Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in vitro.

Abstract

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IκB (rAd-dnIκB). This blockage of the NF-κB signal pathway in STO cells led to a significant decrease in [(3)H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IκB (rAd-dnIκB). These results suggested that the NF-κB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIκB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

Full text

Page 1

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 44, No. 7, 457-463, July 2012
Copyright ⓒ 2012 by the Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Overexpression of bone morphogenetic protein 4 in STO
fibroblast feeder cells represses the proliferation of mouse
embryonic stem cells in vitro
Gu-Hee Kim1, Gong-Rak Lee2, Hyung-Im Choi1,
Neung-Hwa Park3, Hun Taeg Chung1
and In-Seob Han1,4
1School of Biological Sciences
2Department of Medical Science
School of Medicine
University of Ulsan
Ulsan 680-749, Korea
3Department of Internal Medicine
Ulsan University Hospital and School of Medicine
University of Ulsan
Ulsan 680-749, Korea
4Corresponding author: Tel, 82-52-259-2352;
Fax, 82-52-259-1694; E-mail, hanis@ulsan.ac.kr
http://dx.doi.org/10.3858/emm.2012.44.7.052
Accepted 10 May 2012
Available Online 17 May 2012
Abbreviations: BMP, bone morphogenetic protein; ESC, embryonic
stem cell; rAd-dnIκB, recombinant adenovirus-dominant negative
IκB
Abstract
Embryonic stem cells (ESCs) can be propagated in vi-
tro on feeder layers of mouse STO fibroblast cells. The
STO cells secrete several cytokines that are essential
for ESCs to maintain their undifferentiated state. In this
study, we found significant growth inhibition of mouse
ESCs (mESCs) cultured on STO cells infected with ad-
enovirus containing a dominant-negative mutant form
of Iκ κB (rAd-dnIκ κB). This blockage of the NF-κ κB signal
pathway in STO cells led to a significant decrease in
[3H]thymidine incorporation and colony formation of
mESCs. Expression profile of cytokines secreted from
the STO cells revealed an increase in the bone morpho-
genetic protein4 (BMP4) transcript level in the STO
cells infected with adenoviral vector encoding domi-
nant negative Iκ κB (rAd-dnIκ κB). These results sug-
gested that the NF-κ κB signaling pathway represses ex-
pression of BMP4 in STO feeder cells. Conditioned me-
dium from the rAd-dnIκ κB-infected STO cells also sig-
nificantly reduced the colony size of mESCs. Addition
of BMP4 prevented colony formation of mESCs cul-
tured in the conditioned medium. Our finding sug-
gested that an excess of BMP4 in the conditioned me-
dium also inhibits proliferation of mESCs.
Keywords: bone morphogenetic protein 4; culture
media, conditioned; embryonic stem cells; feeder
cells; NF-κB
Introduction
Several growth factors that are required for self-
renewal of embryonic stem cells (ESCs) are usually
provided either exogenously or by feeder cells
(Smith, 2001; Ahn et al., 2010). Different embryonic
fibroblast cell types have been tested as feeder cells
for culture of undifferentiated ESCs. These cells
secrete several cytokines that are essential for
maintaining mouse ESCs (mESCs) in an un-
differentiated state. STO, a transformed mouse
fibroblast line, is commonly used as feeder cells to
support mESC growth (Smith et al., 1988). mESCs
depend on bone morphogenetic protein (BMP) and
leukemia inhibitory factor (LIF) to maintain their
pluripotent status (Brons et al., 2007). Conditioned
medium from STO cell culture has been studied as
an economical alternative to these cytokines
commonly used in mESC growth media.
BMPs are members of the transforming growth
factor beta (TGF-β) family and have been implicated
in embryonic development, including bone formation
and repair, organogenesis, pattern formation in the
early embryo, and epithelial-mesenchymal interactions
(Jones et al., 1991; Vainio et al., 1993; Fainsod et al.,
1994; De Robertis and Sasai, 1996). BMP4 is
required at a very early step as an important regulator
of the growth of hematopoietic stem cells (HSC),
participating in the control of their proliferation,
expansion and differentiation (Pearson et al., 2008).
The major effect of BMP4 on the self-renewal of

