Enhancement of human embryonic stem cell pluripotency through inhibition of the mitochondrial respiratory chain.

Abstract

Human embryonic stem cell (hESC) pluripotency has been reported by several groups to be best maintained by culture under physiological oxygen conditions. Building on that finding, we inhibited complex III of the mitochondrial respiratory chain using antimycin A or myxothiazol to examine if specifically targeting the mitochondria would have a similar beneficial result for the maintenance of pluripotency. hESCs grown in the presence of 20 nM antimycin A maintained a compact morphology with high nuclear/cytoplasmic ratios. Furthermore, real-time PCR analysis demonstrated that the levels of Nanog mRNA were elevated 2-fold in antimycin A-treated cells. Strikingly, antimycin A was also able to replace bFGF in the media without compromising pluripotency, as long as autocrine bFGF signaling was maintained. Further analysis using low-density quantitative PCR arrays showed that antimycin A treatment reduced the expression of genes associated with differentiation, possibly acting through a ROS-mediated pathway. These results demonstrate that modulation of mitochondrial function results in increased pluripotency of the cell population, and sheds new light on the mechanisms and signaling pathways modulating hESC pluripotency.

Full text

Page 1

Enhancement of Human Embryonic Stem Cell Pluripotency
Through Inhibition of The Mitochondrial Respiratory Chain
S. Varum1,3, O. Momcilovic1,4, C. Castro1,2, A. Ben-Yehudah1,2, J. Ramalho-Santos3,*, and
C. S. Navara5,*
1Pittsburgh Development Center, Gynecology and Reproductive Sciences, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania
2Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania
3Center for Neuroscience and Cell Biology, Department of Life Sciences, School of Science and
Technology, University of Coimbra, Portugal
4Department of Human Genetics, Graduate School of Public Health, University of Pittsburg
5Biology Department, University of Texas, San Antonio
Abstract
Human embryonic stem cell pluripotency has been reported by several groups to be best maintained
by culture under physiological oxygen conditions. Building on that finding, we inhibited complex
III of the mitochondrial respiratory chain using Antimycin A or Myxothyazol to examine if
specifically targeting the mitochondria would have a similar beneficial result for the maintenance of
pluripotency. hESC’s grown in the presence of 20nM Antimycin A maintained a compact
morphology with high nuclear/cytoplasmic ratios. Furthermore, Real Time PCR analysis
demonstrated that the levels of Nanog mRNA were elevated two fold in Antimycin A treated cells.
Strikingly, Antimycin A was also able to replace bFGF in the media without compromising
pluripotency, as long as autocrine bFGF signaling was maintained. Further analysis using low density
quantitative PCR arrays showed that Antimycin A treatment reduced the expression of genes
associated with differentiation possibly acting through a ROS-mediated pathway. These results
demonstrate that modulation of mitochondrial function results in increased pluripotency of the cell
population, and sheds new light on the mechanisms and signaling pathways modulating hESC
pluripotency.
Keywords
human embryonic stem cells; pluripotency; mitochondria; mitochondrial complex III; Antimycin A
© 2009 Elsevier B.V. All rights reserved.
*Co-Corresponding Authors (christopher.navara@utsa.edu; jramalho@ci.uc.pt).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting
proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could
affect the content, and all legal disclaimers that apply to the journal pertain.
NIH Public Access
Author Manuscript
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
Published in final edited form as:
Stem Cell Res. 2009 ; 3(2-3): 142–156. doi:10.1016/j.scr.2009.07.002.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 2

