultured O
indefinitely O
in O
an O
undifferentiated O
state O
[5-7]. O
Thus, O
these O
cells O
provide O
a O
renewable O
source O
of O
pluripotent O
stem B-CellType
cells I-CellType
from O
which O
many O
types O
of O
differentiated O
cells O
could O
be O
produced O
for O
experimental O
and O
therapeutic O
purposes. O
Cell O
differentiation O
protocols O
currently O
exist O
for O
the O
derivation O
of O
neurons, O
cardiomyocytes, O
endothelial B-Anatomy
cells, O
hematopoietic B-Anatomy
progenitor I-CellType
cells, O
keratinocytes, O
osteoblasts, O
and O
hepatocytes B-CellType
to O
name O
a O
few O
[2,3,8,9]. O
In O
addition O
to O
providing O
for O
potential O
cellular O
replacement O
therapies, O
opportunities O
exist O
in O
programming O
hES B-CellType
cells I-CellType
to O
correct O
a O
genetic O
defect O
and/or O
to O
express O
a O
therapeutic O
transgene O
of O
interest. O
Using O
such O
approaches, O
many O
possibilities O
exist O
for O
treating O
a O
number O
of O
genetic O
and O
immune B-Anatomy
system I-Anatomy
disorders O
[1]. O
Many O
novel O
applications O
can O
be O
foreseen O
for O
hES B-CellType
cells I-CellType
in O
infectious O
disease O
research. O
AIDS O
is O
a O
potential O
disease O
that O
can O
benefit O
from O
exploiting O
hES B-CellType
cells I-CellType
for O
cell O
replacement O
therapy O
as O
they O
have O
the O
capacity O
to O
differentiate O
into O
various O
hematopoietic B-Anatomy
cells. O
HIV B-Species
continues O
to O
be O
a O
major O
global O
public O
health O
problem O
with O
infections O
increasing O
at O
an O
alarming O
rate O
[10,11]. O
Given O
the O
present O
lack O
of O
effective O
vaccines O
and O
the O
ineffectiveness O
of O
drug O
based O
therapies O
for O
a O
complete O
cure, O
new O
and O
innovative O
approaches O
are O
essential. O
Gene O
therapy O
through O
intracellular O
immunization O
offers O
a O
promising O
alternative O
approach O
and O
possible O
supplement O
to O
current O
HAART O
therapy O
[12-14]. O
HIV B-Species
mainly O
targets O
cells O
of O
the O
hematopoietic B-Anatomy
system, O
namely, O
T B-CellType
cells, O
macrophages, O
and O
dendritic B-CellType
cells I-CellType
[15]. O
As O
infection O
progresses, O
the O
immune B-Anatomy
system I-Anatomy
is O
rendered O
defenseless O
against O
other O
invading O
pathogens O
and O
succumbs O
to O
opportunistic O
infections. O
There O
is O
a O
great O
deal O
of O
progress O
in O
the O
area O
of O
stem O
cell O
gene O
therapy O
for O
AIDS O
[12]. O
A O
primary O
goal O
of O
many O
ongoing O
studies O
is O
to O
introduce O
an O
effective O
anti-HIV B-Species
gene O
into O
hematopoietic B-Anatomy
stem I-CellType
cells I-CellType
[16-18]. O
As O
these O
cells O
possess O
the O
ability O
to O
self O
renew, O
they O
have O
the O
potential O
to O
continually O
produce O
HIV B-Species
resistant O
T B-CellType
cells I-CellType
and O
macrophages B-CellType
in O
the O
body O
thus O
providing O
long O
term O
immune O
reconstitution. O
These O
approaches O
use O
CD34 B-GeneProtein
hematopoietic B-Anatomy
stem I-CellType
cells I-CellType
for O
anti-HIV B-Species
gene O
transduction O
via O
integrating O
viral B-Species
vectors O
such O
as O
lentiviral B-Species
vectors O
[16-18]. O
Lentiviral B-Species
vectors O
have O
several O
advantages O
over O
conventional O
retroviral O
vectors O
since O
higher O
transduction O
efficiencies O
can O
be O
obtained O
and O
there O
is O
less O
gene O
silencing. O
The O
CD34 B-GeneProtein
cells I-CellType
currently O
used O
for O
many O
therapies O
are O
primarily O
obtained O
from O
bone B-Anatomy
marrow I-Anatomy
or O
mobilized O
peripheral B-Anatomy
blood I-Anatomy
[1,19]. O
Thus, O
CD34 B-GeneProtein
progenitor I-CellType
cells I-CellType
are O
an O
essential O
ingredient O
for O
HIV B-Species
gene O
therapy. O
In O
view O
of O
the O
need O
for O
CD34 B-GeneProtein
cells I-CellType
for O
HIV B-Species
gene O
therapy O
as O
well O
as O
for O
other O
hematopoietic B-Anatomy
disorders, O
if O
one O
can O
produce O
these O
cells O
in O
unlimited O
quantities O
from O
a O
renewable O
source, O
it O
will O
overcome O
the O
limitations O
of O
securing O
large O
numbers O
of O
CD34 B-GeneProtein
cells I-CellType
for O
therapeutic O
purposes. O
In O
this O
regard, O
progress O
has O
been O
made O
in O
deriving O
CD34 B-GeneProtein
cells I-CellType
from O
hES B-CellType
cells I-CellType
(hES-CD34). O
Different O
methods O
currently O
exist O
to O
derive O
CD34 B-GeneProtein
cells I-CellType
from O
hES B-CellType
cells I-CellType
with O
varying O
efficiencies O
[20-27]. O
Recent O
reports O
have O
indicated O
the O
capacity O
of O
hES O
cell O
derived O
CD34 B-GeneProtein
cells I-CellType
to O
give O
rise O
to O
lymphoid B-Anatomy
and O
myeloid B-Anatomy
lineages I-Anatomy
thus O
paving O
the O
way O
for O
utilization O
of O
these O
cells O
for O
hematopoietic B-Anatomy
cell O
therapy O
[20,27-29]. O
For O
the O
effective O
utilization O
of O
hES-CD34 B-CellType
cells I-CellType
for O
HIV B-Species
gene O
therapy, O
a O
number O
of O
parameters O
need O
to O
be O
examined. O
First, O
one O
has O
to O
demonstrate O
that O
hES-CD34 B-CellType
cells I-CellType
can O
give O
rise O
to O
macrophages B-CellType
and O
helper B-CellType
T I-CellType
cells I-CellType
which O
are O
the O
main O
cells O
that O
need O
to O
be O
protected O
against O
HIV B-Species
infection. O
Recent O
evidence O
has O
shown O
that O
hES-CD34 B-CellType
cells I-CellType
can O
give O
rise O
to O
myelomonocytic B-Anatomy
cells I-CellType
[21]. O
However, O
thorough O
phenotypic O
or O
functional O
characterization O
of O
these O
cells O
is O
lacking. O
It O
is O
also O
not O
clear O
if O
these O
cells O
are O
susceptible O
to O
HIV B-Species
infection. O
Similarly, O
although O
the O
hES-CD34 B-CellType
cells I-CellType
were O
shown O
to O
have O
lymphoid B-Anatomy
progenitor I-CellType
capacity, O
only O
B B-CellType
cell I-CellType
and O
natural B-CellType
killer I-CellType
(NK) I-CellType
cell O
differentiation O
has O
been O
examined O
so O
