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Leukemia inhibitory factor (LIF) is capable of
maintaining embryonic stem (ES) cells in a pluripotent state through
promoting self-renewal or suppressing stem cell
differentiation[1-5]. It has become a standard protocol to use LIF to
maintain murine ES cell pluripotency, whereas withdrawal of
LIF allows ES cells to undergo cell
differentiation[1-7]. Upon withdrawal of both LIF and feeder cells, ES cells are able to
differentiate spontaneously into various cell types in three
primitive layers[8-11].
As a pleiotropic factor, LIF has been proven to have a
wide array of actions during the process of neural
progenitor cell differentiation into neuronal and glial
cells[12-19]. In the central nervous system, the LIF signaling pathway
synergistically functions with other signaling pathways to
inhibit the differentiation of neural stem cells along a glial
lineage[12,13]. However, LIF downstream factor STAT3, in
combination with other factors, selectively promotes fetal neural
stem cell differentiation toward a glial
fate[14-16]. During neural crest cell differentiation, LIF enhances the survival of
sensory neurons and stimulates their formation from neural
crest progenitor cells[17-19]. When ES cells are cultured in a
chemically defined serum-free medium designed for neuronal
cell culture, LIF alone is insufficient to sustain ES cell renewal[5]. Furthermore, when cultivated in low-density and
serum-free conditions, ES cells with neural progenitor
properties can be identified. It seems that LIF is critically
required under these conditions for ES cells to undergo
neuronal colony formation[20]. These observations form the
basis for the hypothesis that LIF might have a role in
mediating the neural development of ES cells.
ES cell transition from pluripotency towards a specific
cell type involves the actions of multiple cytokines.
Removing LIF from culture medium seems to allow ES cells to
differentiate into nearly all kinds of cell types, but it is unclear if
LIF plays a role during ES cell differentiation towards a
specific cell type[8-11,21]. In previous studies, either in retinoic
acid (RA)-induced or non-induced ES cell differentiation,
LIF was removed from culture medium. However, whether
LIF plays a role in inhibiting neural differentiation during
this process remains
unknown[22-24,27,28].
To better understand whether LIF potentially exerts a
role during the conversion of ES cells to a neuronal fate,
embryoid bodies (EB) were obtained from ES cells and plated
for further culture in a serum-containing medium either with
(LIF+) or without LIF (LIF-), and then analyzed for cell
differentiation.
Materials and methods
ES cell culture and embryoid body formation The mouse
ES cell line D3 was obtained from the ATCC (American Type
Culture Collection) organization, and was maintained in an
undifferentiated state with feeder cells in the presence of LIF
(ESGRO; Chemicon USA)[1-5]. The standard culture medium
used in this work for maintaining ES cells contained the
following components: high glucose Dulbecco¡¯s modified
Eagle¡¯s medium (DMEM; Gibco, USA) supplemented with
15% fetal calf serum (Hyclone USA or Sijiqing China), 0.1
mmol/L b-mercaptoethanol (Gibco USA), 1 mmol/L sodium
pyruvate (Gibco USA), 1% non-essential amino acids (Gibco,
USA), 2 mmol/L glutamine (Gibco, USA), 0.1 mg/mL
penicillin-streptomycin (Gibco, USA). LIF was added into the
culture medium at a final concentration of 1000 U/mL before
use. For cell differentiation induction, ES cells were
dispersed into a single cell suspension with 0.25% trypsin
(Gibco, USA). Hanging drops, each of which contained
approximately 1000 cells in a 20 µL volume of culture medium,
were maintained for 3 d on the lids of dishes filled with
phosphate-buffered saline (PBS)[22-24]. EB that formed in the
hanging drops were then plated and cultured on uncoated Petri
dishes or cover-slips (Fisher) for 3 d. Prior to hanging drop
culture, the expression of pluripotent marker genes Oct3/4,
alkaline phosphatase (AP) and SSEA-1 was examined to
ensure that the ES cells were indeed in an undifferentiated state
(data not shown)[1-2]. The optimal procedure, shown in
Figure 1A, includes suspension of ES cells for 3 d to form an EB
(3 d EB), followed by plating EB cells for an additional 3 d (6
d EB). To assess the potential role of LIF during EB
differentiation, the same concentration of LIF as that used in
ES cell-maintaining medium was either included (LIF+, 1000
U/mL) or excluded (LIF-) in culture medium during the
suspension and plating processes.
