Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures
Koji Shimozaki1,
Kinichi Nakashima1,*,
Hitoshi Niwa2 and
Tetsuya Taga1,*
1 Department of Cell Fate Modulation, Institute of Molecular Embryology and
Genetics, Kumamoto University, Kumamoto, 860-0811, Japan
2 Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental
Biology, Kobe, 650-0047, Japan
*
Authors for correspondence:
taga{at}kaiju.medic.kumamoto-u.ac.jp
and
kin{at}kaiju.medic.kumamoto-u.ac.jp
Accepted 10 March 2003
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SUMMARY
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Oct3/4 plays a critical role in maintaining embryonic stem cell
pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES
cells cultured in serum-free LIF-deficient medium caused accelerated
differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2
and subsequently differentiated into neurons. Neurogenesis of ES cells is
promoted by SDIA (stromal cell-derived inducing activity), which accumulates
on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained
by SDIA whereas without it expression was promptly downregulated. Suppression
of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA.
In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated
neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm
formation and subsequent neuronal differentiation from ES cells.
Key words: Oct3/4, SDIA, ES cells, Neurogenesis, LIF, Mouse
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INTRODUCTION
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During mammalian development, the initial step in the generation of the
nervous system is the process of neural induction. However, little is known
about the regulatory mechanisms governing mammalian neural induction. Mouse
embryonic stem (ES) cells, which can contribute in vivo to the formation of
all tissues, have provided a useful means to answer this question. It has
recently been reported that neurogenesis of ES cells in vitro without the
formation of embryoid bodies and retinoic acid treatment can be achieved by
SDIA (stromal cell-derived inducing activity), which accumulates on the
surface of the stromal cell line PA6
(Kawasaki et al., 2000
). In
character, ES cells resemble the inner cell mass (ICM) cells formed on
embryonic day (E) 3.5, from which ES cells are derived
(Bradley et al., 1984
). The
neural fate of the ICM cells in vivo is thought to be determined later in
development on E6.5-E8 during gastrulation, when neural precursor cells start
to be formed (Schoenwolf and Smith,
1990
). The time course of neural marker induction in
SDIA-stimulated ES cells in vitro was shown to correlate well with that seen
in the embryo (Kawasaki et al.,
2000
). Thus, the neurogenesis-inducing system with SDIA is thought
to mimic the neurogenesis that occurs in vivo, but its molecular basis remains
unknown.
Addition of leukemia inhibitory factor (LIF) is sufficient to establish and
maintain mouse ES cells without feeder cells in the presence of fetal calf
serum (Nichols et al., 1990
),
and that activation of the transcription factor STAT3 in the signaling is
sufficient to maintain the undifferentiated state of mouse ES cells
(Matsuda et al., 1999
;
Niwa et al., 1998
). Besides
STAT3, another transcription factor, Oct3 (also known as Oct4; hereafter
referred to as Oct3/4) is known to be important for the maintenance of ES
cells. Oct3/4 is a POU-family transcription factor which has been confirmed to
function specifically in pluripotent cell populations
(Okamoto et al., 1990
;
Rosner et al., 1990
;
Scholer et al., 1990b
). Oct3/4
expression has been detected in the oocyte, blastocyst and embryonic ectoderm
before gastrulation, is down-regulated in the neural tube from approximately
E8.5, and thereafter becomes restricted to germ cells
(Rosner et al., 1990
;
Scholer et al., 1990a
).
Targeted gene disruption has revealed the essential role of Oct3/4 in mouse
development (Nichols et al.,
1998
). Oct3/4-deficient embryos fail to initiate fetal development
by a loss of the establishment of a pluripotent population of cells in the
preimplantation embryo. These cells give rise to tissue-specific stem cells in
mammalian embryogenesis, but very few experimental studies have suggested
possible mechanisms underlying early neural differentiation.