far O
[21,28]. O
The O
capacity O
to O
generate O
T B-CellType
cells I-CellType
remains O
to O
be O
evaluated. O
With O
this O
background, O
as O
a O
first O
step, O
our O
primary O
goal O
in O
these O
studies O
is O
to O
examine O
the O
capacity O
of O
hES-CD34 B-CellType
cells I-CellType
to O
give O
rise O
to O
phenotypically O
and O
functionally O
normal O
macrophages B-CellType
and O
whether O
such O
cells O
are O
susceptible O
to O
productive O
HIV B-Species
infection. O
Since O
lentiviral B-Species
vectors O
have O
been O
shown O
to O
successfully O
transduce O
hES B-CellType
cells I-CellType
[30-33], O
we O
further O
investigated O
the O
ability O
of O
transduced O
hES B-CellType
cells I-CellType
to O
differentiate O
into O
transgenic O
macrophages B-CellType
that O
can O
support O
HIV-1 B-Species
infection. O
Demonstration O
of O
HIV-1 B-Species
productive O
infection O
in O
these O
cells O
will O
permit O
future O
efficacy O
evaluations O
of O
anti-HIV B-Species
genes O
in O
this O
system. O
Here O
we O
show O
that O
normal O
and O
lentiviral B-Species
vector O
transduced O
hES-CD34 B-CellType
cells I-CellType
can O
give O
rise O
to O
phenotypically O
and O
functionally O
normal O
macrophages B-CellType
that O
support O
HIV B-Species
infection O
thus O
paving O
the O
way O
for O
many O
novel O
approaches O
to O
evaluate O
their O
potential O
for O
HIV B-Species
gene O
therapy. O
Derivation O
of O
macrophages B-CellType
from O
hES B-CellType
cellsUndifferentiated O
hES O
cell O
colonies B-Anatomy
grown O
in O
media O
supplemented O
with O
4 O
ng/ml O
bFGF B-GeneProtein
displayed O
normal O
morphology O
of O
pluripotent O
human B-Species
embryonic B-CellType
stem I-CellType
cells I-CellType
with O
tight O
and O
discreet O
borders O
on O
the O
MEF B-CellType
feeder B-Anatomy
layers I-Anatomy
(Fig O
1A). O
Similarly, O
lentiviral B-Species
vector O
transduced O
hES O
cell O
colonies, O
also O
displayed O
normal O
morphology O
and O
growth O
characteristics O
(Fig O
1A). O
As O
expected, O
the O
vector B-Anatomy
transduced I-Anatomy
colonies I-Anatomy
displayed O
green O
fluorescence O
due O
to O
the O
presence O
of O
the O
GFP B-GeneProtein
reporter O
gene. O
When O
cultured O
on O
irradiated O
S17 B-CellLine
mouse I-CellType
bone I-CellType
marrow I-CellType
stromal I-CellType
cells, O
both O
nontransduced O
and O
transduced O
hES B-CellType
cells I-CellType
developed O
into O
embryonic B-Anatomy
cystic I-Anatomy
bodies I-Anatomy
(Fig O
1A). O
FACS O
analysis O
of O
single O
cell O
suspensions O
of O
the O
cystic B-Anatomy
bodies I-Anatomy
showed O
levels O
of O
CD34 B-GeneProtein
cells I-CellType
which O
ranged O
from O
7–15%. O
Figure O
1B O
displays O
a O
representative O
FACS O
profile O
of O
hES-CD34 B-CellType
cells. O
Purified O
CD34 B-GeneProtein
cells I-CellType
were O
later O
cultured O
in O
semi-solid O
methylcellulose O
medium O
to O
derive O
myeloid B-Anatomy
colonies. O
Both O
nontransduced O
(denoted O
as O
ES O
in O
figures) O
and O
vector O
transduced O
(denoted O
as O
GFP B-GeneProtein
ES O
in O
figures) O
hES B-CellType
cell I-CellType
derived I-CellType
CD34 I-CellType
cells I-CellType
gave O
rise O
to O
normal O
myelomonocytic B-CellType
colonies I-Anatomy
similar O
to O
human B-Species
fetal I-Anatomy
liver I-Anatomy
derived O
CD34 B-GeneProtein
cells I-CellType
(denoted O
as O
CD34 B-GeneProtein
in O
figures) O
(Fig O
1A). O
When O
pooled O
colonies O
were O
cultured O
further O
in O
liquid O
cytokine O
media O
for O
12–15 O
days O
for O
differentiation, O
the O
cells O
developed O
into O
morphologically O
distinct O
macrophages B-CellType
(Fig O
1A). O
When O
compared, O
the O
morphology O
of O
macrophages B-CellType
derived O
from O
all O
stem B-CellType
cell I-CellType
progenitor I-CellType
populations O
appeared O
similar. O
These O
results O
were O
found O
to O
be O
consistent O
in O
replicative O
experiments. O
The O
transgene O
GFP B-GeneProtein
expression O
was O
also O
maintained O
during O
the O
differentiation O
of O
hES B-CellType
cells I-CellType
into O
mature O
macrophages. O
GFP B-GeneProtein
expression O
in O
cystic B-Anatomy
body I-Anatomy
derived O
CD34 B-GeneProtein
cells I-CellType
was O
around O
80% O
(data O
not O
shown) O
with O
similar O
levels O
seen O
in O
differentiated O
macrophages B-CellType
(Fig O
2).Figure O
1Derivation O
of O
macrophages B-CellType
from O
lentiviral B-Species
vector O
transduced O
and O
normal O
hES B-CellType
cells. O
A) O
Transduced O
and O
non-transduced O
H1 B-CellLine
hES I-CellType
cells I-CellType
were O
cultured O
on O
mouse B-Species
S17 I-CellType
bone I-CellType
marrow I-CellType
stromal I-CellType
cell I-CellType
layers B-Anatomy
to O
derive O
cystic B-Anatomy
bodies. O
Cystic B-Anatomy
body I-Anatomy
derived O
CD34 B-GeneProtein
cells I-CellType
were O
purified O
by O
positive O
selection O
with O
antibody O
conjugated O
magnetic O
beads O
and O
placed O
in O
methocult O
media O
to O
obtain O
myelomonocytic B-CellType
colonies. O
Pooled O
colonies O
were O
cultured O
in O
liquid O
cytokine O
media O
supplemented O
with O
GM-CSF B-GeneProtein
and O
M-CSF B-GeneProtein
to O
promote O
macrophage O
growth. O
For O
comparison, O
fetal B-Anatomy
liver I-Anatomy
derived O
CD34 B-GeneProtein
cells I-CellType
were O
cultured O
similarly O
to O
derive O
macrophages. O
Representative O
ES B-CellType
cell I-CellType
colonies, O
cystic B-Anatomy
bodies, O
methocult O
colonies, O
and O
derivative O
macrophages B-CellType
are O
shown O
with O
GFP B-GeneProtein
expressing O
cells O
fluorescing O
green O
under O
UV O
illumination. O
B) O
Representative O
FACS O
profile O
of O
hES B-CellType
cell I-CellType
derived I-CellType
CD34 I-CellType
cells I-CellType
stained O
with O
PE O
conjugated O
antibodies. O
Percent O
positive O
CD34 B-GeneProtein
cells I-CellType
are O
shown O
with O
isotype O
control O
shown O
in O
the O
left O
panel.Figure O
2Phenotypic O
FACS O
analysis O
of O
hES O
cell O
derived B-GeneProtein
macrophages. O
A) O
Macrophages B-CellType