Introduction
The mechanisms underlying the maintenance of hESC self renewal and pluripotency are
complex and poorly understood. Furthermore, the mechanisms by which hESC and mouse
embryonic stem cells (mESC) maintain pluripotency differ. Both Leukemia Inhibitory Factor
(LIF) and Bone Morphogenetic Protein 4 (BMP 4) play important roles in the maintenance of
mESC self renewal via activation of Stat3 [1-3]. Conversely, LIF is not sufficient to maintain
pluripotency of hESC [4] and addition of BMP 4 to the culture media results in rapid
differentiation [5]. Instead, the combined actions of basic Fibroblast Growth Factor (bFGF,
FGF2) and TGFβ/Activin/Nodal signaling pathways are believed to be critical to hESC self
renewal. Nearly all formulations for the routine culture of hESC include exogenous bFGF,
demonstrated to increase the cloning efficiency of hESC maintained in serum free conditions
[6]. When added at high concentrations bFGF supports hESC self renewal in feeder-free
conditions in the presence [7-8] or absence of conditioned media [8]. Several studies have also
shown that the combined activities of Noggin and bFGF maintain hESC self renewal in the
absence of conditioned medium due to the suppression of BMP activity [9-10]. Furthermore,
Activin or Nodal appear to cooperate with bFGF to maintain hESC pluripotency in chemically
defined media [11].
In addition to media constituent requirements Ezashi et al. [12] demonstrated that culture of
hESC under low oxygen (O2) tension (5%) reduced the appearance of spontaneous
differentiation. This may be the normal physiological state, as early-stage mammalian embryos
also develop under low O2 concentrations (1.5%-5.3%) until they implant in the uterine
endometrium, when O2 levels increase with vascularization [13]. When cultured under low
O2 tension, mammalian cells decrease ATP production via oxidative phosphorylation in the
mitochondria and increase glycolytic functions in order to meet energy demands. Studies of
mitochondrial number and morphology in hESC have demonstrated that undifferentiated hESC
have relatively few mitochondria in the cytoplasm and these mitochondria have few christae,
an indication of immature morphology [14-16]. As hESC differentiate the number of
mitochondria with a mature morphology increases, concomitant with the ATP levels produced
by oxidative phosphorylation [16]. Taken together, these results suggest that inhibition of
mitochondrial function may prevent differentiation, and thus modulate the maintenance of
pluripotency.
In this study we tested this hypothesis by specifically inhibiting complex III of the
mitochondrial respiratory chain using Antimycin A, an antibiotic isolated from streptomyces
sp. Antimycin A specifically blocks the flow of electrons from semiquinone to ubiquinone in
the quinone cycle of complex III, thus disrupting the proton gradient across the inner membrane
of mitochondria and preventing O2 consumption at complex IV as well as ATP formation.
Complex III is also known to be a source of Reactive Oxygen Species (ROS) production in the
cell; predominantly superoxide anion (O2-), which when produced in moderate amounts
activates hypoxic signaling pathways in the cell. Consequently, complex III is considered the
O2 sensor of the cell [17-18]. Antimycin A treatment simultaneously decreases ATP production
via oxidative phosphorylation and increases ROS formation.
Materials and Methods
Human embryonic stem cell culture
WA07 cells (WiCell) were cultured under normoxic conditions (21% O2 and 5% CO2) in
Knockout medium containing 80% Knockout Dulbecco’s modified Eagle’s medium (DMEM)
(Invitrogen, Carslbad, CA) supplemented with 20% Knockout Serum Replacer (Invitrogen),
1mM L-glutamine, 4ng/ml basic human recombinant FGF and 0.1mM MEM non essential
amino acids, 50μg/ml penicillin, and 50μg/ml streptomycin (all from Invitrogen, Carlsbad,
Varum et al. Page 2
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 3