Oct3/4 has been suggested to be a candidate for the master regulator of
initiation, maintenance and differentiation of pluripotent cells. A recent
investigation using the conditional Oct3/4 repression or expression system in
ES cells has revealed that the precise level of this transcription factor is
important for the maintenance of stem cell self-renewal
(Niwa et al., 2000
). It
remains an interesting question whether up- or down-regulation of Oct3/4
affects neuroectoderm differentiation of ES cells.
In this report, we show that sustained upregulation of Oct3/4 in ES cells
in serum-free LIF-deficient medium leads to efficient neuroectoderm formation
and subsequent neuronal differentiation. We further show that ES cells
cultured on a PA6 monolayer to induce neurogenesis continue to express Oct3/4.
It is thus suggested that Oct3/4 promotes neuronal differentiation of ES cells
under neurogenesis-inducing conditions.
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MATERIALS AND METHODS
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Cell culture
Undifferentiated ES cells (EB5, ZHTc6 and ZHBTc4) were maintained in an
undifferentiated state on gelatin-coated dishes as described previously
(Kawasaki et al., 2000
;
Niwa et al., 1998
;
Niwa et al., 2000
). For
differentiation, ES cells were cultured on a fixed monolayer of PA6 feeder
cells or poly-L-ornithine and fibronectin (O/F)-coated dishes in G-MEM medium
supplemented with 10% KSR (Gibco-BRL), 2 mM glutamine, 1 mM pyruvate, 0.1 mM
nonessential amino acids, and 0.1 mM 2-ME. PA6 cells were grown to confluency,
fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature, and
rinsed with PBS several times before plating ES cells on them. In this
SDIA-stimulation method, ES cells were plated on fixed PA6 cells at a density
of 2x104 cells per 6 cm dish. In the ZHTc6 cell
differentiation culture without PA6, the cells were seeded at a density of
1x105 cells per 6 cm O/F-coated dish in the ES-maintaining
medium overnight, washed twice with PBS, and then cultured in N2-supplemented
DMEM/F-12 medium in the presence or absence of 10% FBS with or without 1
µg/ml of tetracycline-HCl (Sigma) on O/F-coated dishes. In some cases 10
ng/ml of Bmp2 (Yamanouchi Pharmaceutical) or 150 ng/ml of BmpßR-Fc
(R&D Systems) were added to the medium, and the Bmp2- or
BmpßR-Fc-containing medium was exchanged for fresh medium every other day
till the end of the culture.
Immunocytochemistry
Immunofluorescent staining was performed with the following antibodies: an
anti-MAP2 monoclonal antibody (Sigma) and an anti-nestin polyclonal antibody
(provided by K. Yoshikawa, Osaka Univ.). The following secondary antibodies
were used: an Alexa 488-conjugated goat anti-mouse IgG antibody (Molecular
Probes), and a rhodamine-conjugated donkey anti-rabbit IgG antibody (Chemicon,
Temecula, CA). The cells were counterstained with Hoechst 33258 to identify
the nuclei. Images were obtained using fluorescence microscopy (AX70
microscope; Olympus, Tokyo).
RNA analysis
For RT-PCR analysis, we performed oligo(dT)-primed reverse transcription on
aliquots (5 µg) of total RNA and used 1/100 of the resultant single strand
cDNA products for each PCR amplification. Primer sets, with which all cDNAs
were amplified in a quantitable range, are listed in
Table 1.
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RESULTS
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Upregulation of Oct3/4 expression in ES cells induces effective
neurogenesis under serum-free LIF-deficient conditions
For the maintenance of ES cell self-renewal it has been demonstrated that
Oct3/4 expression must remain within plus or minus 50% of the normal level,
and that up- or down-regulation of Oct3/4 beyond this range triggers
differentiation into primitive endoderm/mesoderm and trophectoderm,
respectively (Niwa et al.,
2000
). Since neuroectoderm differentiation of ES cells under such
conditions has not been examined, we first attempted to analyze neuronal
marker expression in ZHTc6 ES cells with a regulatable Oct3/4 transgene, which
had been established previously (Niwa et
al., 2000
). For this, ZHTc6 cells were cultured in either the
presence or absence of Tc on poly-L-ornithine/fibronectin (O/F)-coated dishes
in N2-supplemented serum-free DMEM/F12 without LIF. We applied this culture
condition to ES cells because this is the one under which we had routinely
induced neuronal differentiation from fetal mouse neuroepithelial cells in
vitro (Takizawa et al., 2001
).