derived O
from O
transduced O
and O
nontransduced O
hES B-CellType
CD34 I-CellType
and O
fetal B-Anatomy
liver I-Anatomy
CD34 I-CellType
cells I-CellType
were O
stained O
with O
antibodies O
to O
CD14, O
HLA-DR, O
CD4, O
CCR5, O
and O
CXCR4 B-GeneProtein
and O
the O
expression O
of O
these O
surface O
markers O
was O
analyzed O
by O
FACS. O
B) O
Isotype O
controls O
for O
PE O
and O
PE-CY5 O
antibodies. O
Percent O
positive O
cells O
are O
displayed O
in O
the O
plots O
for O
each O
respective O
cell O
surface O
marker O
staining. O
Dot O
plots O
are O
representative O
of O
triplicate O
experiments. O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
display O
a O
normal O
phenotypic O
profileMacrophages O
play O
a O
critical O
role O
in O
immune O
system O
function O
and O
are O
also O
major O
target O
cells O
for O
many O
viral B-Species
infections O
including O
HIV-1. O
Distinct O
surface O
phenotypic O
markers O
exist O
on O
these O
cells O
and, O
thus O
far, O
there O
has O
been O
no O
thorough O
evaluation O
of O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages. O
Therefore O
we O
analyzed O
hES O
cell O
derived O
macrophages B-CellType
for O
the O
presence O
of O
characteristic O
cell O
surface O
markers O
and O
compared O
these O
to O
the O
phenotypic O
profile O
displayed O
on O
fetal B-Anatomy
CD34 I-CellType
cell I-CellType
derived O
macrophages. O
The O
surface O
markers O
analyzed O
were O
CD14, O
a O
monocyte/macrophage O
specific O
marker, O
HLA-DR B-GeneProtein
(a O
class B-GeneProtein
II I-GeneProtein
MHC I-GeneProtein
molecule I-GeneProtein
found O
on O
antigen O
presenting O
cells), O
CD4, O
the O
major O
receptor O
for O
HIV-1 B-Species
infection, O
and O
CCR5 B-GeneProtein
and O
CXCR4, O
chemokine O
receptors O
which O
are O
critical O
coreceptors O
essential O
for O
HIV-1 B-Species
entry. O
EGFP B-GeneProtein
expression O
was O
also O
analyzed O
to O
determine O
the O
levels O
of O
transduction O
and O
any O
transgene O
silencing O
that O
may O
occur O
during O
differentiation. O
Fetal B-Anatomy
liver I-Anatomy
(CD34), O
nontransduced O
(ES), O
and O
vector O
transduced O
(GFP O
ES) O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
were O
all O
positive O
for O
the O
monocyte/macrophage O
marker O
CD14 B-GeneProtein
(99.3%, O
88.7%, O
and O
99.2%, O
respectively) O
(Fig O
2A). O
However, O
the O
mean O
fluorescent O
intensity O
(MFI) O
was O
found O
to O
be O
lower O
on O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages. O
Surface O
expression O
of O
HLA-DR B-GeneProtein
was O
observed O
at O
similar O
levels O
between O
macrophages B-CellType
derived O
from O
fetal B-Anatomy
liver I-Anatomy
CD34 I-CellType
cells I-CellType
(99.6%), O
nontransduced O
hES B-CellType
cells I-CellType
(92.8%), O
and O
transduced O
hES B-CellType
cells I-CellType
(98.2%) O
(Fig O
2A). O
CD4 B-GeneProtein
levels O
were O
comparable O
for O
all O
stem B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
(99.2%, O
83.3%, O
and O
88.7%, O
respectively) O
(Fig O
2A). O
CCR5 B-GeneProtein
and O
CXCR4 B-GeneProtein
cell O
surface O
expression O
was O
also O
observed O
for O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cell O
(99.6% O
and O
99.3%), O
nontransduced O
hES O
cell O
(91.9% O
and O
92.6%), O
and O
transduced O
hES B-CellType
cell O
(98.9% O
and O
99.3%) O
derived O
macrophages B-CellType
(Fig O
2A). O
As O
compared O
to O
fetal B-Anatomy
liver I-Anatomy
CD34 I-CellType
cell I-CellType
derived I-CellType
macrophages, O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
displayed O
a O
higher O
level O
of O
expression O
of O
CXCR4. O
Isotype O
controls O
for O
both O
PE O
and O
PECY5 B-GeneProtein
stains O
are O
shown O
in O
Fig O
2B. O
The O
above O
phenotypic O
data O
are O
representative O
of O
triplicate O
experiments. O
Transgenic O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
are O
functionally O
normalThe O
antigen O
presenting O
cell O
surface O
specific O
marker O
HLA-DR B-GeneProtein
(MHC O
II) O
on O
normal O
macrophages B-CellType
is O
critical O
for O
presenting O
antigen O
to O
CD4 B-GeneProtein
T B-CellType
cells. O
A O
second O
co-stimulatory O
molecule, O
B7.1 B-GeneProtein
is O
present O
at O
low O
basal O
levels O
on O
resting O
macrophages B-CellType
and O
is O
necessary O
to O
activate O
T B-CellType
cells. O
Its O
expression O
is O
elevated O
upon O
activation O
with O
certain O
stimuli O
such O
as O
LPS. O
Our O
results O
of O
LPS B-GeneProtein
stimulation O
of O
respective O
macrophages B-CellType
have O
shown O
upregulation O
of O
B7.1 B-GeneProtein
with O
values O
for O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cell O
(CD34) O
(27.9% O
to O
75.4%) O
nontransduced O
(ES) O
(17.8% O
to O
49.4%) O
and O
transduced O
(GFP O
ES) O
(35.6% O
to O
65.7%) O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
(Fig O
3A). O
These O
values O
represent O
a O
significant O
upregulation O
of O
B7.1 B-GeneProtein
for O
all O
three O
macrophage O
populations.Figure O
3Functional O
analysis O
of O
hES B-CellType
cell I-CellType
derived I-CellType
macrophages I-CellType
for O
B7.1 B-GeneProtein
costimulatory O
molecule O
upregulation O
and O
phagocytosis O
of O
E. B-Species
coli I-Species
particles: O
A) O
Mature O
macrophages B-CellType
were O
stimulated O
with O
LPS B-GeneProtein
to O
determine O
B7.1 B-GeneProtein
upregulation. O
Twenty-four O
hours O
post-stimulation, O
macrophages B-CellType
were O
labeled O
with O
a O
PE-CY5 O
conjugated O
anti-B7.1 O
antibody O
and O
analyzed O
by O
FACS. O
B7.1 B-GeneProtein
upregulation O
data O
are O
representative O
of O
triplicate O
experiments. O
Isotype O
control O
is O
shown O
in O
the O
left O
panel. O
B) O
To O
assess O
phagocytic B-CellType
function, O
E. B-Species
coli I-Species
Bioparticles® O
were O
added O
directly O
to O
the O
cultured O
macrophages. O
Twenty O
four O
hours O
post-addition, O
cells O
were O
analyzed O
by O
FACS. O
Percent O
positive O
cells O
are O
displayed O
in O
the O
plots O