CA). Cells were passaged manually (day 0) using a pulled glass needle and then plated onto
Mitomycin C inactivated mouse embryonic fibroblast feeder cells - MEF’s (Specialty Media,
Phillipsburgh, NJ). On day two, 20 nM Antimycin A (Sigma-Aldrich, St Louis, MO) was added
and media was changed every other day and new drug was added. hESC culture on Matrigel
(BD Biosciences) was performed as described previously [19] briefly, human knockout
medium was conditioned in MEF’s and a total of 8ng bFGF/ml was used to supplement the
media. Matrigel was diluted according to manufacturer’s instructions and was allowed to coat
dishes for at least 30 min at room temperature before cells were added. In order to allow cells
to plate, treatments were initiated at day two after scraping and fresh media and drugs were
added every other day. MnTbap and PD173074 [20-21] were used at 50μM and 100nM,
respectively.
Immunocytochemistry
Immunocytochemistry (ICC) for the standard pluripotency markers: Oct-4, Nanog and SSEA-4
was assessed as previously described [22]. Only cells in which Oct-4 was localized in the
nucleus were considered positive for this marker, whereas cytoplasmatic localization was
considered as negative.
RNA extraction, RT PCR and TaqMan Low Density Arrays
Total RNA extraction and PCR mixtures were prepared as previously described [23]. Real
Time PCR was performed using an ABI Prism 7700 (Applied Biosystems Incorporated, Foster
City, CA). Taqman gene expression assays (Applied Biosystems) were used for Nanog, Oct-4
and β actin. Water and no RT samples were used as negative controls, all samples were run in
triplicate. The TaqMan® Array human stem cell Pluripotency Panel (Applied Biosystems) was
used following manufacturer’s instructions. Nine (FGF5, KRT1, GCM1, HBB, WT1, GCG,
INS, IPF1, MYF5) genes were excluded from our analysis due to very poor amplification in
any sample. mRNA fold changes were calculated using the -ΔΔCt method and normalized
using β actin expression as endogenous control.
Western Blotting
hESC were collected manually in PBS and pelleted. Total protein extract was collected using
RIPA buffer (Sigma) supplemented with 1mM PMSF. Protein quantification was carried out
using the Bradford assay (Bio Rad laboratories Inc, CA) and 10 μg of protein was separated
by 12% SDS-PAGE. Primary antibodies used: Nanog (Kamiya Biomedical Company) and
Oct-4 (Santa Cruz Biotechnology). ECL Advance Western Blot Detection kit (Amersham
Biosciences, Piscataway, NJ) was used for detection.
Teratoma Formation
hESC treated for prolonged periods of time with 20nM of Antimycin A either in the presence
or in the absence of bFGF were injected into the testis of non-obese diabetic/severe combined
immune deficient (NOD/SCID) mice. Three mice were injected per experiment as previously
described [24].
Apoptosis/Necrosis assay
hESC were maintained on Matrigel and treated with Antimycin A as previously described and
Apoptosis/Necrosis rates were detected using Annexin V/ PI apoptosis detection kit (BD
Biosciences) following manufacturer’s instructions. Briefly, cells were washed with PBS,
dissociated with Accutase followed by two washes in cold (4°C 1×106 cells were resuspended
in 100μl of binding buffer and 5μl of both Annexin V and PI were added. The mixture was
incubated for 15 min at 25°C in the dark followed by the addition of 400μl binding buffer.
Labeled cells were analyzed by flow cytometry (BD LSR II, BD Biosciences).
Varum et al. Page 3
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 4

Measurement of hESC proliferation
hESC proliferation was determined by BrdU (Roche) incorporation into the genomic DNA
during the S phase of the cell cycle. hESC maintained on Matrigel were treated with Antimycin
A as previously described. At day six after plating hESC were incubated in medium containing
BrdU for three hours and washed three times with PBS. Cells were fixed and incorporated
BrdU was detected with Anti-BrdU according to manufacturer’s instructions. Cells were
imaged by confocal microscopy and at least 2500 cells were counted to determine number of
cycling cells.
ATP measurement by HPLC
hESC were maintained on Matrigel and Antimycin A treated as previously described.
Intracellular adenine nucleotides (ATP, ADP and AMP), were determined as previously
described [25]. In brief, adenine nucleotides were extracted with 0.6 M Perchloric acid
supplemented with 25 mM EDTA-Na. Cell supernatants were neutralized with 3M KOH in
1.5M Tris followed by centrifugation. Supernatants were assayed by high-performance liquid
chromatography (HPLC). The detection wavelength was 254nm, and the column was a
Licophesfere100 RP108 5μM (Merk). Adenylate Energy Charge was calculated according to
the following formula: ATP+0.5×ADP/(ATP+ADP+AMP).
Lactate Dehydrogenase (LDH) activity
Lactate Dehydrogenase activity was determined using the QuantiChrom Lactate
Dehydrogenase Kit (Bio Assay Systems, CA) following manufacturer’s instructions. Cells
were mechanically dissociated and lysed in 100mM potassium phosphate containing 2mM
EDTA buffer, centrifuged and the resulting supernatants were assayed using the working
reagent. Optical density was read at 565nm immediately after the mixture of the sample and
the working reagent, and also 25min after addition. LDH activity was determined based on the
following formula: LDH activity = 43.68 × (OD S25−ODS0)/(OD calibrator − OD H2O) × dilution
factor.
Superoxide anion detection by MitoSox Red
hESC were maintained on Matrigel for 7 days and treatments (Antimycin A and /or MnTbap)
were initiated at day two after scraping. Fresh media and drugs were added every other day.
On day seven, cells were washed with PBS and dissociated with Accutase followed by two
washes in PBS. Superoxide was detected using MitoSox Red (Invitrogen). Cells were
suspended in media and incubated with 2.5μM of MitoSox Red for 30 min at 37°C followed
by one wash in PBS and subsequent analysis by Flow cytometry.
Statistical Analysis
Means and Standard Error of the Mean were calculated and statistically significant differences
were determined by paired t-test, Chi-Square test, or One-Way ANOVA followed by Dunnett’s
Multiple Comparison test. n refers to sample size. Significance was determined at p< 0.05.
Results
Antimycin A (20nM) was added 48 hours after passaging to hESC cultures (WA07) growing
on MEF’s in standard culture media, and this treatment was maintained for 5 days (Fig. 1).
hESC grown under these conditions maintained a typical compact morphology with high
nuclear-cytoplasmic ratio and colonies with well-defined borders, comparable to those in
control cells (Fig. 1d). To quantify the levels of pluripotency, we used Real Time PCR for the
well characterized pluripotency transcription factors Nanog and POU5F1 (also known as
Oct-4). hESC treated with Antimycin A showed a two fold increase in Nanog mRNA levels
Varum et al.Page 4
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 5