Page 2

458 Exp. Mol. Med. Vol. 44(7), 457-463, 2012
Figure 1. Inhibition of NF-κB signaling in STO feeder cells by infection
with rAd-dnIκB prevents colony formation of mESCs. Photographs of
representative colonies were taken on day 4 of treatment. (A)
Morphological changes of mESCs cultured on STO cells infected by
rAd-dnIκB at the indicated MOI. (B) The morphology of mESC colonies
cultured on STO feeder cells. (C) The number and size of colonies of
mESCs cultured on STO cells infected by rAd-dnIkB. The colony number
was counted under a microscope, and the colony size was determined
by measuring the colony area with the Image J program (NIH). All assays
were performed in duplicate (scale bar, 50 μm).
ESCs is accomplished by means of the inhibition of
both extracellular receptor kinase (ERK) and p38
mitogen-activated protein kinase (MAPK) pathways
(Qi et al., 2004). Human BMP4 and hematopoietic
cytokines have been shown to modulate the
proliferative and differentiative potentials of definitive
fetal, neonatal, and adult hematopoietic progenitors
(Bhatia et al., 1997, 1999; Bhardwaj et al., 2001;
Murdoch et al., 2002). ESCs cultured on embryonic
feeder cells can be induced into trophoblastic
differentiation by collagen IV or BMP4 (Xu et al.,
2002; Schenke-Layland et al., 2007). Early exposure
to BMP4 inhibits the neurogenic differentiation of
ESCs, whereas later exposure causes induction of
peripheral neuronal differentiation (Schulz et al.,
2004). These studies indicate that BMP4 is required
for self-renewal of ESCs or for differentiation into a
specific lineage if they are provided with the correct
cues. BMP4 regulation is complex and precisely
controlled by various transcription factors and inter-
and intra- cellular signaling pathways. A recent report
showed that TNF-α represses transcription of human
BMP4 in lung epithelial cells through the nuclear
factor κB (NF-κB) signaling pathway (Zhu et al.,
2007). Sequence analysis has suggested that the 5’
upstream region of BMP4 contains a putative NF-κB
binding site. However, few functional results are
currently available regarding the regulation of BMP4
gene expression in any organ.
Here we blocked the NF-κB signaling pathway in
STO feeder cells by infection with recombinant
adenovirus encoding a dominant-negative mutant of
IκB (rAd-dnIκB). As shown in lung epithelial cells
(Zhu et al., 2007), this blockade induced high
expression of BMP4 in the STO cells and
consequently resulted in a decreased survival of
mouse ESCs in co-cultures. The addition of
exogenous BMP4 to ES culture medium also
inhibited colony formation from mESCs. These
findings suggest that the regulated expression of
BMP4 in feeder cells is important for self-renewal of
mESCs in vitro.
Results
Inhibition of the NF-κ κB signaling pathway in STO cells
decreases proliferation of mESCs
To investigate the cellular and molecular con-
sequences of IκB in STO cells, we constructed an
adenovirus plasmid expressing a dominant-negative
mutant of IκB (rAd-dnIκB) that cannot be phosphory-
lated and thus abrogates the NF-κB signaling path-
way and a negative control plasmid expressing
EGFP (rAd-GFP). We infected STO feeder cells by
incubation with rAd-dnIκB virus at a multiplicity of
infection (MOI) values of 3, 5, and 10 and then used
these feeder cells for culture of mESCs. Infection of
STO cells by the rAd-dnIκB reduced the number and
colony size of co-cultured mES cells as MOI
increased, while the number and colony size of control
STO cells infected by rAd-GFP was unaffected
(Figure 1). These data suggest that inhibition of the
NF-κB signaling pathway in STO cells prevents
growth of mESCs. Using reverse transcription
(RT)-PCR analysis with rAd-dnIκB specific primers,
we confirmed that the reduced proliferation was
caused by STO cells but not by mESCs themselves
(Supplemental Data Figure S1).
To investigate whether inhibition of the NF-κB
pathway in STO cells affected proliferation of
mESCs, we performed a 3H-thymidine incorporation
assay. The STO cells expressing dnIκB markedly
inhibited cellular proliferation of mESCs at 24 h, 48 h,
and 72 h (Figure 2A). These data indicated that the