for O
each O
experiment. O
These O
data O
are O
representative O
of O
triplicate O
experiments.Another O
important O
function O
of O
macrophages B-CellType
is O
their O
ability O
to O
phagocytose B-CellType
foreign O
material O
and O
present O
antigenic O
peptides O
on O
their O
cell B-CellComponent
surface. O
To O
evaluate O
phagocytic B-CellType
function, O
fluorescently O
labeled O
E. B-Species
coli I-Species
Bioparticles® O
were O
added O
to O
macrophage O
cultures O
followed O
by O
FACS O
analysis. O
Nontransduced O
(94.6%) O
as O
well O
as O
lentiviral B-Species
vector O
transduced O
(98.7%) O
hES B-CellType
cell O
derived O
macrophages B-CellType
were O
found O
to O
be O
capable O
of O
phagocytosing O
the O
Bioparticles® O
in O
comparison O
to O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cell O
derived O
macrophages B-CellType
(95.8%) O
(Fig O
3B). O
These O
values O
are O
representative O
of O
triplicate O
experiments. O
Magi-CXCR4 B-CellLine
cells I-CellType
with O
no O
phagocytic B-CellType
capacity O
were O
used O
as O
non-phagocytic O
cell I-CellType
controls O
and O
similarly O
exposed O
to O
E. B-Species
coli I-Species
Bioparticles® O
(Fig O
3B). O
No O
uptake O
of O
the O
bacteria O
could O
be O
seen. O
Thus, O
uptake O
of O
E. B-Species
coli I-Species
Bioparticles® O
by O
macrophages B-CellType
is O
indicative O
of O
active O
ingestion.Macrophages, O
as O
effector B-CellType
cells, O
play O
a O
key O
role O
in O
the O
inflammatory O
response. O
Activated O
macrophages B-CellType
secrete O
various O
cytokines, O
two O
of O
the O
major O
ones O
being O
IL-1 B-GeneProtein
and O
TNF-α. O
To O
determine O
if O
hES B-CellType
cell O
derived O
macrophages B-CellType
have O
such O
a O
capacity, O
cells O
were O
stimulated O
with O
LPS. O
On O
days O
1, O
2, O
and O
3 O
post-stimulation, O
culture O
supernatants O
were O
analyzed O
by O
ELISA O
to O
detect O
IL-1 B-GeneProtein
and O
TNF-α. O
As O
seen O
in O
figure O
4A, O
there O
were O
no O
significant O
differences O
in O
IL-1 B-GeneProtein
secretion O
between O
the O
three O
sets O
of O
macrophages. O
Similarly, O
nontransduced O
and O
transduced O
hES B-CellType
cell O
derived O
macrophages B-CellType
were O
also O
capable O
of O
TNF-α B-GeneProtein
secretion O
upon O
LPS B-GeneProtein
stimulation. O
However, O
levels O
of O
the O
respective O
cytokines O
detected O
were O
slightly O
lower O
than O
those O
from O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cell O
derived O
macrophages B-CellType
(Fig O
4B). O
The O
values O
of O
cytokine O
secretion O
levels O
represent O
triplicate O
experiments.Figure O
4Cytokine O
IL-1 B-GeneProtein
and O
TNFα B-GeneProtein
secretion O
by O
stimulated O
hES B-CellType
cell O
derived O
macrophages: O
Macrophages B-CellType
derived O
from O
transduced O
and O
nontransduced O
hES B-CellType
and O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cells I-CellType
were O
stimulated O
with O
5 O
μg/ml O
LPS. O
On O
days O
1, O
2, O
and O
3 O
post-stimulation, O
supernatants O
were O
collected O
and O
assayed O
by O
ELISA O
for O
(A) O
IL-1 B-GeneProtein
and O
(B) O
TNFα. O
Experiments O
were O
done O
in O
triplicate. O
hES B-CellType
cell O
derived O
macrophages B-CellType
support O
productive O
HIV-1 B-Species
infectionThe O
above O
data O
have O
shown O
that O
hES B-CellType
cell O
derived O
macrophages B-CellType
are O
very O
similar O
to O
normal O
human B-Species
macrophages I-CellType
based O
on O
phenotypic O
and O
functional O
analysis. O
In O
addition O
to O
being O
important O
cells O
of O
the O
immune B-Anatomy
system, O
macrophages B-CellType
are O
among O
the O
major O
target O
cells O
for O
certain O
viral B-Species
infections, O
particularly O
for O
HIV-1. O
We O
wanted O
to O
determine O
if O
hES B-CellType
cell O
derived O
macrophages B-CellType
were O
susceptible O
to O
HIV-1 B-Species
infection O
compared O
to O
standard O
macrophages. O
In O
these O
studies, O
we O
only O
used O
an O
R5-tropic O
strain O
of O
HIV-1 B-Species
since O
macrophages B-CellType
are O
natural O
targets O
for O
this O
virus. O
Our O
results O
from O
challenge O
studies O
of O
these O
cells O
clearly O
indicated O
the O
capacity O
of O
hES B-CellType
cell O
derived O
macrophages B-CellType
in O
supporting O
a O
productive O
infection. O
Levels O
of O
virus B-Species
increased O
up O
to O
15 O
days O
similar O
to O
non-hES O
derived O
macrophages B-CellType
showing O
that O
the O
initial O
viral B-Species
input O
was O
amplified O
in O
productive O
viral B-Species
infection. O
However, O
the O
levels O
of O
viral B-Species
yield O
were O
found O
to O
be O
slightly O
lower O
for O
the O
ES B-CellType
cell I-CellType
derived O
macrophages. O
In O
the O
case O
of O
GFP-ES O
macrophages, O
there O
was O
a O
decline O
in O
viral B-Species
titer. O
This O
could O
be O
due O
to O
possible O
lower O
numbers O
of O
cells O
present O
in O
the O
initial O
cultures. O
As O
a O
first O
step O
towards O
the O
use O
of O
hES B-CellType
cells I-CellType
for O
hematopoietic B-Anatomy
stem O
cell O
and O
HIV O
gene O
therapies, O
we O
have O
shown O
here O
that O
phenotypically O
and O
functionally O
normal O
macrophages B-CellType
could O
be O
derived O
from O
hES-CD34 B-CellType
cells. O
Both O
non O
transduced O
and O
lentiviral B-Species
vector O
transduced O
hES B-CellType
cells I-CellType
were O
found O
to O
be O
capable O
of O
generating O
CD34 B-GeneProtein
cells I-CellType
that O
give O
rise O
to O
macrophages B-CellType
which O
could O
support O
productive O
HIV-1 O
infection. O
Current O
sources O
of O
CD34 B-GeneProtein
cells I-CellType
consist O
of O
human B-Species
bone B-Anatomy
marrow, O
cytokine O
mobilized B-Anatomy
peripheral I-Anatomy
blood, O
fetal B-Anatomy
liver, O
and O
cord B-Anatomy
blood I-Anatomy
[34]. O
However, O
the O
number O
of O
cells O
that O
can O
be O
obtained O
for O
manipulations O
is O
not O
unlimited. O
Therefore, O
deriving O
CD34 B-GeneProtein
cells I-CellType
for O
therapeutic O
and O