compared with control cells (Fig. 1a, p<0.01, n= 6). Oct-4 mRNA levels were not statistically
different (Fig. 1a). Similar results were obtained with the complex III inhibitor, Myxothiazol,
used at a concentration of 20 nM (data not shown).
To determine if the effects observed after addition of Antimycin A to the culture media were
sustained or only transient, hESC were maintained for several passages (1-2 months) in the
presence of Antimycin A. hESC grown under these conditions maintained good morphology
throughout and maintained a two-fold elevation of Nanog mRNA (p<0.05, n=3) relative to
controls. Again, Oct-4 mRNA levels were not significantly different from controls (Fig. 1a).
Western blot analysis of these extended treatment cells showed that Nanog protein levels were
also increased in Antimycin A treated cells (Fig. 1b); Oct-4 protein levels were not significantly
different between treatments (Fig. 1c). To ensure that prolonged Antimycin A treatment did
not negatively impact the pluripotent phenotype, we characterized these cells by ICC for the
pluripotency markers Oct-4, Nanog, and SSEA-4. Nanog and Oct-4 were both found in the
nucleus of Antimycin A treated cells, and the cell surface marker SSEA-4 labeled colonies
(Fig. 1d) similar to control cells.
To determine if Antimycin A treated cells retained the potential to differentiate in vivo, we
injected hESC treated with Antimycin A for 9 passages into NOD/SCID mice to test their
ability to form teratomas. Similar to hESC maintained under standard conditions (not shown),
Antimycin A treated cells were able to form teratomas exhibiting tissues of all three germ layers
(Fig. 1e), including gastrointestinal tissue (endoderm), cartilage (mesoderm) and neuro ganglia
(ectoderm). Finally, cells grown for extended periods (19 passages) in the presence of
Antimycin A also maintained a stable 46 XX karyotype (data not shown).
To exclude the possibility that the effect of Antimycin A on Nanog expression was due to an
increase in cell survival of pluripotent stem cells relative to differentiated cells, the levels of
Annexin V/PI positive cells were determined by flow cytometry (Fig. 2 a-c). There was no
statistically significant difference in the percentage of viable cells between Control (83.25
±2.15, Fig. 2a, c) and Antimycin A (81.2±9.9, Fig. 2b, c) treated cells. Based on these results
Antimycin A does not appear to affect cell survival.
In addition, proliferation of hESC was determined by BrdU incorporation into genomic DNA
during S phase of the cell cycle (Fig.2 d-e). No statistically significant difference was observed
in the percentage of cells that incorporate BrdU between control and Antimycin A treated cells.
In combination with the above results, Antimycin A does not seem to affect cell death or
proliferation of hESC.
As discussed above, bFGF is a well described factor important in the maintenance of
pluripotency in hESC, even supporting pluripotency in the absence of feeder cells or
conditioned media when present at high concentrations [9-10]. In order to better understand
the beneficial effect of Antimycin A we cultured hESC on mouse feeders for 7 days using
different combinations of Antimycin A and bFGF and assayed Oct-4 expression by ICC.
Colonies were divided into three categories according to the pattern of Oct-4 expression and
quantified: totally positive colonies in which the majority of cells were positive for Oct-4 (Fig.
3a bottom left); partially positive colonies containing a significant quantity of both positive
and negative cells (Fig. 3a bottom center); and negative colonies in which the majority of cells
were negative for Oct-4 (Fig. 3a bottom right). At least 200 colonies were counted per
treatment. As expected, removal of bFGF from the media resulted in a significant decrease in
the number of totally positive colonies and an increase in the number of partially positive
colonies (Fig.3b; p<0.001), confirming the importance of this growth factor in the maintenance
of healthy hESC colonies. Interestingly, when Antimycin A was added to culture media lacking
bFGF the number of totally positive colonies (65.9% vs. 67.0%) and partially positive colonies
Varum et al. Page 5
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 6

