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Cytotechnology 36: 137–144, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands. 137
Neural stem cells lose telomerase activity upon differentiating into
astrocytes
Takumi Miura1, Yoshinori Katakura1∗, Katsuhiko Yamamoto1, Norihisa Uehara1, Toshie
Tsuchiya2, Eun-Ho Kim3& Sanetaka Shirahata1
1Department of Genetic Resources Technology, Kyushu University, Fukuoka 812-8581, Japan; 2Division of
Medical Devices, National Institution of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan;
3Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
(∗Author for correspondence; E-mail: katakura@grt.kyushu-u.ac.jp; Fax: +81 92 642 3050)
Received 12 December 2000; accepted 9 June 2001
Key words: astrocytes, differentiation, neural stem cells, telomerase, TGF-β
Abstract
Serum-free mouse embryo (SFME) cells were established by D. Barnes et al., and are known to be a neural stem
cell line, which differentiate into astrocytes upon treatment with TGF-β. Therefore, SFME cells is thought to be a
model well suited to analyze the differentiation mechanism of neural stem cells. Until now, we have investigated the
regulation mechanisms of telomerase activity and telomere length in human cancer and normal cells. Telomerase
is the enzyme responsible for the synthesis and maintenance of telomere repeats located at chromosomal ends and
is normally expressed in embryonic and germline cells, but not in most normal cells. Here, using SFME cells, we
attempted to analyze the regulation mechanism of telomerase activity in neural stem cells and to detect a change
upon differentiation into astrocytes. When SFME cells were cultured in the presence of TGF-β, cells showed an
elongated morphology and decreased its growth to 50% of control culture. Cells also expressed the glial fibrillary
acidic protein (GFAP), a marker for astrocytes, indicating that TGF-βinduced differentiation in SFME cells from
neural stem cells into astrocytes. At the same time, TGF-βalso inhibited telomerase activity and repressed the
expression of the mouse telomerase reverse transcriptase (mTERT), demonstrating that SFME cells was vested
with a finite replicative life span upon treatment with TGF-β. To understand the mechanisms regulating mTERT
levels during differentiation into astrocytes, we have estimated the expression level of c-myc, which is known to
be a key molecule in activating the TERT promoter. As a result, TGF-β-treated SFME cells were shown to repress
the expression of c-myc. Furthermore, promoter analysis, using the 5-region of the mTERT gene, which possess
two E-box elements bound to c-Myc/Max, demonstrated that mTERT promoter activity greatly decreased in TGF-
β-treated SFME cells as compared to non-treated SFME cells. These suggest that c-myc might play a critical role
in the expression of mTERT, and that down-regulation of c-myc dependent upon the astrocytic differentiation in
SFME cells might cause the repression of mTERT in TGF-β-treated SFME cells.
Abbreviations: hTERT – human telomerase reverse transcriptase; mTERT – mouse telomerase reverse tran-
scriptase; BMP2 – bone morphogenetic protein 2; CNS – central nervous system; GAPDH – glyceraldehyde-
3-phosphate dehydrogenase; GFAP – glial fibrillary acidic protein; LIF – leukemia inhibitory factor; RT-PCR –
reverse transcription-polymerase chain reaction; SFME – serum-free mouse embryo; TGF-β– transforming growth
factor-β; TRAP – telomeric repeat amplification protocol.
Introduction
Telomeres are highly conserved sequences at the ends
of eukaryotic chromosome. In most mammalian cells,
telomeric DNA consists of tandem repeats of the
sequence TTAGGG. Telomeres are widely thought
to play a role in the maintenance of chromosome
structure and stability (Greider, 1996). Telomerase
138
is a ribonucleoprotein enzyme that synthesizes the
telomeric DNA sequence onto the 3ends of chro-
mosomes. Embryonic cells and germline cells have
been shown to express telomerase activity. In contrast,
most normal somatic cells express low or undetect-
able levels of telomerase, and as a consequence their
telomeres shorten with each cell division cycle (Har-
ley et al., 1990; Hastie et al., 1990), and eventually
reach the threshold length that leads to replicative sen-
escence as a result of genomic instability. On the other
hand, it has been suggested that multipotent cells, such
as embryonic and germline cells, acquire their immor-
tality by maintaining telomerase activity (Thomson et
al., 1998).