EB treatment For RA induction, 6 d EB were treated with
10-6-10-5 mol/L all-trans RA (Sigma) for an additional 3 d and
then transferred to fresh medium without
LIF[22-24]. To block the LIF downstream signaling pathway, MEK1/2 specific
inhibitor PD98059 (Calbiochem, USA) was dissolved into
100% EtOH and was added into culture medium with LIF at a
final concentration of 2 µmol/L during either EB formation or
EB plating; JAK/STAT3 specific inhibitor cucurbitacin I
(Calbiochem, USA) was also dissolved into 100% EtOH and
used to inhibit JAK/STAT3 function at a concentration of
300 nmol/L. The 6 d EB were fixed and analyzed with
immunostaining.
Immunocytochemical assays SSEA-1 was obtained from
the Developmental Studies Hybridoma Bank (University of
Iowa, Iowa City, IA, USA). The monoclonal antibody Tuj1,
a neuron-specific marker, was obtained from Sigma (St Louis,
MO, USA), and the antibodies GFAP (Dako), a glial marker,
and the antibodies against GATA-4, an extraembryonic
endoderm marker, and brachyury, a mesoderm marker, were from
Santa Cruz (Santa Cruz, CA, USA). For
immunocytochemical assays, cells were fixed with 4% paraformaldehyde (PFA)
in PBS for 10 min, permeabilized with 0.1% Triton X-100/PBS
for 1 min, washed with PBS 3 times, and blocked with 5%
normal goat serum in PBS for 1.5 h. Samples were then
incubated with primary antibodies in 10% NGS (normal goat
serum)-PBS at 4°C for 12 h. After extensive washing with
PBS, samples were incubated with secondary antibodies in
10% NGS-PBS for 1 h, washed again with PBS and then
mounted in mowiol (Sigma USA). Specimens were analyzed
under a confocal microscope (Zeiss L510 or Leica confocal
microscope).
Cell proliferation and apoptosis assays
Bromode-oxyuridine (BrdU; Sigma USA) was used to label
proliferating cells in 5 d, 8 d (RA treated for 2 d) and 10 d (RA treated
for 3 d and cultured for an additional 1 d) EB. BrdU was
added into the culture medium at a concentration of 10
nmol/L for 12-16 h. Cells were washed with PBS for 10 min and fixed
with 4% PFA for 15-20 min, then washed with PBS again for
15 min. Samples were then incubated in 2 N HCl for 5-0 min,
followed by PBS washing for 15 min. EB were rinsed with
Na2B4O7 (0.1 mol/L) for 5-10 min to neutralize the HCl.
Samples were then immunostained with BrdU antibodies and
analyzed with a confocal microscope. TUNEL
(Promega, USA) staining was performed according to the manufacturer¡¯s
instructions. Specimens were mounted by mowiol and
analyzed with a confocal microscope.
Statistics of apoptosis and proliferation
EB were scanned into stacks of images (5 µm/images) with a confocal laser
microscope. The middle image for each EB was used, and
the number of TUNEL- or BrdU-labeled cells in the image
was automatically counted by IPP (Image-Pro-Plus) software
using the same parameters. The area of EB on each image
was enclosed with the lasso or marquee tool of Photoshop
software. We read out the pixel value of the enclosed area in
a histogram. The number of BrdU- or TUNEL-labeled cells
divided by the pixel value is the relative density of BrdU- or
TUNEL-labeled cells per image, respectively. Thirty EB were
counted in each case. Student¡¯s t-test was used on these
data.
Results
Production of neuronal cells from ES cells was enhanced
by LIF under various differentiation
conditions ES cells in hanging culture formed spheroid aggregates, known as EB,
that can differentiate into a large variety of cell
types[8,9,21-29]. In previous reports, EB formation and differentiation occurred
under LIF-free conditions[22-24]. However, studies regarding
neural progenitor cell differentiation and ES cell neuronal
fate commitment imply that LIF might play roles in ES cell
neuronal transition. To assess this potential function we
cultured LIF under various conditions and investigated
neuronal differentiation thereafter.