As shown in Fig. 1A, a large
number of MAP2-positive neurons emerged in the culture without Tc, in which
ZHTc6 cells are known to exhibit sustained expression of Oct3/4 from the
transgene. In contrast, only a small number of MAP2-positive neurons were
differentiated from ZHTc6 cells with no transgene expression, when Tc was
present in the medium. Cells positive for other neuronal markers, ßIII
tubulin and neurofilament-M, also emerged efficiently by the Oct3/4
expression. Astrocytes and oligodendrocytes, however, did not appear within
the 10-day culture, either with or without Tc (data not shown). The results
suggest that sustained upregulation of Oct3/4 expression leads to efficient
neuronal differentiation of ZHTc6 cells under the serum-free LIF-deficient
culture condition. A time course study showed that continuous Oct3/4
upregulation in the culture without Tc led to earlier and more extensive
differentiation of neurons (Fig.
1B). In this culture system, Oct3/4 did not induce any detectable
apoptosis. These data imply that sustained upregulated expression of Oct3/4
accelerates the neuronal differentiation of ES cells under these culture
conditions.

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Fig. 1. Efficient neurogenesis of ES cells under serum-free LIF-deficient
conditions. ZHTc6 ES cells were established
(Niwa et al., 2000 ) in which
expression constructs for a tetracycline (Tc)-regulated transactivator (tTA)
transgene and a tTA-responsive Oct3/4 transgene were introduced. The ZHTc6
cells with no transgene expression in the presence of Tc showed induced Oct3/4
expression the level of which is comparable to that from the endogenous
allele. Withdrawal of Tc triggers the transgene expression, which induces
Oct3/4 expression at a level 50% above that of Oct3/4 expression in normal ES
cells. (A) ZHTc6 cells were cultured in serum-free N2-supplemented DMEM/F-12
without LIF for 8 days on poly-L-ornithine/fibronectin (O/F)-coated dishes in
the presence (a) or absence (b) of Tc. Cells were stained with an anti-MAP2
antibody (green) and Hoechst 33258 (blue). Upregulation of Oct3/4 induced by
withdrawal of Tc promoted neurogenesis of ES cells. Scale bar, 50 µm. (B)
ZHTc6 cells were cultured as in A with or without Tc for 10 days. The
frequency of MAP2-positive cells with respect to the total number of cells was
analyzed every 2 days. Vertical bars indicate the s.d.
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Continuous upregulation of Oct3/4 expression induces neuroectoderm
differentiation from ES cells under serum-free LIF-deficient conditions
We next examined the expression of neural and non-neural marker genes in
ZHTc6 cells described above cultured with or without Tc. Sox2 is one of the
earliest known transcription factors to be expressed in ICM and its expression
continues throughout neural tube development
(Wood and Episkopou, 1999
). As
shown in Fig. 2A, Sox2 mRNA was
highly expressed in the differentiated ZHTc6 cells cultured without Tc to
induce continuous upregulated expression of Oct3/4. The neural markers Otx1
(Acampora et al., 1998
) and
Emx2 (Simeone et al., 1992
)
were also obviously expressed in ZHTc6 cells cultured without Tc. To examine
the non-neural cell fate of ZHTc6 cultured without Tc, we analyzed the
expression of an epidermal marker, cytokeratin-17 (CK-17)
(McGowan and Coulombe, 1998
),
and an early endodermal marker, Gata4
(Arceci et al., 1993
). As shown
in Fig. 2, CK-17 expression was
not detectable in ZHTc6 cells cultured without Tc and Gata4 expression was
lower than in ZHTc6 cells cultured with Tc. These results suggest that
continuous expression of Oct3/4 may play a role in guiding the fate of ES
cells towards neural differentiation under certain conditions where there are
no neural inhibitors, such as those contained in serum, e.g. bone
morphogenetic proteins (Bmps).