Multipotent stem cells would be an invaluable
source for regeneration studies of the injured or dam-
aged cells and organs. Recently, to regenerate and/or
rescue the damaged central nervous system (CNS),a
worldwide attention has been focused on the stud-
ies of neural stem cells. Neural stem cells exist not
only in the developing mammalian nervous system
but also within the adult nervous system of all mam-
malian organisms (McKay, 1997; Alvarez-Buylla et
al., 1998). Mouse embryonic neuroepithelial cells are
known to contain cells that have the potential to differ-
entiate into both neurons and glial cells like astrocytes
(Reynolds et al., 1992; Johe et al., 1996; Rajan and
McKay, 1998). Serum-free mouse embryo (SFME)
cells have been characterized as a multipotent neural
stem cell line. SFME cells cultured in the serum-
free medium continues to grow without reaching the
crisis stage or showing gross genomic alteration. Fur-
thermore, SFME cells do not show tumorigenicity in
syngeneic and athymic mice (Loo et al., 1987). In
addition, SFME cells differentiates into astrocytes in
the presence of TGF-βor serum (Sakai et al., 1990).
Therefore, SFME cells are thought to be valuable for
investigating the differentiation mechanism of neural
stem cells.
As mentioned above, SFME cells can be main-
tained for extended period, thus is thought to be
immortal, which suggests that these cells maintain its
immortality by telomerase. Previously, the total brain
of a murine embryo has been shown to express sig-
nificant level of telomerase activity (Greenberg et al.,
1998; Martin-Rivera et al., 1998). However, little is
known about the regulation of telomerase regulation
during brain development and neural differentiation.
In this report, we attempted to study the regulation
mechanisms of telomerase activity during the differen-
Figure 1. The effect of TGF-βon SFME cell proliferation. SFME
cells were cultured for 9 days in the presence of 10 ng ml−1TGF-β
() or the normal growth medium (, control). Cells were counted
using a cell counter (Sysmex F-300, TOA, Kobe, Japan) every day.
tiation of neural stem cells into astrocytes using SFME
cells.
Materials and methods
Cells culture and reagents
SFME cells were cultured in Dulbecco’s modified
Eagle’s medium/F12 (Life Technologies, Gainthers-
burg, MD) supplemented with 100 µgml
−1insulin
(Sigma, St. Louis, MO), 10 µgml
−1transferrin
(Sigma), 10 µgml
−1high-density lipoprotein (Bio-
medical Technologies Inc., Stoughton, MA), 50 ng
ml−1EGF (Sigma), 25 µgml
−1ampicillin (WAKO,
Osaka, Japan), 200 U ml−1penicillin (Meiji, Tokyo,
Japan), 200 µgml
−1streptomycin (Meiji) and 10 nM
sodium selenite (Sigma) in a humidified atmosphere
with 5% CO2at 37 ◦C. SFME cells were treated for
the indicated periods of time with 10 ng ml−1TGF-β
(AUSTRAL Biologicals, San Ramon, CA).
RT-PCR
Total RNA was prepared by using TRIzol reagent
(Life Technologies). 2.5 µg of total RNA was an-
nealed with 500 ng of oligo (dT)20 at 70 ◦Cfor
10 min, followed by reverse transcription at 42 ◦C
for 50 min using SuperScript II reverse transcriptase
(Life Technologies). PCR was performed by using
1µl of the RT-reaction mixture for the amplifica-
tion of either mTERT (mouse telomerase reverse tran-
scriptase), c-myc,GFAP (glial fibrillary acidic protein)
or mGAPDH in a total volume of 10 µl contain-
ing 0.25 units of Ta q polymerase (Roche Diagnostics,
139
Indianapolis, IN) and 10 pmol of primers spe-
cific for mTERT (5-TAGAGGATTGCCACTGGCTC-
3and 5-TCCAATGCTCTGCAGCTTGC-3), or for
c-myc (5-TTCTCTCCTTCCTCGGACTC-3and 5-
TGGCAGCTGGATAGTCCTTC-3), or for GFAP (5-
ATGATGGAGCTCAATGACCG-3and 5-GGTTT-
CATCTTGGAGCTTCT-3)orformGAPDH (5-
CCGTAGACAAAATGGTGAAGGT-3and 5-GTG-
GTGCAGGAGGCATTGCTGA-3). Amplifications
were performed 31 cycles (30 sec at 94 ◦C, 30 sec
at 60 ◦Cand1minat72◦C) for mTERT,27cycles
(30 sec at 94 ◦C, 30 sec at 58 ◦C and 1 min at 72 ◦C)
for c-myc, 27 cycles (30 sec at 94 ◦C, 30 sec at 55 ◦C
and 1 min at 72 ◦C) for GFAP and mGAPDH.All
PCR reactions were done under hot-start conditions
and analyzed by electrophoresis on 3% agarose gel.