investigative O
purposes O
from O
hES B-CellType
cells I-CellType
with O
unlimited O
growth O
potential O
has O
the O
advantage O
of O
a O
consistent O
and O
uniform O
source. O
The O
ability O
to O
obtain O
phenotypically O
normal O
and O
functionally O
competent O
macrophages B-CellType
from O
hES B-CellType
cells I-CellType
is O
important O
to O
evaluate O
their O
potential O
therapeutic O
utilities O
in O
the O
future. O
Additionally, O
testing O
of O
transgenic O
hES B-CellType
cells I-CellType
derived O
via O
lentiviral B-Species
vector O
gene O
transduction O
is O
also O
helpful O
to O
determine O
the O
stability O
of O
the O
transgene O
expression O
and O
their O
capacity O
for O
differentiation O
into O
end B-CellType
stage I-CellType
mature I-CellType
cells I-CellType
such O
as O
macrophages. O
Based O
on O
these O
considerations, O
both O
non- O
transduced O
and O
lentiviral O
vector O
transduced O
hES B-CellType
cells I-CellType
were O
evaluated O
for O
their O
capacity O
to O
give O
rise O
to O
CD34 B-GeneProtein
progenitor I-CellType
cells. O
In O
colony O
forming O
assays O
using O
semisolid O
methylcellulose O
medium, O
the O
morphology O
of O
myelomonocytic B-CellType
colonies O
derived O
from O
hES B-CellType
CD34 I-CellType
cells I-CellType
appeared O
similar O
to O
that O
of O
fetal B-Anatomy
liver I-Anatomy
CD34 I-CellType
cells. O
When O
subsequently O
cultured O
in O
cytokine O
media O
that O
promotes O
macrophage O
differentiation, O
morphologically O
normal O
macrophages B-CellType
were O
obtained O
with O
hES-CD34 B-CellType
cells I-CellType
similar O
to O
that O
of O
fetal B-Anatomy
liver I-CellType
CD34 I-CellType
cells. O
At O
higher O
magnification, O
the O
macrophages B-CellType
displayed O
flat O
projecting O
cellular O
borders O
with O
fried O
egg O
appearance O
with O
distinct O
refractory O
lysosomal B-CellComponent
granules I-CellComponent
in O
the O
cytoplasm B-CellComponent
(data O
not O
shown). O
Lentiviral B-Species
vector O
transduced O
hES B-CellType
cells I-CellType
also O
did O
not O
display O
any O
abnormal O
growth O
or O
differentiation O
characteristics O
as O
compared O
to O
nontransduced O
hES-CD34 B-CellType
cells I-CellType
indicating O
no O
adverse O
effects O
due O
to O
vector O
integration O
and O
expression. O
Transduced O
cells O
gave O
rise O
to O
cystic B-Anatomy
bodies I-Anatomy
with O
similar O
CD34 B-GeneProtein
cell O
content O
and O
profiles O
upon O
development. O
The O
transduced O
hES-CD34 B-CellType
cells I-CellType
also O
gave O
rise O
to O
apparently O
normal O
macrophages B-CellType
that O
expressed O
the O
transgene O
as O
shown O
by O
GFP B-GeneProtein
expression. O
These O
results O
are O
consistent O
with O
those O
of O
others O
that O
showed O
normal O
differentiation O
of O
hES B-CellType
cells I-CellType
to O
other O
cell O
types O
following O
lentiviral B-Species
transduction O
[32]. O
A O
requirement O
for O
successful O
cellular O
and O
HIV-1 B-Species
gene O
therapy O
is O
that O
mature B-CellType
end I-CellType
stage I-CellType
cells I-CellType
derived O
from O
CD34 B-GeneProtein
progenitor I-CellType
cells I-CellType
be O
phenotypically O
and O
functionally O
normal O
to O
maintain O
and O
restore O
the O
body's O
immunological O
function. O
Accordingly, O
hES O
cell O
derived O
macrophages B-CellType
were O
evaluated O
to O
determine O
if O
they O
met O
these O
criteria. O
Macrophages B-CellType
display O
distinct O
cell O
surface O
markers O
upon O
end O
stage O
differentiation. O
To O
determine O
whether O
hES O
cell O
derived O
macrophages B-CellType
display O
these O
surface O
markers, O
FACS O
analysis O
was O
performed O
to O
detect O
the O
presence O
of O
CD14, O
HLA-DR B-GeneProtein
(MHCII), O
CD4, O
CCR5, O
and O
CXCR4. O
As O
observed O
in O
Fig O
2A, O
both O
nontransduced O
and O
transduced O
hES O
cell O
derived O
macrophages B-CellType
expressed O
all O
of O
these O
markers O
with O
some O
differences O
in O
their O
levels O
of O
expression. O
HLA-DR, O
CD4, O
and O
CCR5 B-GeneProtein
expression O
profiles O
were O
comparable O
between O
all O
cell O
types O
analyzed. O
Even O
though O
all O
cell O
types O
analyzed O
stained O
positive O
for O
CD14, O
relative O
expression O
of O
CD14 B-GeneProtein
was O
slightly O
lower O
on O
hES O
cell O
derived O
macrophages B-CellType
compared O
to O
fetal B-Anatomy
liver I-Anatomy
CD34 B-GeneProtein
cell O
derived O
macrophages. O
On O
the O
contrary, O
the O
levels O
of O
CXCR4, O
a O
chemokine O
receptor O
involved O
in O
cellular O
homing, O
were O
found O
to O
be O
higher O
on O
hES-CD34 O
cell O
derived O
macrophages. O
This O
may O
be O
due O
to O
inherent O
differences O
in O
the O
cell O
types O
and/or O
due O
to O
their O
physiological O
state O
at O
the O
time O
of O
harvest O
[35]. O
Additional O
hES O
cell O
lines O
need O
to O
be O
evaluated O
in O
the O
future O
to O
establish O
if O
these O
differences O
are O
consistent. O
A O
major O
functional O
role O
of O
macrophages B-CellType
in O
vivo O
is O
their O
ability O
to O
serve O
as O
professional O
antigen B-CellType
presenting I-CellType
cells. O
During O
this O
process O
macrophages B-CellType
present O
antigen O
peptide O
fragments O
complexed O
with O
both O
classes O
of O
MHC B-GeneProtein
molecules I-GeneProtein
and O
deliver O
a O
costimulatory O
signal O
through O
the O
expression O
of O
B7 B-GeneProtein
molecules. O
Upon O
stimulation O
with O
LPS, O
hES-CD34 O
cell O
derived O
macrophages B-CellType
had O
shown O
upregulation O
of O
the O
costimulatory O
molecule O
B7.1 B-GeneProtein
similar O
to O
cells O
derived O
from O
fetal B-Anatomy
liver. O
Furthermore, O
the O
hES-CD34 O
cell O
derived O
macrophages B-CellType
also O
showed O
a O
normal O
capacity O
to O
ingest O
foreign O
particles O
in O
phagocytosis O
assays O
using O
E.coli B-Species
Bioparticles®. O
In O
addition O
to O
antigen O
presentation O
and O
phagocytosis, O
macrophages B-CellType
also O
play O
a O
critical O
role O
in O
inflammation O
and O
secrete O