(33.3% vs. 32.6%) was indistinguishable from standard culture conditions (Fig. 3b), suggesting
that addition of Antimycin A maintains Oct-4 expression levels in culture conditions lacking
bFGF. Surprisingly, the addition of both Antimycin A and bFGF to the culture increased the
number of totally positive colonies (86.8% vs 67.07%, p<0.001). This answer to this apparent
contradiction is unknown but several factors may be involved. Firstly, western blotting and
Real Time PCR are unbiased quantitative analyses that probe an entire population of cells,
while ICC as carried out here was inherently qualitative in terms of signal intensity, colony
size and colony heterogeneity (i.e., more or less positive cells in a colony), all of which could
have played a role. Furthermore, subcellular localization of Oct-4 should also be considered.
It is understood that during early differentiation of hESC Oct-4 localizes primarily in the
cytoplasm instead of the nucleus, as is observed with undifferentiated hESC. During human
development Oct-4 is first observed cytoplasmically before localizing to the nucleus in the
inner cell mass [53]. In this case cells in which Oct-4 localizes to the cytoplasm will be counted
as negative in our ICC analysis but would still be positive by western blotting. Subcellular
localization of Oct-4 may thus be a better assay of pluripotency than total cellular protein.
Importantly, when performing double labeling experiments for both Oct-4 and Nanog, the most
striking effect of Antimycin treatment within single colonies was an increase of Oct-4+/Nanog
+ cells (97% vs. 70%) at the expense of the Oct-4+/Nanog-subpopulation (0.4% vs. 23%).
Finally, we performed Real Time PCR for Nanog in both WA07 (Fig.3c) and WA09 (Sup Fig.
1, carried out in a separate facility) for the conditions above. We observed that both cell lines
are responsive to bFGF withdrawal demonstrated by a decrease in Nanog expression. In both
cell lines Antimycin A maintains Nanog expression upon bFGF withdrawal, suggesting that
this effect is not related to the particular characteristics of one hESC line.
One possible explanation for these results is that Antimycin A induces bFGF secretion by
feeders and/or hESC. In order to address this point, bFGF concentration in the culture media
was measured by ELISA (Sup Fig. 2). No bFGF secretion by mitomycin inactivated MEF’s
was detected. The bFGF initially present in the media (4ng/ml), was rapidly degraded/
processed by the cells, as demonstrated in media concentrations of 65.92±5.01 pg/ml and 24.41
±3.58 pg/ml after 6 hr or 24 hr of culture, respectively (Sup Fig. 2 a). These results are in
accordance with the findings of Eiselleova et al.[26] that showed, in contrast to human feeders,
MEFs do not produce bFGF. Antimycin A treatment does not promote bFGF secretion by the
MEFs as media concentrations are indistinguishable from controls (59.04± 3.94pg/ml and
27.31± 7.89pg/ml) after 6 hr and 24 hr of culture, respectively. In contrast to MEFs, hESCs
appear to secrete bFGF into the media, since concentrations of 64.40±36.72 pg/ml of bFGF
were detected after 24 hr of culture in media not supplemented with bFGF (Supp Fig.2b). These
results are in accordance with the findings of Dvorak et al.[27]. bFGF production by hESCs
was not affected by Antimycin A treatment. These results indicate the effect of Antimycin A
on pluripotency was not due to an increase in bFGF secretion by the feeders or by hESC
themselves.
These findings raise questions regarding the involvement of the bGF pathway in this effect.
To address this issue we used the bFGF receptor inhibitor PD173074 [20]. hESC were
maintained on Matrigel for 7 days in different combinations of bFGF, PD173074 and
Antimycin A (Fig. 3d). Similar to what we observed for hESC cultured on feeders, bFGF
removal reduced Nanog expression (1.6 fold decrease) when compared to control cells (bFGF
alone). Moreover, the addition of PD173074 promoted a significant decrease in Nanog and
Oct-4 expression when compared to control cells (2.8 and 2.1 fold decrease, respectively).
These results are in accordance with the findings of Dvorak et al.[28] who demonstrated a
crucial role for the autocrine bFGF signaling pathway in the maintenance of hESCs self-
renewal. Treatment with Antimycin A in the absence of bFGF maintained Nanog and Oct-4
mRNA expression at similar levels to controls; however, the expression of these markers
decreased significantly upon the addition of PD173074 (1.8 and 1.96 fold decrease for Nanog
Varum et al.Page 6
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 7