Page 3

Repression of mESC proliferation by BMP4 459
Figure 2. Proliferation of mES cells was inhibited by the STO cells in-
fected with rAd-dnIκB. (A) The mESCs were cultured on STO cells as in-
dicated, and proliferation of mESCs was measured by [3H] thymidine in-
corporation assay. (B) RT-PCR analysis of the ESCs marker genes.
GAPDH mRNA was measured as a control. (C) The relative density of
gene expression was determined by dividing the density of each gene by
that of GAPDH. **A statistically significant difference (P ＜ 0.01) com-
pared to the rAd-GFP control.
Figure 3. Effect of the conditioned media from STO cells infected with
rAd-dnIκB. Conditioned medium from STO cells infected by rAd-C was
mixed with that of STO cells infected by rAd-GFP at the indicated ratio
(rAd-GFP only, 9:1, 3:1, 1:1, 1:3, 1:9, and rAd-dnIκB only). ESCs were
cultured in the conditioned medium for three days. (A) Morphology of
representative colonies and (B) comparison of colony sizes of mESCs.
The colony size (＞ 10 colonies/sample) was determined by measuring
the colony area with the Image J program (NIH). All assays were per-
formed in duplicate (scale bar, 25 μm).
NF-κB signaling pathway was essential for STO
cells to function as feeder cells in inducing self-
renewal of mES cells.
We analyzed transcription of proliferation-related
genes of ESCs when these cells were co-cultured
with the STO cells infected by rAd-dnIκB. Figure 2B
showed that mRNA levels of Oct 4 and Nanog were
increased in the ESCs, whereas the expression
levels of Id genes were unchanged.
Conditioned medium from the STO cells infected by
rAd-dnIκ κB reduces the colony size of mESCs
To investigate whether inhibition of the NFκB
signaling pathway destroyed the characteristic of
STO cells to secrete ESC proliferation-stimulating
factors, we analyzed colony formation of mESCs
after culture with the conditioned medium collected
from the rAd-dnIκB-infected STO cell cultures
(Figure 3). The colony size decreased significantly
as conditioned medium from rAd-dnIκB-infected
STO cells was increased but remained unchanged
in control medium from rAd-GFP-infected STO cells.
This suggests that STO cells secrete a factor which
stimulates proliferation of mESCs mediated by the
NFκB signaling pathway.
Inhibition of the NFκ κB signal transduction pathway
increases BMP4 expression in STO feeder cells
To identify factor which changed upon inhibition of
the NFκB signaling pathway in STO cells, we
analyzed the mRNA levels of BMP4, LIF, and Wnts
genes because they are known as critical factors for
maintaining the characteristics of mESCs (Hao et
al., 2006). RT-PCR analysis detected higher
expression of BMP4 in the STO cells infected by
rAd-dnIκB, whereas mRNA levels of LIF and Wnts
genes were not significantly different (Figure 4A).
Real-time PCR confirmed level of BMP4 mRNA was
increased in the rAd-dnIκB-infected STO cells
(Figure 4B).
These data suggest that the enhanced level of
BMP4 in STO feeder cells might inhibit the
proliferation of mESCs.