The expression of neuronal specific marker Tuj1 was
analyzed by immunostaining primary 6 d EB cultured in either
LIF+ or LIF- medium. As shown in Figure 1, numerous
Tuj1-positive cells were seen surrounding or within LIF+ 6 d EB
(Figure 1B), whereas Tuj1-positive cells were rarely observed
in LIF- 6 d EB (Figure 1C). The percentage of the primary EB
that contained Tuj1-positive cells in LIF+ medium (27/31)
was approximately two times greater than that for EB
cultured in LIF- medium (12/40) (Figure 1D). Moreover, the
average number of Tuj1-positive cells per EB was only 2 in
LIF- medium (Figure 1E), whereas LIF+ EB had an average
of 8.5 Tuj1-positive cells per EB, with up to 20 positive cells
in some extreme cases (Figure 1E; these results were from 6
replicated experiments). To test whether different batches of
sera, different numbers of cell passages, or different cell lines
could potentially influence the result, we repeated the whole
experiment with different batches of sera, with ES cells with
various numbers of passages (8, 12, 15, 20 passages), and
with the feeder-free E14.1 ES cell line, and obtained similar
results (data not shown)[30,31]. LIF is thus strongly
implicated in promoting the differentiation of ES cells to neuronal
cells.
To verify whether LIF has dose-dependent or time-window effects on ES cell differentiation to neuronal cells,
we cultured EB in media containing different concentrations
of LIF (5000 U/mL, 1000 U/mL, 500 U/mL or 0 U/mL). The
numbers of Tuj1-positive cells within each EB were counted
(the experiment was repeated 3 times). The Tuj1-positive
cells per EB when treated with a higher concentration of LIF
outnumber those observed when treated with a lower
concentra-tion, indicating that the enhancement of neuronal
differentiation is indeed dose-dependent within the limited
concentration threshold (Figure 1F). To investigate the step
at which LIF exerts its action in the course of EB plating, LIF
(1000 U/mL) was included in the culture medium at different
time points. We found that neuronal differentiation was
obviously enhanced by the addition of LIF between d 0 and d
5 of culture, especially between d 3 and d 4 (Figure 1G).
Compared with EB cultured in LIF+ medium, irrespective of
when LIF was added, the LIF- EB had the least neuronal
conversion capacity (Figure 1G).
To investigate whether cell-cell interactions could result
in the promotion of neuronal conversion in LIF+ medium, we
cultured ES cells on cover slides at low density for 6 d to
form a monolayer either with or without LIF. After 6 d in
culture, some ES cells in LIF+ medium differentiated into
Tuj1-positive cells, either in groups or individually (Figure
2A), although the ratio was very low. In contrast, there was
no Tuj1-positive cell observed in the culture with LIF-free
medium (Figure 2B). In classical RA-induced neuronal
differentiation experiments, ES cells are cultured under LIF-
conditions, although there is no evidence indicating that
LIF inhibits the neuronal differentiation of ES
cells[22-24]. To test the potential possibility that LIF also has a positive
effect on the RA-induced neuronal differentiation of ES cells,
we carried out RA induction assays. We found that LIF did
enhance neuronal differentiation in the presence of RA, as
evidenced by the fact that LIF+7 d EB (with RA induction for
1 d) generated more Tuj1-positive cells (Figure 2C) than did
LIF-7 d EB (Figure 2D). Our results strongly imply that the
LIF signaling pathway has a positive role during the
neuronal differentiation of ES cells.
