Accumulating
evidence
suggest
that
pluripotency
–
the
ability
to
generate
all
somatic
cell
types
–
is
not
a
fixed
state.
Pluripotent
cell
populations
encompass
a
heterogeneous
group
of
cells
with
different
phenotypic
and
functional
properties
in
equilibrium.
At
least
two
phases
of
pluripotency,
immature
and
primed
for
differentiation,
have
been
identified
during
early
mouse
development,
and
these
are
typified
by
the
two
distinct
stem
cell
populations
–
mouse
embryonic
(mES)
and
epiblast-­‐derived
(mEpiS)
stem
cells
–
isolated
from
epiblast
layers
of
pre-­‐
and
post-­‐implantation
embryo,
respectively.
Many
lines
of
human
ES
(hES)
cells
have
also
been
established
in
vitro
from
the
inner
cell
mass
of
pre-­‐
implantation
blastocysts
yet
the
precise
in
vivo
lineage
affiliation
of
these
cells
remains
largely
unresolved.
Profiling
chromatin
in
a
particular
cell
line
has
proven
to
be
a
valuable
signature
for
cell
identity
and
developmental
stage.
One
approach
has
been
to
assay
the
timing
of
DNA
replication
during
S-­‐phase
of
the
cell
cycle
across
a
panel
of
loci,
as
an
indicator
of
chromatin
accessibility.
This
replication
timing
profiling
was
notably
capable
of
discriminating
pluripotent
mES
cells
from
cells
with
a
more
restricted
differentiation
capacity.
This
study
sought
to
address
whether
distinct
pluripotent
states
could
be
reliably
discriminated
at
the
chromatin
level.
In
particular,
the
replication
timing
profiles
of
a
number
of
hES
cell
lines
were
characterised
and
compared
to
those
of
mES
and
mEpiS
cell
lines
derived
from
different
genetic
backgrounds.
Profiles
of
undifferentiated
H1,
H7
and
H9
hES
cell
lines
typically
harboured
an
increased
proportion
of
late-­‐replicating
loci
during
S-­‐
phase
when
compared
to
mES
cells,
which
were
confirmed
to
have
a
steady
and
mostly
early-­‐replicating
profile
regardless
of
their
genetic
background.
Moreover,
hES
cell
replication
profile
greatly
varied
between
cultures
and
cell
lines;
a
level
of
replication
timing
variability
also
observed
among
mEpiS
cells,
as
opposed
to
mES
cells.
These
results
highlight
that
hES
and
mEpiS
cells
most
likely
share
a
common
unstable
epigenetic
state
or
transitional
state
primed
on
the
verge
of
differentiation.
The
epigenetic
state
of
hES
cell
lines
was
further
interrogated
by
analysing
cells
grown
under
two
different
culture
conditions,
both
however
similarly
relying
on
TGF-­‐β/Activin
signalling.
Results
demonstrated
that
hES
cells
could
adopt
distinct
yet
reversible
epigenetic
signatures
while
retaining
their
pluripotency.
In
particular,
extensive
and
dynamic
shifts
of
replication
timing,
from
late-­‐to-­‐early,
were
consistently
observed
at
many
target
loci
in
hES
cells
as
well
as
in
human
induced
pluripotent
cells
(iPS)
cells,
upon
increased
SMAD2/3-­‐associated P300/CBP
histone
acetyltransferase
(HAT)
activity.
This
was
accompanied
by
fluctuations
in
the
expression
of
NANOG
and
REX1
(also
known
as
ZFP42),
and
a
change
in
hES
cell’s
functional
properties,
as
judged
by
their
responsiveness
to
differentiation-­‐inducing
signals.
Interestingly,
inhibiting
P300/CBP
HAT
activity
by
curcumin
treatment
in
undifferentiated
hES
cells
was
sufficient
to
revert
back
to
a
late-­‐replicating
profile
associated
with
a
histone
hypoacetylated
state.
Stable
knockdown
of
P300
in
hES
cell
cultures,
however,
resulted
in
a
gradual
loss
of
the
pluripotent
identity
through
differentiation
or
apoptosis,
preventing
further
analysis
of
effects
on
replication
timing.
Collectively,
these
data
strongly
support
the
view
that
different
but
interchangeable
pluripotent
states
exist
within
hES
cell
cultures
and
suggest
a
role
for
P300/CBP
HAT
activity
in
determining
distinct
epigenetic
states
in
hES
cells.
As
mentioned
above,
REX1
was
also
significantly
upregulated
in
H1
hES
cells
upon
culture
condition
change.
REX1
is
a
developmental
stage-­‐specific
marker
that
is
expressed
in
the
ICM
of
both
mouse
and
human
embryos,
as
well
as
in
pluripotent
mES,
hES
and
iPS
cells.
However,
its
function
in
hES
cells
remains
largely
unclear.
Human
ES
cells
overexpressing
REX1
were
here
generated
to
investigate
the
role
of
REX1
in
regulating
hES
cell
pluripotent
identity.
These
cells
expressed
similar
levels
of
key
pluripotency
markers
than
their
normal
counterparts
and
remained
capable
of
self-­‐renewing
and
differentiating
into
the
three
germ
layers
in
vitro.
Interestingly,
however,
upon
withdrawal
of
exogenous
Activin
A,
REX1
overexpressing
hES
cells
in
contrast
to
control
cells
retained
a
comparatively
high
level
of
OCT4
and
stained
positive
for
alkaline
phosphatase,
a
known
marker
of
undifferentiated
hES
cells.
Taken
together,
these
results
point
to
a
possible
role
for
REX1
in
sustaining
hES
cell
self-­‐renewal
ability.