Page 4

460 Exp. Mol. Med. Vol. 44(7), 457-463, 2012
Figure 4. Overexpression of Bmp4 in STO cells infected with rAd-dnIκB
inhibits mESC proliferation. (A) RT-PCR analysis of the STO feeder cells
infected by rAd-GFP and rAd-dnIκB. The rAd-dnIκB infection induced
overexpression of Bmp4 gene in the STO cells. GAPDH mRNA was
measured as a control. (B) The relative density of genes expression was
determined by dividing the density of each gene by that of GAPDH. The
level of BMP4 mRNA was analyzed by using real time PCR. Results are
representative of three independent experiments. (C) The number of col-
onies of mESCs was counted after culturing in the conditioned medium
produced from STO cells plus exogenous BMP4. Addition of BMP4 re-
sulted in a decrease in the number of colonies produced from the con-
ditioned medium of rAd-GFP medium compared to that of rAd-dnIκB
medium. (D) Morphology of mESC colonies cultured in the conditioned
medium plus exogenous BMP4.
Over-production of BMP4 in STO feeder cells leads to
decreased mESC proliferation
To confirm whether over-production of BMP-4 led to
inhibition of mESC proliferation, we added recombi-
nant BMP4 to the mESC culture medium. Addition of
exogenous BMP4 (2.5-25 ng/ml) reduced the colony
number of mESCs co-cultured with rAd-GFP-infected
STO feeder cells (P ＜ 0.01) to a similar level as in
non-treated mESCs co-cultured with the rAd-dnIκB-
infected STO cells (Figures 4C and 4D). These
results indicate that BMP4 is required in appropriate
amounts to maintain mESC self-renewal.
Discussion
Mammalian feeder cells continue to be broadly
accepted as the method for maintaining ESC culture
because these cells seem to produce some
unidentified factor that makes them very effective.
Extensive cell proliferation must be carefully
addressed in stem cell research before theoretical
possibilities of stem cells are translated into clinical
applications (Park et al., 2008). One active area of
current stem cell research includes identification of
the molecular processes underlying the uniform
maintenance of proliferation, the undifferentiated
state and pluripotency. Defining some of the signals
important in the self-renewal of human ESCs is
necessary to eliminate the need to include
animal-derived materials. BMP4 has been identified
as a good candidate for extensive ex vivo production
of hESCs for therapeutic applications (Bhatia et al.,
1999; Hollnagel et al., 1999; Ying et al., 2003;
Vicente Lopez et al., 2011). Suppression of BMP
signaling has been beneficial and not deleterious to
hESC culture (Xu et al., 2005). In contrast, BMP4
was not responsible for the activity of STO cells that
support ES self-renewal or proliferation (Hao et al.,
2006). Here we more closely evaluated the ability of
BMP-4 from feeder cells to maintain mESC culture.
We found that a dominant-negative catalytically
inactive construct (rAd-dnIκB) led to profound
attenuation of NF-κB signaling, which translated into
an increase of Bmp4 expression in STO feeder
cells. Similarly, down-regulation of Bmp4 by
activation of the NF-κB signaling pathway was
found in other kinds of cells (Muraoka et al., 2000;
Zhu et al., 2007). This suggests that production of
BMP4 in feeder cells is possibly induced by
inhibition of the NF-κB pathway.
The major effect of BMP4 on the self-renewal of
ESCs is accomplished by means of the inhibition of
both extracellular receptor kinase (ERK) and p38
mitogen-activated protein kinase (MAPK) pathways
(Qi et al., 2004). Suppression of the MAPK pathway
by BMP4 maintains pluripotency of ES cells by
regulating gene expression of the ESC markers,
Oct4 and Nanog. In our study, enhanced levels of
BMP4 in STO cells inhibited colony formation and
increased expression of Oct4 and Nanog genes of
mESCs in an undifferentiated culture system,
whereas id gene expression was unchanged in
mESCs. Since a critical level of Oct4 is known to be
essential to maintain pluripotency of ESCs (Niwa et
al., 2000), induced expression of Oct4 in our system
may have contributed to inhibition of ESC
self-renewal. Addition of exogenous BMP4 also
decreased the colony number of undifferentiated
mESCs in a dose-dependent manner. When CHO