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Fig. 2. Upregulation of Oct3/4 expression in ES cells induces neural marker gene
expression under serum-free LIF-deficient conditions. (A) ZHTc6 cells were
cultured for 8 days in the presence or absence of Tc. Total RNA was extracted
from each culture and subjected to RT-PCR analysis for the genes indicated on
the right. (B) RNA was extracted from each ZHTc6 cell culture on days 2, 4 and
8, and subjected to RT-PCR analysis for Fgf5 expression.
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In mouse embryogenesis, the neuroectoderm is formed after the primitive
ectoderm stage (Tam, 1989
).
The results described above suggest that sustained upregulation of Oct3/4
expression in ES cells might have accelerated the formation of primitive
ectoderm and forced these cells to go beyond this transit stage towards
further developmental stages. To evaluate whether sustained upregulation of
Oct3/4 expression has any effect on the primitive ectoderm formation in our
culture system, we examined the expression of Fgf5, a marker of primitive
ectoderm (Hebert et al., 1991
).
In a time course study, the expression kinetics of Fgf5 in ZHTc6 cells
cultured in the presence or absence of Tc was different
(Fig. 2B). In the absence of
Tc, the expression of Fgf5 was significant on day 2 but subsequently decreased
and disappeared on day 8, whereas Fgf5 expression in the presence of Tc was
significant from day 2 until day 4 and decreased but still remained detectable
on day 8. These results suggest that ES cells with sustained upregulated
expression of Oct3/4 tend to go beyond the primitive ectoderm stage to the
neuroectoderm stage more efficiently.
We next examined whether the Oct3/4-induced neuronal differentiation of ES
cells is affected by anti-neurogenic factors. Neural inducers have been shown
to act by antagonizing Bmps in the extracellular space
(Hemmati-Brivanlou and Melton,
1997
; Sasai and De Robertis,
1997
). As shown in Fig.
3D, addition of 10 ng/ml Bmp2 resulted in complete suppression of
the neuronal differentiation of ZHTc6 cells in serum-free LIF-deficient
culture without Tc. Upregulated expression of Oct3/4 by omission of Tc does
not therefore appear to sufficiently antagonize the Bmp signaling. Bmp
antagonists such as chordin and noggin are known to be neural inducers
(Hemmati-Brivanlou and Melton,
1997
). We examined the effect of a BmpR-Fc fusion protein, which
has the ability to block Bmp signaling, on the ZHTc6 cell culture. BmpR-Fc did
not affect the neurogenesis from ZHTc6 cells either with or without Tc
(Fig. 3E,F). Thus, the
neurogenic effect of sustained upregulation of Oct3/4 does not appear to be
simply due to the induction of Bmp-neutralizing factors. It should be noted
that, in contrast to the serum-free conditions, no MAP2-positive cells were
detected in the serum-containing culture regardless of the sustained
upregulation of Oct3/4 expression (Fig.
3G,H). Addition of LIF also inhibited the Oct3/4-induced neuronal
differentiation of ES cells even under serum-free conditions (K.S. and T.T.,
unpublished data), indicating that the withdrawal of both serum and LIF is
necessary for Oct3/4-induced neurogenesis of ES cells.

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Fig. 3. Neurogenic function of Oct3/4 is not explained by the inhibition of cell
autonomic Bmp signaling. MAP2 staining (green) of ZHTc6 cells cultured for 8
days with (A,C,E,G) or without (B,D,F,H) Tc. In some cultures, Bmp2 (10 ng/ml;
C,D), BmpßR-Fc (150 ng/ml; E,F), or 10% serum (G,H) were added. Nuclei
were stained with Hoechst 33258 (blue). Scale bar, 50 µm.