Measurement of telomerase activity
Telomerase activity was measured by the PCR-based
TRAP (Telomeric Repeat Amplification Protocol) as-
say (Kim et al., 1994) with some modifications
(Katakura et al., 1997).
Primers and conditions for PCR were as previously
described (Katakura et al., 1997). Quantification of te-
lomerase activity was performed by the Kodak EDAS
system (Eastman Kodak Co., Rochester, NY).
Measurement of mTERT promoter activity
Genomic DNA was extracted from SFME cells us-
ing the DNA Extractor WB Kit (WAKO). Using this
mouse genomic DNA as template, the mTERT pro-
moter region (–1560 to –27) was isolated by PCR us-
ing specific primers (5-CCCCTCCCTCCTCTTCT-
TTG-3and 5-CACGTGCGGGAACCAAGATG-3)
as shown in Figure 5. This fragment containing the
mTERT promoter was inserted into Nhe I site of
the pGL3-Basic luciferase (firefly luciferase) plasmid
(Promega, Madison, WI). As a positive control, pGL3-
TK, which has the firefly gene under the transcription
control of TK promoter, was used. For promoter
assays, approximately 1 ×105cells/well in 24-well
plates were co-transfected with the mTERT promoter-
luciferase construct (300 ng/well) and the pRL-TK
(150 ng/well, Promega) which expresses the Renilla
luciferase and was used as an internal control for trans-
fection efficiency. All transfections were performed
using the LipofectAMINE PLUS Reagent (Life Tech-
nologies). In TGF-βtreated cells, cells were treated
with TGF-β(10ngml
−1) after transfection. The cells
were harvested at 48 hr after transfection and the luci-
ferase assay was done using the Dual-Luciferase Re-
porter Assay System (Promega). Samples were coun-
ted for 10 sec in a LUMINESCENCE READER BRL-
301 (ALOKA, Tokyo, Japan), and the data is repres-
ented as the relative ratio of firefly luciferase/Renilla
luciferase light unit.
Results and discussions
TGF-βinduced differentiation of neural stem cells
into astrocytes
We used SFME cells, which is a cell line established
from 16-day-old Balb/c mouse embryo cells, to invest-
igate the change in telomerase activity during the dif-
ferentiation of neural stem cells. SFME cells is known
to retain neural stem cell phenotypes when cultured in
serum free media, and >80% of SFME cells differenti-
ate into astrocytes upon treatment with TGF-β(Sakai
et al., 1990). Here, we confirmed that differentiation
of SFME cells into astrocytes does indeed occur upon
treatment with TGF-β.TGF-βweakly inhibited the
growth of SFME cells (Figure 1), which was observed
from day 4 after treatment with TGF-β.
Furthermore, TGF-β-treated SFME cells showed
extended cell morphology characteristic to astrocytes
(Figure 2). RT-PCR analysis demonstrated that GFAP
was induced in TGF-β-treated SFME cells, but not in
non-treated SFME cells (Figure 2). These results sug-
gest that TGF-βinduced cell differentiation in SFME
cells to become astrocytes.
It has previously been shown that TGF-βinhib-
ited the growth of primary astrocytes (Toru-Delbauffe
et al., 1990; Vergelli et al., 1995), although TGF-
βinduced differentiated SFME cells continued to
grow at the reduced growth rate. It is unclear at
present why astrocytes differentiated from SFME cells
lacks the mechanisms of growth inhibition by TGF-
β. On the other hand, SFME cells were maintained
in serum-free medium supplemented with EGF and
other growth factors (Ernst et al., 1991). SFME cells
arrested growth at the G1 phase of cell cycle and sub-
sequently underwent apoptosis upon the withdrawal
of EGF (Rawson et al., 1990). Slinskey et al. (1999)
has reported that p53, p21 and MDM-2 are not re-
quired for this G1 arrest and apoptosis in SFME cells
by the withdrawal of EGF. According to these stud-
ies, p53 pathway in SFME cells does not appear to
mediate G1 arrest and apoptosis. It has been previ-
ously shown that TGF-βincreases the expression of
140
Figure 2. Effect of TGF-βon SFME cell differentiation. Photomicrographs of SFME cells grown for 2 days in the absence (A) or presence
of TGF-β(10ngml
−1) (B). (C) Expression of GFAP mRNA during TGF-β-induced astrocytic differentiation. SFME cells were treated with
TGF-β(10ngml
−1) for 2 days, and RT-PCR assays were performed to detect GFAP and GAPDH mRNA.