cytokines O
in O
response O
to O
external O
stimuli. O
When O
exposed O
to O
LPS, O
the O
hES-CD34 O
cell O
derived O
macrophages B-CellType
secreted O
two O
important O
cytokines O
IL-1 B-GeneProtein
and O
TNF-α B-GeneProtein
similar O
to O
that O
of O
fetal B-Anatomy
liver B-Anatomy
derived O
cells. O
The O
above O
data O
has O
established O
that O
phenotypically O
and O
functionally O
normal O
macrophages B-CellType
could O
be O
derived O
from O
hES-CD34 B-CellType
cells. O
Macrophages B-CellType
in O
addition O
to O
playing O
important O
physiological O
roles O
are O
also O
major O
cell O
targets O
for O
certain O
viral B-Species
infections, O
particularly O
HIV-1. O
Here O
we O
evaluated O
the O
susceptibility O
of O
hES-CD34 O
cell O
derived O
macrophages B-CellType
to O
be O
productively O
infected O
with O
HIV-1. O
Similar O
to O
that O
of O
fetal B-Anatomy
liver I-Anatomy
CD34 I-CellType
cell I-CellType
derived I-CellType
cells, O
the O
hES-CD34 O
macrophages B-CellType
also O
supported O
HIV-1 B-Species
infection O
although O
the O
levels O
of O
viral B-Species
yield O
differed O
somewhat. O
However O
this O
should O
not O
be O
a O
major O
concern O
for O
testing O
anti-HIV B-Species
genes O
in O
these O
cells. O
In O
all O
the O
above O
experiments, O
the O
vector O
transduced O
transgenic O
macrophages B-CellType
also O
behaved O
similarly O
to O
that O
of O
nontransduced O
cells O
showing O
that O
they O
were O
also O
physiologically O
normal. O
The O
lack O
of O
vector O
toxicity O
on O
cellular O
maturation O
is O
encouraging O
for O
future O
work O
with O
transduced O
hES-CD34 B-CellType
cells I-CellType
to O
derive O
other O
important O
differentiated O
cells O
like O
T B-CellType
cells I-CellType
and O
dendritic B-CellType
cells I-CellType
relevant O
for O
HIV B-Species
studies. O
Although O
there O
are O
numerous O
studies O
on O
hES O
cell O
differentiation O
into O
many O
important O
end B-CellType
stage I-CellType
mature I-CellType
cells, O
systematic O
work O
on O
hES O
cell O
hematopoietic B-Anatomy
differentiation O
and O
thorough O
characterization O
of O
end B-CellType
stage I-CellType
mature I-CellType
cells I-CellType
that O
participate O
in O
critical O
immune O
responses O
has O
just O
begun O
[21,27-29]. O
Our O
current O
results O
established O
that O
physiologically O
normal O
macrophages B-CellType
could O
be O
derived O
from O
hES B-CellType
cells I-CellType
and O
that O
these O
cells O
have O
the O
potential O
for O
use O
in O
cellular O
and O
gene O
therapies. O
To O
our O
knowledge O
this O
is O
the O
first O
demonstration O
that O
hES B-CellType
cell I-CellType
derivatives I-CellType
can O
be O
used O
for O
infectious O
disease O
research. O
Due O
to O
the O
extensive O
ability O
for O
hES B-CellType
cells I-CellType
to O
self-renew, O
large O
numbers O
of O
differentiated O
cells O
can O
be O
derived O
so O
that O
infection O
studies O
and O
evaluation O
tests O
can O
be O
carried O
out O
in O
a O
more O
standardized O
way. O
Our O
results O
showing O
that O
both O
normal O
and O
transgenic O
derivative O
macrophages B-CellType
support O
HIV-1 B-Species
infection O
points O
out O
to O
their O
utility O
for O
testing O
anti-HIV B-Species
constructs O
transduced O
into O
hES-CD34 B-CellType
cells I-CellType
and O
pave O
the O
way O
for O
their O
application O
in O
stem O
cell O
based O
HIV B-Species
gene O
therapy. O
So O
far O
a O
number O
of O
studies O
including O
our O
own O
have O
tested O
many O
gene O
therapeutic O
constructs O
in O
CD34 B-GeneProtein
cells O
from O
conventional O
sources. O
These O
constructs O
include O
anti-HIV B-GeneProtein
ribozymes, O
RNA O
decoys, O
transdominant O
proteins, O
bacterial O
toxins, O
anti-sense O
nucleic O
acids, O
and O
most O
recently O
siRNAs O
[36-50]. O
In O
addition, O
a O
number O
of O
cellular O
molecules O
that O
aid O
in O
HIV-1 B-Species
infection O
such O
as O
cellular O
receptors O
and O
coreceptors O
CD4, O
CCR5 B-GeneProtein
and O
CXCR4 B-GeneProtein
have O
also O
been O
successfully O
tested O
in O
CD34 B-GeneProtein
cell O
derived O
macrophages B-CellType
and O
T B-CellType
cells I-CellType
[16,18,38]. O
Some O
of O
these O
approaches O
have O
progressed O
into O
clinical O
evaluations O
as O
well O
[14,51,52]. O
Based O
on O
our O
current O
results, O
many O
of O
these O
novel O
anti-HIV B-Species
constructs O
can O
also O
be O
tested O
in O
hES-CD34 B-CellType
cells I-CellType
for O
their O
potential O
application. O
Although O
there O
are O
advantages O
of O
using O
hES O
cell O
derived O
CD34 B-GeneProtein
cells I-CellType
for O
potential O
cellular O
therapies, O
transplantation O
of O
these O
cells O
constitutes O
an O
allogenic O
source O
with O
immune O
rejection O
as O
a O
major O
issue. O
However, O
a O
recent O
study O
using O
human B-Species
leukocyte I-Species
reconstituted I-Species
mice I-Species
suggested O
that O
hESCs B-CellType
and O
their O
derivative O
cell O
types O
were O
less O
prone O
to O
invoking O
an O
allogeneic O
response O
[53]. O
Other O
recent O
studies O
demonstrated O
successful O
engraftment O
of O
primary O
and O
secondary O
recipients O
with O
hES O
cell O
derived O
hematopoietic B-CellType
cells I-CellType
in O
both O
immunodeficient O
mice B-Species
and O
in O
vivo O
fetal B-Anatomy
sheep B-Species
models O
adding O
further O
support O
that O
any O
obstacles O
could O
be O
overcome O
[23,54,55]. O
Moreover, O
multiple O
novel O
strategies O
to O
avoid O
immune-mediated O
rejection O
of O
hES B-CellType
cell-derived I-CellType
cells I-CellType
have O
been O
proposed O
[56,57]. O
It O
is O
not O
too O
far O
in O
the O
future O
that O
even O
autologous B-CellType
hES I-CellType
cells I-CellType
may O
be O
derived O
from O
specific O
individuals O
for O
deriving O
CD34 B-GeneProtein
cells I-CellType
which O
can O
be O
used O
for O
cell O
replacement O
therapy. O
Phenotypically O
normal O
and O
functionally O
competent O
macrophages B-CellType
could O
be O
derived O
from O
hES-CD34 B-CellType