Page 5

Repression of mESC proliferation by BMP4 461
cells overexpressing BMP4 were implanted in the
avascular region of quail embryos, endothelial
growth and capillary plexus formation were
enhanced (Reese et al., 2004). Our data is
consistent with that of a recent study showing a
significant increase in the proliferation rate after
addition of a small amount of BMP4 to bone marrow
stem cells but a decrease to the lowest rate after
adding a larger amount of BMP4 (Mazaheri et al.,
2011). Other reports also demonstrated that high
doses significantly increased apoptosis and
drastically reduced cell proliferation, whereas low
doses of BMP4 significantly increased cultured cell
content, reduced the number of apoptotic cells, and
increased the number of cycling cells (Vicente
Lopez et al., 2011). Taken together, these results
showed that excess BMP4 produced in STO cells
inhibited proliferation of ESCs. Because of a smaller
change in the expression of BMP4 target genes
(ids), it is unlikely that a single signaling pathway
affected by BMP4 activation is capable of entirely
inhibiting self-renewal of mESCs.
Methods
STO feeder cells and mESC growth
STO feeder cells (ATCC, Toronto Ontario, Canada) were
plated in Dulbecco’s Modified Eagle Medium (DMEM;
Invitrogen, Carlsbad, CA) supplemented with 10% fetal bo-
vine serum (FBS; WelGENE, Daegu, Korea), 100 U/ml
penicillin, and 100 mg/ml streptomycin. Murine embryonic
stem D3 cells (mESCs) were maintained on mitomycin
C-pretreated STO feeder cells in DMEM supplemented
with 15% FBS, 1% nonessential amino acid (Sigma, St.
Louis, MO), 0.1 mM β-mercaptoethanol (Sigma), 1% pen-
icillin and streptomycin, and 1 × 106 U LIF (Chemicon,
Temecula, CA). The ESCs were passaged every three
days to maintain their undifferentiated state.
Infection of STO feeder cells with adenovirus
The adenovirus vector containing dominant-negative IκB
(rAd-dnIκB) or control adenovirus containing green fluo-
rescence protein (rAd-GFP) was propagated in 293 cells
and purified by CsCl density gradient. The titer of ad-
enovirus stock was determined as plaque forming units
(pfu)/ml by the limiting dilution assay. The viral prepara-
tions were dialyzed and stored at -80oC until use.
Post-confluent STO cells cultured in tissue culture dishes
were washed once with complete PBS and incubated with
the adenovirus in DMEM (100 μl/well) at 37oC at different
multiplicities of infection (MOI), that is, virus to cell ratio, as
indicated. Fresh DMEM growth medium supplemented with
15% FBS was added after 1.5 h, and cells were main-
tained for two more days. The adenovirus was washed off
with PBS, and ESCs (2 × 104 cells/ml) were seeded into
these STO feeder cell dishes and cultured until assayed.
[3H] Thymidine incorporation assay
ESCs (1 × 104 cells/ml) cultured on the STO feeder cells
were transferred into 96-well plates and cultured for 24 h.
For cell proliferation assays, [3H] thymidine (Amersham
Biosciences KOREA Ltd, Seoul, Korea) was added into the
cell culture plates for 96 h, and cells were harvested.
Thymidine incorporation was determined in a scintillation
counter.
mESC culture in the STO-conditioned medium
The STO feeder cells infected by adenovirus were cultured
in T-flasks containing DMEM supplemented with 10% FBS.
After culture for 24 h, STO-conditioned media were pre-
pared by collecting the supernatant from the STO cell cul-
ture medium. We centrifuged the supernatant at 1,200 rpm
for 5 min followed by filtration through a Minisart filter (pore
size 0.2 μm; Sigma). Filtered supernatant was used as the
conditioned medium after supplementing with glucose,
nonessential amino acid, β-mercaptoethanol, and anti-
biotics to the same concentrations as those in the ESC
growth medium described above. Mouse ESCs (1 × 104
cells/ml) were seeded and cultured in Matrigel (BD
BioSciences, Mountain View, CA)-coated cell culture plates
at 37oC until observation by microscope. Exogenous BMP4
(R&D Systems Inc., Minneapolis, MN) was added into the
mESC culture medium once a day.
RNA purification and RT-PCR
STO cells were cultured to 90-95% confluence in STO me-
dium and then in ES cell medium for 24 h before total RNA
purification using TRI reagent (Sigma). cDNA was synthe-
sized using the Superscript II first-strand synthesis kit
(Invitrogen). PCR amplification was carried out with pri-
mers specific to mouse BMP4, Wnt3, Wnt5a, STAT3, LIF,
Oct4, Nanog, Id1, Id2, Id3, and Id4. RT-PCR for GAPDH
was used as an internal control in each experiment.
Real time PCR
Real time PCR was performed using the StepOnePlusTM
Real-time PCR System (Applied Biosystems Inc., Foster
city, CA) using Eva Green dye. The mRNA expression lev-
els of BMP4 in Ad-dnIκB cells were compared to the ex-
pression levels in Ad-GFP cells. The levels of PCR product
were calculated from standard curves established for each
primer pair (Livak and Schmittgen, 2001).
Supplemental data
Supplemental data include a figure and can be found with
this article online at http://e-emm.or.kr/article/article_files/
SP-44-7-06.pdf.
Acknowledgements
This work was supported by Priority Research Center
Program (2011-0030746) and Basic Research Lab (BRL-
2011-0001570) through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education,