p21WAF1/CI P1, and thereby TGF-βinduces growth
suppression of several cell types (Pardali et al., 2000).
Sun et al. (1998) has reported that the overexpression
of MDM-2 in culture cells could overcome the anti-
proliferative effect of TGF-β. Therefore, incomplete
growth inhibition in response to TGF-βobserved in
differentiated SFME cells may be caused by defects in
p53 signaling pathway. Further studies are needed to
clarify the signaling pathway of TGF-βrelating to the
differentiation and proliferation of SFME cells.
Differentiated SFME cells into astrocytes lost its
telomerase activity
Several reports indicate that neuronal differentiation
leads to the down-regulation of telomerase activity
(Kurk et al., 1996; Chen et al., 1997; Fu et al., 1999,
2000; Tian et al., 1999; Haik et al., 2000). Here, we
also measured the telomerase activity of the TGF-β
induced differentiated SFME cells with the TRAP as-
say. Treatment of SFME cells with TGF-βat 10 ng
ml−1resulted in a remarkable reduction of telomerase
activity, while non-treated SFME cells retained strong
telomerase activity (Figure 3), indicating that SFME
cells are immortal from the standpoint of a replicat-
ive life-span, and that SFME cells are vested with a
finite replicative life-span upon differentiation. There-
fore, the telomere length in differentiated SFME cells
would shorten during long-term culture because of
the loss of telomerase activity. These differentiated
cells would then enter into the senescence and crisis
stage. It has been demonstrated that some cell lines
suppressed telomerase activity during terminal differ-
entiation (Sharma et al., 1995; Albanell et al., 1996;
Bestilny et al., 1996; Savoysky et al., 1996), which
is distinct from our case in that SFME cells main-
tains its growth potential even after differentiation. We
can rephrase these results as that the suppression of
telomerase activity in SFME cells is dependent upon
differentiation into astrocytes, but not upon cell cycle
arrest. Suppression of telomerase activity independent
141
Figure 3. The effect of TGF-βon the telomerase activity of
SFME cells. SFME cells were subcultured in the absence or pres-
ence of TGF-β(10ngml
−1). To measure telomerase activity
semi-quantitatively, 1 ×106cells were harvested, washed and lysed.
Telomerase activity was determined in each cell extract using the
TRAP assay method. The TRAP assay products were visualized by
SYBR Green I staining.
Figure 4. Expression of mTERT and c-myc mRNA during
TGF-β-induced astrocytic differentiation. SFME cells were treated
with TGF-β(10ngml
−1) for 2 days, and RT-PCR assays were
performed to detect mTERT,c-myc and GAPDH mRNA.
of cell cycle arrest was also observed in our previous
study showing that TGF-βdecreased telomerase activ-
ity in human cancer cells with maintaining its growth
potential (Katakura et al., 1999). Both cases suggests
that TGF-βtransmits specific signals to suppress te-
lomerase activity both in cancer cells and neural stem
cells.
Undifferentiated SFME cells expressed high level
of telomerase activity (Figure 3, lane 1). Telomerase
activity has been shown to be responsible for the rep-
licative immortality of human cells, thus exists at high
level in germ lines, embryonic tissues and embryonic
stem (ES) cells (Wright et al., 1996; Thomson et
al., 1998). Therefore, the high level of telomerase
activity observed in SFME cells means that they have
an infinite replicative life-span. In fact, it has been
observed that SFME cells have been continuously pas-
saged without evidence of growth crisis or senescence
for over 200 doublings when cultured in serum-free
medium (Loo et al., 1987). Here, we conclude that
SFME cells are neuronal stem cells and might be a
useful model cell line for identifying the pathways or
regulation mechanisms of telomerase activity during
neuronal cell differentiation.