cells. O
Since O
these O
cells O
are O
susceptible O
to O
HIV-1 B-Species
infection, O
they O
provide O
a O
uniform O
source O
of O
macrophages B-CellType
for O
viral B-Species
infection O
studies. O
Based O
on O
these O
results, O
it O
is O
also O
now O
feasible O
to O
transduce O
hES-CD34 B-CellType
cells I-CellType
with O
anti-HIV B-GeneProtein
genes I-GeneProtein
such O
as O
inhibitory O
siRNAs B-GeneProtein
and O
test O
their O
antiviral B-Species
efficacy O
in O
down O
stream O
differentiated O
cells O
such O
as O
macrophages B-CellType
which O
are O
among O
the O
primary B-CellType
cells I-CellType
that O
need O
to O
be O
protected O
against O
HIV-1 B-Species
infection. O
Thus, O
the O
potential O
utility O
of O
hES B-CellType
derived I-CellType
CD34 I-CellType
hematopoietic I-CellType
cells I-CellType
for O
HIV-1 B-Species
gene O
therapy O
can O
be O
evaluated. O
Growth, O
propagation O
and O
lentiviral O
transduction O
of O
hES B-CellType
cellsThe O
NIH O
approved O
human B-Species
ES O
H1 B-CellLine
cell O
line O
was O
obtained O
from O
WiCell O
(Madison, O
Wisconsin). O
hES O
cell O
colonies B-Anatomy
were O
cultured O
on O
mouse B-Species
embryonic I-CellType
fibroblasts I-CellType
(MEF) O
(Chemicon, O
Temecula, O
CA) O
in O
the O
presence O
of O
DMEM-F12 O
(Invitrogen, O
Carlsbad, O
CA) O
supplemented O
with O
20% O
KNOCKOUT O
serum O
replacement O
with O
1 O
mM O
L-glutamine, O
1% O
Nonessential O
Amino O
Acids, O
0.1 O
mM O
β-mercaptoethanol, O
0.5% O
penicillin/streptomycin, O
and O
4 O
ng/ml O
human B-GeneProtein
basic I-GeneProtein
fibroblast I-GeneProtein
growth I-GeneProtein
factor. O
Culture O
medium O
was O
replaced O
daily O
with O
fresh O
complete O
DMEM-F12. O
Mature O
colonies B-Anatomy
were O
subcultured O
weekly O
by O
digesting O
with O
collagenase B-GeneProtein
IV I-GeneProtein
as O
previously O
described O
[5]. O
A O
VSV-G O
pseudotyped O
lentiviral B-Species
vector O
(SINF-EF1a-GFP) O
containing O
a O
GFP B-GeneProtein
reporter O
gene O
(kindly O
supplied O
by O
R. O
Hawley, O
George O
Washington O
University) O
was O
used O
for O
hES O
cell O
transductions O
as O
previously O
described O
(30, O
58). O
Generation O
of O
the O
pseudotyped O
vector O
in O
293T B-CellLine
cells I-CellType
and O
its O
concentration O
by O
ultracentrifugation O
were O
described O
previously O
[30,48]. O
For O
vector O
transduction, O
the O
undifferentiated O
hES B-CellType
cells I-CellType
were O
prepared O
into O
small O
clumps B-Anatomy
of O
50–100 O
cells O
with O
enzyme O
digestion O
as O
done O
for O
routine O
passaging O
of O
cells. O
The O
cell B-Anatomy
clumps I-Anatomy
were O
incubated O
with O
the O
vector O
for O
2 O
hrs O
in O
the O
presence O
of O
polybrene O
6 O
ug/ml. O
A O
secondary O
cycle O
of O
transduction O
was O
done O
by O
adding O
fresh O
vector O
and O
incubating O
for O
another O
2 O
hrs. O
The O
general O
vector O
titers O
were O
1 O
× O
107 O
and O
the O
multiplicity O
of O
infection O
was O
10. O
The O
transduction O
efficiency O
was O
about O
50%. O
The O
transduced O
colonies O
were O
cultured O
on O
MEF B-CellType
like O
above. O
Derivation O
and O
purification O
of O
CD34 B-GeneProtein
cells I-CellType
from O
hES B-CellType
cellsUndifferentiated O
hES B-CellType
cells I-CellType
were O
cultured O
on O
S17 B-CellLine
mouse B-Species
bone B-Anatomy
marrow I-Anatomy
stromal B-Anatomy
cell O
monolayers B-Anatomy
to O
derive O
cystic B-Anatomy
bodies I-Anatomy
containing O
CD34+ B-CellType
hematopoietic I-CellType
progenitor I-CellType
stem I-CellType
cells. O
hES O
cell O
cultures O
were O
treated O
with O
collagenase B-GeneProtein
IV(1 O
mg/ml) O
for O
10 O
minutes O
at O
37°C O
and O
subsequently O
detached O
from O
the O
plate O
by O
gentle O
scraping O
of O
the O
colonies. O
The O
hES O
cell O
clusters B-Anatomy
were O
then O
transferred O
to O
irradiated O
(35 O
Gy) O
S17 B-CellLine
cell I-CellLine
layers B-Anatomy
and O
cultured O
with O
RPMI O
differentiation O
medium O
containing O
15% O
FBS O
(HyClone, O
Logan, O
UT), O
2 O
mM O
L-glutamine, O
0.1 O
mM O
β-mercaptoethanol, O
1% O
MEM-nonessential O
amino O
acids, O
and O
1% O
penicillin/streptomycin. O
Media O
was O
changed O
every O
2 O
to O
3 O
days O
during O
14–17 O
days O
of O
culture O
on O
S17 B-CellLine
cells I-CellType
[20].After O
allowing O
adequate O
time O
for O
differentiation, O
hES B-CellType
cystic B-Anatomy
bodies I-Anatomy
were O
harvested O
and O
processed O
into O
a O
single O
cell O
suspension O
by O
collagenase B-GeneProtein
IV I-GeneProtein
treatment O
followed O
by O
digestion O
with O
trypsin/EDTA O
supplemented O
with O
2% O
chick B-Species
serum O
(Invitrogen, O
Carlsbad, O
CA) O
for O
20 O
minutes O
at O
37°C. O
Cells O
were O
washed O
twice O
with O
PBS O
and O
filtered O
through O
a O
70 O
uM O
cell O
strainer O
to O
obtain O
a O
single O
cell O
suspension. O
To O
assess O
the O
levels O
of O
CD34 B-GeneProtein
cells I-CellType
in O
the O
bulk O
cell O
suspension, O
cells O
were O
labeled O
with O
PE O
conjugated O
anti-CD34 O
antibody O
(BD O
Biosciences, O
San O
Jose, O
CA) O
and O
analyzed O
by O
FACS. O
To O
purify O
the O
CD34 B-GeneProtein
cells, O
Direct O
CD34 B-GeneProtein
Progenitor I-CellType
Cell I-CellType
Isolation O
Kit O
(Miltenyi O
Biotech, O
Auburn, O
CA) O
was O
used O
following O
the O
manufacturer's O
protocol. O
Isolated O
CD34 B-GeneProtein
hematopoietic I-CellType
progenitor I-CellType
stem I-CellType
cells I-CellType
were O
then O
analyzed O
by O
FACS O
as O
mentioned O
above O
to O
determine O
cell O
purity. O
For O
comparative O
experiments, O
human B-Species
CD34 I-CellType
hematopoietic I-CellType
progenitor I-CellType
cells I-CellType
were O
also O
purified O
from O
fetal B-Anatomy
liver I-Anatomy
tissue B-Anatomy
as O
described O
above. O
Derivation O
of O
macrophages B-CellType
from O
hES O
cell O
derived O
and O
human B-Species
fetal I-CellType
CD34 I-CellType
cellsCD34 O
cells I-CellType
were O
cultured O
initially O
in O
semisolid O
media O
to O
derive O