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Maintenance of Oct3/4 expression in mouse ES cells during
SDIA-induced neurogenesis
The above results reminded us of the SDIA-induced neurogenesis of ES cells
in which neurogenic differentiation is dramatically promoted by culture on PA6
stromal cells under serum-free LIF-deficient conditions. EB5 ES cells, which
have normal Oct3/4 alleles rather than the Tc-regulated Oct3/4 transgene, were
cultured in serum-free LIF-deficient medium on dishes precoated with either
O/F or a monolayer of PA6 cells. As shown in
Fig. 4A-F, when ES cells were
cocultured on a PA6 monolayer, the cells expressed nestin, a marker of neural
progenitors, very efficiently after 4 days. MAP2-positive neurons frequently
emerged after 8 days, as has been previously reported
(Kawasaki et al., 2000
). When
the cells were cultured on the O/F-coated dishes, only a few MAP2-positive
cells were detected. When EB5 cells were cultured on a gelatin-coated dish (a
negative control for the dish used for preparing the PA6 monolayer), the cells
differentiated into neurons at a very low frequency, as in the case of the
culture on the O/F-coated dishes (data not shown). Taken together with the
observations of the ZHTc6 ES cell cultures, these results prompted us to
examine the Oct3/4 expression during SDIA-induced neurogenesis. As shown in
Fig. 4G, the Oct3/4 expression
was rapidly downregulated in ES cells when the cells were cultured on
O/F-coated dishes. In contrast, the expression of Oct3/4 was maintained when
the cells were cultured on PA6 cells. Consistent with the previous report
(Kawasaki et al., 2000
), the
expression of the mesencephalic dopaminergic neuron marker Nurr1 was
effectively induced in ES cells cultured on PA6 cells
(Fig. 4G).

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Fig. 4. Effective SDIA-induced neurogenesis of ES cells accompanied by maintenance
of Oct3/4. Expression EB5 ES cells were cultured on O/F-coated dishes (A,C,E)
or a monolayer of PA6 cells (B,D,F). Cells were stained with anti-nestin (red)
and anti-MAP2 (green) antibodies on each day indicated in the figure. Scale
bar, 50 µm. (G) RT-PCR analysis of the expression of Oct3/4 and a
mesencephalic dopaminergic neuron marker, Nurr1, in EB5 ES cells cultured as
in A-F.
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Maintenance of Oct3/4 expression is involved in SDIA-induced
neurogenesis
Considering the above results, we wanted to know the consequences of
suppression of Oct3/4 expression during SDIA-mediated neurogenesis using
ZHBTc4 ES cells. This line of ZHBTc4 cells was established by disrupting the
remaining endogenous Oct3/4 allele in ZHTc6 cells using a targeting
construct with a Tc-regulated transgene consisting of Oct3/4 driven by hCMV*-1
promoter. In ZHBTc4 cells, Oct3/4 expression is completely shut down by Tc
(Niwa et al., 2000
). The
undifferentiated pluripotential state of ZHBTc4 cells can be maintained in
serum-containing medium with LIF in the absence of Tc. When ZHBTc4 cells were
stimulated with SDIA by culturing on a PA6 monolayer without Tc, the cells
differentiated into neurons as effectively as ZHTc6 cells in the absence of
Tc. Interestingly, there were no MAP2-positive cells when ZHBTc4 cells were
cultured with Tc to shut off the Oct3/4 expression
(Fig. 5B and the second column
in 5E). These data indicated
that Oct3/4 expression was essential for the generation of neurons by
SDIA-mediated neurogenesis. The MAP2-negative cells were flat, which is
reminiscent of trophectodermal cells, although we have not yet investigated
these cells further. We also examined the effect of SDIA on the neurogenesis
of ZHTc6 cells cultured without Tc to induce upregulated expression of Oct3/4
from the regulatable transgene. As shown in
Fig. 5C, D and the right half of
E, neurogenesis of ZHTc6 cells induced on a PA6 monolayer was
promoted by the upregulation of Oct3/4 expression caused by the withdrawal of
Tc. In order to show the time course of the appearance of nestin-positive
cells and MAP2-positive cells from SDIA-stimulated ZHTc6 cells, the cells were
stimulated by SDIA with or without Tc, and subjected to immunostaining every 2
days with anti-nestin and anti-MAP2 antibodies. As shown in
Fig. 6, the sustained
upregulation of Oct3/4 expression caused by Tc withdrawal further accelerated
and enhanced the SDIA-induced differentiation of nestin-positive neural
progenitors and MAP2-positive neurons.