TGF-βrepressed the expression of mTERT and c-myc
mRNA
To elucidate the detailed mechanism for the down-
regulated telomerase activity in the differentiated
SFME cells, we next measured the expression level
of mTERT mRNA in the TGF-βtreated-SFME cells
using the RT-PCR analysis. mTERT expression was
shown to be repressed in SFME cells exposed to TGF-
βat 10 ng ml−1for 2 days when compared with
the expression of the internal control gene GAPDH
(Figure 4). This result is consistent with the report
that expression of TERT was tightly linked to the en-
zymatic activity of telomerase (Greenberget al., 1998;
Martin-Rivera et al., 1998). Moreover, it has been re-
ported that mTERT mRNA levels parallel a marked
decrease in telomerase activity during terminal differ-
entiation of the mouse erythroleukemia cells (Green-
berg et al., 1998). Therefore, we conclude that the
down-regulated telomerase activity as shown in Fig-
ure 3 might be caused by the transcriptional repression
of mTERT mRNA. The regulation mechanisms of
mTERT expression must be elucidated to understand
the molecular events that lead to neural differenti-
ation. These findings suggest that the expression of
mTERT is also tightly associated with the differenti-
ation of SFME cells into astrocytes. However, very
little is known about the expression and regulation of
the mTERT gene during neural differentiation. It has
recently been reported that the repression of TERT
expression during differentiation was correlated with
the loss of c-myc expression (Armstrong et al., 2000).
In agreement with their report, the expression of c-
myc in SFME cells was also shown to decrease upon
differentiation by treatment with TGF-β(Figure 4).
These results suggested that the repression of c-myc
may trigger the repression of mTERT expression dur-
142
Figure 5. The effect of TGF-βon the transcriptional activity of mTERT promoter in SFME cells. A diagram of the mTERT promoter is shown
by fixing the ATG codon as +1 (A). Moreover, two E-box elements are shown in box (Greenberg et al., 1999). (B) The reporter constructs were
transfected into SFME cells, and then treatment with TGF-βwas done for 2 days. Two days after transfection, luciferase expression levels
for each reporter construct were assessed using the Dual-Luciferase Reporter Assay System (Promega). pGL3-Basic is a negative control, and
pGL3-TK is a positive control. Data for each reporter construct is shown as the mean and standard deviation for samples done in triplicate.
ing the neural differentiation process. As shown in
Figure 5A, the mTERT promoter possesses two E-box
elements bound to c-Myc/Max which are located at –
837 and –32 bp from the translation start point (+1).
Thus, we isolated mTERT promoter by PCR using
mouse genomic DNA as the template, then measured
the mTERT promoter activity in differentiated SFME
cells by luciferase assay. Figure 5B clearly demon-
strated that the mTERT promoter activity in TGF-β
induced differentiated SFME cells greatly decreased
as compared to non-differentiated SFME cells. Con-
sidering together with the fact that c-myc is known
to be a strong transcriptional activator for hTERT via
E-box elements (Horikawa et al., 1999; Takakura et
al., 1999; Wu et al., 1999), our results suggest that
down-regulation of c-myc may lead to the repres-
sion of mTERT promoter activity. Down-regulation
mechanisms of c-myc may be dependent upon neural
differentiation and/or be a response to the TGF-β
signaling. These questions must be clarified to elucid-
ate the mechanisms for mTERT repression dependent
upon neural differentiation.
Furthermore, mTERT expression in SFME cells
was not completely repressed when treated with TGF-
βfor 2 days (Figure 4). In addition, the growth
potential partially remained in TGF-βtreated-SFME
cells (Figure 1).
These results suggested that the entry of SFME
cells into a state of terminal differentiation might need
other various molecules than TGF-β, or the continu-
ously treatment of TGF-β. In fact, it has been reported
that there are multiple routes for differentiation of
CNS stem cells into astrocytes (Rajan and McKay,
1998). Nakashima et al. have demonstrated that LIF
and BMP2 function in synergy to induce differenti-
ation into astrocyte, but BMP2 or LIF alone did not in-
duce astrocyte development (Nakashima et al., 1999).
Thus, different growth factors may be necessary for
the complete neuronal differentiation of SFME cells.
Further studies on neural differentiation of SFME cells
may provide valuable information about the neuronal
aging, neuronal dysfunction and neuronal death, such
as Alzheimer’s disease and Parkinson’s disease.
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