LIF signaling pathway is involved in the differentiation
of ES cells to a neural fate LIF plays various roles in
different cell types through gp130-mediated JAK/STAT3 and
ERK/MEK signaling pathways[32-37]. If LIF does have a positive
role in the transition of ES cells to a neuronal fate, this can be
validated by inhibition of the LIF-activated signaling
pathway. The MEK and STAT3 pathways can be inhibited
by different specific chemical
inhibitors[37,38]. When the JAK/STAT3 pathway was blocked by the specific inhibitor
cucurbitacin I (300 nmol/L)[38] during EB formation or EB
plating, neuronal differentiation was largely abolished (Figure
3B). Furthermore, when ES cells were treated with MEK1/2
specific inhibitor PD98059[37] (2 mmol/L) during either EB
formation or EB plating, Tuj1-positive cells appeared within
or surrounding EB, but these Tuj1-positive cells had a round
shape without a long neurite (Figure 3A). Thus, blocking
either LIF downstream signaling pathway interfered with
LIF-enhanced ES cell neuronal differentiation. However,
glial fibrillary acidic protein (GFAP)-positive glial cells are slightly
reduced in the presence of LIF (Figure 3C,3D). Inhibition of
the STAT3 pathway increased the production of
GFAP-positive cells (Figure 3B), whereas there was no GFAP-positive
cell observed when the MEK pathway was inhibited (Figure
3A). This result indicates that both LIF-activated signaling
pathways are involved in the regulation of glial
differentiation from EB.
LIF inhibits cell apoptosis and enhances cell
proliferation during EB differentiation To examine whether apoptosis
and proliferation are also involved in the course of
LIF-enhanced ES cell neuronal differentiation, TUNEL and BrdU
incorporation assays[1,39] were performed in 5 d either in LIF+
(Figure 4A,4D) or in LIF- medium (Figure 4B,4E), respectively.
In the presence of LIF, cell apoptosis was inhibited (Figure
4A-4C) and cell proliferation was enhanced (Figure 4D-4F)
during both EB plating and RA treatment. However, LIF did
not selectively suppress neuroectodermal precursor
apo-ptosis and promote neuroectodermal precursor proliferation.
In BrdU and TUNEL assays with nestin staining, we
observ-ed that nestin-positive cells exhibited signs of apoptosis
and proliferation (data not shown).
LIF selectively enhances the commitment of neural
progenitor from ES cells ES cells are capable of differentiating
into various types of cells, whereas the differentiation of
cardiomyocytes derived from EB can be hindered by
LIF[29]. It remains unclear whether LIF selectively promotes the
production of neuroectodermal precursor cells, or if LIF also
has a role in mesoderm or extraembryonic endoderm cell
lineage commitment. Under LIF+ conditions, a large number of
nestin-positive cells appeared within or surrounding EB
(Figure 5A), whereas EB cultured without LIF had a small
number of nestin-positive cells (Figure 5B), indicating that
LIF promotes the production of neural progenitors from ES
cells. We also evaluated the expression levels of the
mesoderm marker Bra (Brachyury) in 6 d EB cultured either with or
without LIF[40]. The number of brachyury-positive cells in 6
d EB with LIF was significantly smaller than that for 6 d EB
without LIF (Figure 5B,5C). In addition, it has been reported
that ectopic GATA-4 expression is sufficient to induce ES
cell differentiation into extraembryonic
endoderm[41]. We found that in the absence of LIF the 6 d EB had a larger
number of GATA-4-expressing cells surrounding the EB,
whereas GATA-4 expression was dramatically reduced in 6 d
EB with LIF (Figure 5D,5E). We therefore suggest that LIF
potentiates the differentiation of ES cells to a neural fate,
and might suppress both the mesoderm and extraembryonic
endoderm fates.
Discussion
In this work, we observed that neuronal differentiation
of ES cells was enhanced in the presence of LIF. This
enhancement cauld be abolished when the JAK/STAT3
pathway was inhibited. Furthermore, inhibition of the MEK
signaling pathway impaired the differentiation of ES cells
toward a glial lineage. During this process, LIF inhibited cell
apoptosis and promoted cell proliferation, and EB
differentiation to form both mesodermal and extraembryonic
endodermal cells ws inhibited, whereas the production of neural
progenitor cells was increased. Thus our observations
imply that the LIF signaling pathway is involved in ES cell
differentiation into a neuronal cell fate, and provide cues for
further investigations of this pathway.