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Fig. 5. Requirement of Oct3/4 for SDIA-induced neurogenesis. (A,B) ZHBTc4 and (C,D)
ZHTc6 cells were cultured on a PA6 monolayer in serum-free LIF-deficient
medium with (B,C) or without (A,D) Tc for 8 days. The cells were stained with
an anti-MAP2 antibody (green) and Hoechst 33258 (blue). In ZHBTc4 Oct3/4
expression can be shut off by adding Tc. (E) MAP2-positive colonies were
counted. Scale bar, 100 µm.
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Fig. 6. Enhancement of SDIA-induced neurogenesis by upregulation of Oct3/4
expression. ZHTc6 cells were cultured with (open symbols) or without (closed
symbols) Tc on a PA6 monolayer. The frequency of nestin-positive (circles) and
MAP2-positive (triangles) colonies with respect to the total number of
colonies on each day, as indicated, was calculated.
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We next attempted to determine the duration of the expression of Oct3/4
sufficient to mediate SDIA-induced neurogenesis. During the culture of ZHBTc4
cells on a PA6 monolayer, Tc was added to the medium on days 1, 2, 3, 4 and 5
until the end of the 8 day culture, to shut off the Oct3/4 expression on and
after the respective time points (Fig.
7A, yellow bars). As shown in
Fig. 7B, MAP2-positive colonies
and nestin-positive colonies were consistently observed even when the Oct3/4
expression was shut off on and after day 5. No nestin- or MAP2-positive cells
were detected in the culture in which Oct3/4 expression was shut off on and
after day 1 (Fig. 7B). Although
we could detect nestin- or MAP2-positive cells in the culture of ZHBTc4 cells
in which the Oct3/4 expression was shut off on and after day 2 or day 3, the
frequency of such colonies and their size were small
(Fig. 7B,C). When the Oct3/4
expression was prolonged until day 4, the colony size as well as the number of
cells positive for nestin or MAP2 was increased. Even under these culture
conditions, however, the frequency of colonies containing nestin- or
MAP2-positive cells with respect to the total number of colonies was
approximately half of that in the culture without shutting off the Oct3/4
expression. Thus, Oct3/4 expression for 5 days from the beginning of the
culture period appears to be necessary for SDIA to induce efficient
neurogenesis in ES cells. To evaluate the susceptibility of ZHBTc4 cells to
the anti-neurogenic cytokine Bmp2 under the SDIA-induced neurogenic culture
conditions, the cells were cultured on a PA6 monolayer with Bmp2 added at
different time points. As shown in Fig.
8, differentiation of nestin-positive cells and MAP2-positive
cells was sensitive to Bmp2 when this cytokine was included in the culture on
days 2, 3 or 4 until day 8. MAP2-positive colonies and nestin-positive
colonies were observed when Bmp2 was added from day 5 on. It appears that a 5
day stimulation of ZHBTc4 cells by SDIA is sufficient for committing to
neuronal lineages.

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Fig. 7. Temporal requirement of Oct3/4 for SDIA-induced neurogenesis. ZHBTc4 cells
were cultured on a PA6 monolayer for 8 days and stained with anti-nestin and
anti-MAP2 antibodies. (A) Tc was added to the medium on and after the
indicated days to shut off the Oct3/4 expression until the end of culture. (B)
Frequencies of nestin- and MAP2-positive colonies with respect to the total
number of colonies are indicated. Vertical bars indicate the s.d. (C)
Representative views of cultured ZHBTc4 cells immunostained on indicated days
(anti-nestin, red; anti-MAP2, green). Scale bar, 100 µm.