myelomonocytic B-CellType
colonies I-Anatomy
followed O
by O
liquid O
culture O
in O
cytokine O
supplemented O
media O
as O
described O
below. O
Purified O
CD34+ B-CellType
progenitor I-CellType
cells I-CellType
(~2.5 O
× O
105 O
to O
4.0 O
× O
105) O
were O
placed O
directly O
into O
Methocult O
semisolid O
medium O
(Stem O
Cell O
Technologies, O
Vancouver, O
BC), O
mixed, O
and O
cultured O
in O
35 O
mm O
plates. O
Myeloid B-CellType
colonies I-Anatomy
were O
allowed O
to O
develop O
for O
12–15 O
days. O
Upon O
differentiation O
and O
proliferation, O
myelomonocytic B-CellType
colonies I-Anatomy
were O
harvested O
by O
the O
addition O
of O
5 O
ml O
DMEM O
containing O
10% O
FBS, O
10 O
ng/ml O
each O
GM-CSF B-GeneProtein
and O
M-CSF. O
Cells O
(~106) O
were O
placed O
in O
a O
35 O
mm O
well O
and O
allowed O
to O
adhere O
for O
48 O
hours. O
At O
two O
and O
four O
days O
post-harvest, O
medium O
was O
replaced O
with O
fresh O
complete O
DMEM O
supplemented O
with O
10 O
ng/ml O
GM-CSF B-GeneProtein
and O
M-CSF. O
By O
4–5 O
days, O
cells O
developed O
into O
mature O
macrophages B-CellType
which O
were O
used O
for O
subsequent O
phenotypic O
and O
functional O
characterization. O
Phenotypic O
analysis O
of O
hES B-CellType
cell O
derived O
macrophagesTo B-CellType
determine O
if O
nontransduced O
and O
lentiviral B-Species
vector O
transduced O
hES O
cell O
derived O
macrophages B-CellType
display O
normal O
macrophage O
surface O
markers, O
FACS O
analysis O
was O
performed O
using O
respective O
fluorochrome O
conjugated O
antibodies. O
Fetal B-Anatomy
liver I-Anatomy
derived O
CD34+ B-GeneProtein
cells I-CellType
as O
well O
as O
nontransduced O
and O
transduced O
hES O
cell O
derived O
macrophages B-CellType
were O
evaluated O
in O
parallel. O
Cells O
were O
scraped O
from O
their O
wells, O
washed O
two O
times O
with O
PBS, O
and O
stained O
with O
the O
following O
antibodies: O
PE-CD14, O
PE-HLA-DR, O
PECY5-CD4, O
PECY5-CCR5, O
PECY5-CXCR4 B-GeneProtein
(BD O
Biosciences, O
San O
Jose, O
CA). O
A O
blocking O
step O
was O
first O
performed O
by O
incubating O
the O
cells O
with O
the O
respective O
isotype O
control O
for O
30 O
minutes O
at O
4C O
before O
staining O
with O
the O
respective O
cell O
surface O
marker O
antibodies. O
Isotype O
control O
staining O
was O
used O
to O
determine O
background O
levels. O
FACS O
analysis O
was O
performed O
on O
a O
Beckman-Coulter O
EPICS O
® O
XL-MCL O
flow O
cytometer O
with O
data O
analysis O
using O
EXPO32 O
ADC O
software O
(Coulter O
Corporation, O
Miami, O
FL). O
A O
minimum O
of O
8,000 O
cells O
were O
analyzed O
in O
each O
FACS O
evaluation. O
Functional O
analysis O
of O
hES O
cell O
derived O
macrophagesPhysiological O
roles O
of O
macrophages B-CellType
include O
phagocytic O
and O
immune O
related O
functions. O
To O
determine O
if O
hES O
cell O
derived O
macrophages B-CellType
were O
functionally O
normal, O
a O
stimulation O
assay O
to O
determine O
upregulation O
of O
the O
costimulatory B-GeneProtein
molecule I-GeneProtein
B7.1 I-GeneProtein
was O
performed. O
Activated B-CellType
macrophages B-CellType
upregulate O
the O
expression O
of O
B7.1 B-GeneProtein
upon O
activation O
with O
various O
stimuli. O
Accordingly, O
fetal B-Anatomy
liver I-Anatomy
CD34, O
nontransduced O
hES, O
and O
GFP-alone O
transduced O
hES O
cell O
derived O
macrophages B-CellType
were O
stimulated O
by O
the O
addition O
of O
LPS B-GeneProtein
(5 O
ug/ml) O
to O
the O
cell O
culture O
medium. O
Twenty-four O
hours O
post-stimulation, O
cells O
were O
stained O
with O
an O
anti-B7.1 B-GeneProtein
antibody I-GeneProtein
labeled O
with O
PE-Cy5 O
(BD O
Biosciences, O
San O
Jose, O
CA) O
and O
analyzed O
by O
FACS. O
To O
assess O
the O
hES O
cell O
derived O
macrophages' O
phagocytic O
function, O
5 O
ug/ml O
of O
fluorescently O
labeled O
E. B-Species
coli I-Species
Bioparticles® O
(Invitrogen, O
Carlsbad, O
CA) O
were O
added O
directly O
to O
the O
cell O
culture O
medium. O
Four O
hours O
later, O
macrophages B-CellType
were O
washed O
six O
times O
with O
PBS O
and O
fresh O
medium O
with O
10 O
ng/ml O
GM-CSF B-GeneProtein
and O
M-CSF B-GeneProtein
was O
added. O
Twenty-four O
hours O
later, O
cells O
were O
analyzed O
by O
FACS O
for O
the O
presence O
of O
ingested O
Bioparticles® O
which O
can O
be O
detected O
in O
the O
PE O
(FL2) O
channel. O
Lentiviral B-Species
vector O
transduced O
Magi-CXCR4 B-CellLine
cells, O
a O
HeLa B-CellLine
cell I-CellLine
derivative O
with O
no O
phagocytic O
capacity, O
were O
used O
as O
non-phagocytic O
cell O
controls O
and O
similarly O
exposed O
to O
E. B-Species
coli I-Species
Bioparticles®Human O
ES I-CellType
cell I-CellType
derived O
macrophages B-CellType
were O
also O
analyzed O
for O
their O
ability O
to O
secrete O
two O
major O
cytokines, O
IL-1 B-GeneProtein
and O
TNF-α, O
upon O
external O
stimulation. O
Accordingly, O
macrophages B-CellType
were O
stimulated O
with O
5 O
ug/ml O
of O
LPS O
during O
culture. O
On O
days O
1, O
2, O
and O
3 O
post-stimulation, O
cell O
culture O
supernatant O
samples O
were O
collected O
and O
analyzed O
by O
a O
Quantikine® O
ELISA O
kit O
(R&D O
Systems, O
Minneapolis, O
MN). O
Non-stimulated O
supernatants O
were O
also O
analyzed O
for O
basal O
levels O
of O
cytokine O
secretion. O
HIV-1 B-Species
infection O
of O
hES O
cell O
derived O
macrophagesTo O
determine O
if O
hES O
cell O
derived O
macrophages B-CellType
can O
be O
infected O
with O
HIV-1 B-Species
and O
support O
viral O
replication, O
cells O
were O
challenged O
with O
a O
macrophage B-Species
R5-tropic I-Species
BaL-1 I-Species
strain I-Species
of I-Species
HIV-1. O
An O
m.o.i. O
of O
0.01 O
in O
the O
presence O
of O
4 O
ug/ml O
polybrene O
was O
used. O
At O
different O
days O
post-infection, O
culture O
supernatants O
were O
collected O
and O
assayed O
for O
p24 O
antigen O
by O
ELISA. O
To O
quantify O
viral O
p24 O
levels, O
a O
Coulter-p24 O
kit O
(Beckman O
Coulter, O
Fullerton, O
CA) O
was O
used. O