During early vertebrate embryonic development, neural
induction has been thought to be a default process. In the
default model, the inhibition of the BMP (bone
morphogenetic proteins) signaling pathway is necessary for neural
cell fate determination during early embryonic develop-ment[42-44]. However, it remains unknown whether neural
induction requires the participation of other signaling
pathways other than simply BMP depletion. The clue that LIF
might be involved in ES cell neural differentiation comes
from the fact that in low-density and serum-free culture
conditions, ES cells with neural progenitor properties were
identified[20]. Under these culture conditions, LIF was
required for ES cells to undergo neuronal colony formation. In
the present report we provided evidence that LIF could
enhance the production of neural progenitor cells from ES cells,
and support the neuronal differentiation of EB in a
dose-dependent manner (Figure 1). Furthermore, in the presence
of LIF, the commitment of ES cells to both mesodermal and
extraembryonic endodermal fates was suppressed (Figure
5). The LIF pathway also regulates neural differentiation
into neuronal and glial fates. Our results not only indicate
that LIF signaling is indeed involved in ES cell neural
commitment, but also strongly suggest that the LIF pathway
has multiple functions in different stages of ES cell
differentiation.
A recent study has shown that LIF/Stat3 cooperated with
BMP/Smad signaling to maintain ES cells in an
undifferentiated state[5]. It seems that the balance between LIF/Stat3
and BMP/Smad signaling is critical for the choice between
sustaining pluripotency or lineage commitment of ES cells.
Thus the activation of the LIF signaling pathway reported
here may account for promoting more ES cells to adapt to a
neural cell fate, whereas LIF withdrawal leads to an elevated
BMP signaling activity, which further stimulates ES cells to
differentiate into mesodermal and endodermal cells. It would
be of great interest to further investigate how BMP and LIF
signaling pathways cross-talk during ES cell neural
commitment.
Multiple lines of evidence indicate that LIF plays
various roles in different types of neural stem cell
differentia-tion[12-19]. In the central neuronal system, the LIF signaling
pathway synergistically cooperates with other signaling pathways to inhibit neural stem cell differentiation to a glial
fate[12,13]. However, LIF downstream factor STAT3, together
with BMP downstream effector SMAD1, selectively promotes
the glial differentiation of fetal neural stem
cells[14-16]. Furthermore, during neural crest cell differentiation, LIF
promotes sensory neuron differentiation and
survival[17-19]. The LIF pathway requires cooperation with different factors to
achieve its function as a pleiotropic cytokine. In our
experiments, inhibition of the LIF-activated STAT3 pathway
did not interfere with the production of GFAP+ cells, but
abolished the differentiation of Tuj1+ cells from ES cells.
This result is consistent with the finding that Ngn1 inhibits
gliogenesis through inhibiting the activation of STAT
transcription factors during neural differentiation
[12]. Further-more, blocking the LIF-activated MEK pathway abolished
the glial differentiation of ES cells. Our results suggest that
LIF signaling might interact with other pathways to exert
various effects in the differentiation of different neural cell
types. In addition, we found that LIF unselectively inhibited
cell apoptosied and supports cell proliferation during ES cell
neural differentiation. However, we consider that it would
be worthwhile to further investigate the possibility that LIF
affects the survival and proliferation of neuronal cells by
using more specific cell markers.
LIF is not the only factor that has dual roles in ES cell
culture. A recent report indicates that Oct3/4 also has dual
roles in murine ES cell culture: sustaining ES cell self-renewal and taking part in ES cell fate determination. The
precise level of Oct4 in ES cells controls the commitment of
ES cells to undergo 3 different cell
fates[1]. Ectopic expression or overexpression of Oct3/4 also appears to enhance
neuronal differentiation in SDIA (stromal cell-derived
inducing activity)-induced ES cell
differentiation[45,46]. However, it is unclear whether LIF signaling interacts with the Oct3/4
signaling pathway in ES cell neuronal differentiation, but
this certainly warrants further investigation in the future.
Acknowledgements
We thank Dr Hui-zhen SHENG and Dr Nai-he JING for
their technical advice; Dr Peng XIANG for providing the
E14.1 cell line; and Prof Yi-ping CHEN, Prof Peter REINACH
and Dr Shuang-wei LI for comments on the manuscript. We
thank Min-ying LIU for technical assistance.
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