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Fig. 8. Suppression of neural differentiation by Bmp2. ZHBTc4 cells were cultured
on a PA6 monolayer for 8 days. Bmp2 (10 ng/ml) was added to the medium from
the indicated days until the end of culture. On day 8, cells were stained for
nestin and MAP2, and positive colonies were counted.
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DISCUSSION
|
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Pluripotent ES cells can differentiate into a wide variety of cell types,
and the differentiation of ES cells appears to be under the control of
cell-intrinsic nuclear factors and cell-external cues. Regarding the
cell-intrinsic factors, Oct3/4 has important roles in the maintenance of the
pluripotentiality of ES cells (Nichols et
al., 1998
). When the Oct3/4 expression level goes beyond
±50% of the normal level, ES cells lose the pluripotency
(Niwa et al., 2000
). As for
the cell-extrinsic cues, LIF is important for ES cell pluripotency, and under
the LIF-deficient condition, PA6 cell-derived SDIA, for instance, has been
shown to induce the neuronal differentiation of ES cells
(Kawasaki et al., 2000
). It is
likely that PA6 cells provide ES cells with a cue to trigger neurogenic
pathways but its molecular nature and intracellular signaling mechanisms are
unknown. In the present study, we have shown that sustained expression of
Oct3/4 involves PA6-induced neurogenesis, and that forced upregulation of
Oct3/4 in serum-free LIF-deficient medium efficiently induces neurogenesis of
ES cells without feeder cells. This neurogenesis of ES cells was strongly
suppressed by serum or Bmp2. However, addition of a Bmp-antagonist to the
culture did not by itself enhance the neuronal differentiation. Thus,
inhibition of Bmp signals is not the mechanism by which continuous Oct3/4
causes mouse ES cells to differentiate into neural lineages. It is suggested
that Oct3/4 has an effect on ES cells to enhance their neuronal
differentiation pathway under neurogenic culture conditions. It may be worth
noting that the ZHBTc4 cells on O/F-coated dishes without SDIA, under
serum-free LIF-deficient conditions showed efficient neurogenesis in the
absence of Tc; conditions under which Oct3/4 is continuously expressed from
the transgene (data not shown). When ZHBTc4 cells were stimulated by SDIA, the
cells differentiated into neurons as effectively as ZHTc6 cells in the absence
of Tc (Fig. 5), indicating that
persistence, not up-regulation, of Oct3/4 induces neurogenesis. SDIA may
contain a factor that cues ES cells into the neurogenic pathway as well as a
signal that leads to sustained Oct3/4 expression. Although the signaling
pathway that links SDIA and Oct3/4 remains to be elucidated, there are several
orphan nuclear receptors that regulate the repression of Oct3/4 expression
(Fuhrmann et al., 1999
). In
those genes, germ cell nuclear factor (GCNF)
(Chen et al., 1994
) contributes
to the repression of Oct3/4 gene expression during early mouse embyogenesis
(Fuhrmann et al., 2001
). A
possible mechanism whereby SDIA maintains Oct3/4 expression is that the
signals from SDIA might control such negative regulators directly or
indirectly. Although SDIA was reported to induce TH-positive dopaminergic
neurons efficiently, we could not detect TH-positive neurons from ZHTc6 ES
cells cultured on the O/F-coated dish without Tc. The level of Nurr1
expression in ZHTc6 cells that were forced to upregulate Oct3/4 expression was
not as high as that observed in ES cells cultured on a PA6 monolayer (data not
shown). Thus, it is suggested that SDIA promotes the differentiation of
dopaminergic neurons via an unknown additional factor (or factors) besides the
one that leads to the maintenance of Oct3/4 expression.
Recently, differentiation of ES cells into neuroectoderm-like cells was
shown using HepG2 cell-conditioned medium
(Rathjen et al., 2002
).
Maintenance of Oct3/4 expression was detected in ES cells during such
neuroectoderm-like cell formation. Also, recent work on the zebrafish
spiel-ohne-grenzen (spg) mutation, which causes abnomal
hindbrain formation (Burgess et al.,
2002
; Reim and Brand,
2002
), found that the spg mutation disrupts the
pou2 gene, which encodes a POU homeobox transcription factor. This
work also showed by phylogenetic sequence analysis that pou2 is the zebrafish
ortholog of mouse Oct3/4 and human POU5F1 and that mouse Oct3/4 can
functionally replace zebrafish pou2 in a spg mutant rescue
experiment. It is suggested that pou2 functions not only in an early
developmental stage, like Oct3/4 in the inner cell mass of mammals, but also
in a later stage when the brain primordium develops.
It was reported by Kawasaki et al.
(Kawasaki et al., 2000
) that
the time course of neural marker induction by SDIA was, at least in part,
reminiscent of that observed in the developing central nervous system (CNS).
Others have also reported that neural stem cell formation from ES cells was
preceded by a primitive neural stem cell stage
(Tropepe et al., 2001
). The
term primitive neural stem cell has been used to describe a stem cell that
retains a certain degree of pluripotency during the restricted early period of
neural development when neuroectoderm is formed
(Morrison et al., 1997
). In
our present study, sustained expression of Oct3/4 in ES cells promoted the
formation of primitive ectoderm and subsequent neuroectoderm-like cells under
serum-free conditions (Figs 1
and 2). In these processes,
neural commitment seems to be determined during the first 5 days (Figs
7 and
8). The results of this study
suggest that Oct3/4 is involved in the enhancement of neuroectoderm formation
and subsequent neural differentiation.
It is known that the POU family of transcription factors can act as both
transcriptional activators and repressors by cooperating with various
co-factors. Several different co-factors for Oct3/4 have been reported. The
Sry-related factor Sox2 was initially identified as a co-factor for Oct3/4 to
activate the Fgf4 gene promoter (Yuan et
al., 1995
). Mouse Sox2 is expressed in the blastocyst ICM,
embryonic ectoderm, and germ cells. After gastrulation, Sox2 is expressed in
the neural tube from the earliest stage of its formation
(Zappone et al., 2000
). Sox2
is suggested to be essential for early neuroectoderm cells to consolidate
their neural identity during the subsequent steps of neural differentiation.
Oct3/4 and Sox2 are known to cooperate to activate the transcription of
several genes, and it was recently reported that these two genes activate
their own transcription (Tomioka et al.,
2002
). We have also found putative binding sites for Oct3/4 and
Sox2 in both the promoter and enhancer regions of the Sox2 gene, and
observed the synergistic activation of the Sox2 promoter by Sox2 and Oct3/4
transcription factors in mouse E14.5 neuroepithelial cells (Supplemental data:
http://dev.biologists.org/supplemental/).
Sustained expression of Oct3/4 in ES cells under a LIF-deficient condition, as
in our present study, may lead to the effective induction of Sox2 expression,
which could be responsible for the effective induction of neuroectoderm
formation. Thus, a possible role of sustained Oct3/4 expression in such a case
could be to induce Sox2 expression in cells that are on the way to neural
differentiation under serum-free LIF-deficient condition where Smads and STAT3
signals are diminished. Although we have not yet clarified the relationship
between Oct3/4 and other molecules concerned with neural differentiation, we
speculate that Oct3/4 regulates the function of neural cell-inducing
transcription factors in primitive neural development.
 |
ACKNOWLEDGMENTS
|
---|
We thank Yamanouchi Pharmaceutical for the Bmp2 protein and Dr K. Yoshikawa
(Osaka University) for the anti-nestin antibody. We thank Y. Noguchi for
secretarial assistance and K. Kaneko for technical help. This work was
supported by a Grant-in-Aid from the Ministry of Education, Science, Sports
and Culture of Japan, Human Frontier Science Program, and the Virtual Research
Institute of Aging of Nippon Boehringer Ingelheim.
 |
Footnotes
|
---|
Supplemental data available online
 |
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