and Oct-4, respectively). Interestingly, the decrease in Nanog mRNA levels after the addition
of PD173074 was less accentuated in the presence of Antimycin A, perhaps suggesting that
Antimycin A functions through a different pathway than bFGF. These results indicate that,
although Antimycin A can maintain the expression levels of pluripotency markers in absence
of exogenous bFGF, it cannot maintain pluripotency when the endogenous bFGF pathway is
inhibited, reinforcing the importance of the autocrine bFGF signaling pathway. Additionally,
since Antimycin A mitigated the effects of both exogenous bFGF removal and the inhibition
of the endogenous bFGF pathway by PD173074, it is likely that Antimycin A acts in a pathway
separate from bFGF.
To determine if cells treated with Antimycin A remain pluripotent in the absence of bFGF for
prolonged periods of time, hESC were treated with Antimycin A in knockout media without
bFGF for periods greater than one month. The best colonies in all conditions were manually
passaged every 7 days. hESC maintained under these conditions had a similar morphology to
control cells, including tightly packed cells in colonies with well defined borders (Fig. 3e).
Immunocytochemical analysis demonstrated that these cells express the pluripotency markers
Nanog, Oct-4 and SSEA-4 in similar patterns to control cells (Fig. 3e). We also evaluated the
capacity of these cells to differentiate in vivo. Antimycin A treated cells grown in the absence
of exogenous bFGF (33 passages) generated teratomas exhibiting tissues from all three germ
layers (Fig. 3f). These cells maintained a stable 46XX karyotype after prolonged culture (19
passages, data not shown).
To further investigate the role of Antimycin A treatment on gene expression we used the
TaqMan® Low Density Array human stem cell Pluripotency Panel. These arrays contain 90
genes involved in maintenance of pluripotency or promotion of differentiation, along with 6
endogenous control genes [29]. We maintained hESC in different combinations of Antimycin
A and bFGF for more than 20 passages, and analyzed mRNA levels by Real Time PCR. Among
the genes analyzed were Nanog [30-31], POUF51 [32] and TDGF1 [33], all critical for the
maintenance of pluripotency. Consistent with our findings using single gene assays, treatment
with Antimycin A in the presence of bFGF resulted in a statistically significant two fold
increase in Nanog expression, when analyzed with the low density array (Fig. 4a;n=3; p<0.01).
This difference was maintained in the absence of bFGF (p<0.01). TDGF1 was also significantly
elevated after treatment with Antimycin A (1.8-fold increase; P<0.01), while POU5F1 was
elevated but not significantly. Both TDGF1 and POU5F1 were elevated in the absence of bFGF
but did not rise to the level of significance. The other four pluripotency genes (SOX2,
DMNT3B, GABRB3 and GDF3) on the array were not elevated and, surprisingly, removal of
both Antimycin A and bFGF did not significantly reduce expression levels of any of these
genes (Fig. 4a, heatmap) When 32 genes correlated with pluripotency were examined, several
genes were found to be amplified (Fig. 4b, heatmap), including EBAF, LEFTB and NODAL,
but several others decreased with Antimycin A treatment including GBX2 and CRABP2. To
compare these genes as a group across treatments, we averaged the fold changes across all 32
genes. No significant differences in the expression of these pluripotency correlated genes were
observed between any treatments (Fig. 4b).
We also studied the expression of genes involved in differentiation. These genes were grouped
according to their participation in endoderm (Fig. 5a), mesoderm (Figure 5b), trophoblast (Fig.
5c), or ectoderm (Fig. 5d) differentiation and averaged as above. Antimycin A treatment in the
absence of bFGF resulted in no statistical differences in the expression of differentiation genes
in comparison to control cells (Fig. 5a-d). The addition of Antimycin A in the presence of bFGF
reduced the expression of differentiation genes in all categories (Fig. 5a-c), although only the
decrease in ectoderm related genes was statistically significant (Fig. 5d, p<0.01, n=3). These
results are in accordance with the findings of Vallier et al.[34] who reported that Nanog
overexpression prevents neuroectoderm differentiation. In contrast to the pluripotency genes
Varum et al.Page 7
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 8

whose expression did not change after withdrawal of bFGF and Antimycin A, endoderm,
trophoblast and mesoderm genes were all significantly higher in the absence of these
compounds (Fig. 5a-c; p<0.01, n=3). Only the ectodermal genes were unchanged in the absence
of bFGF and Antimycin A (Figure 5d). This last result is in accordance with Stern et al. [35]
who reported that bFGF plays a crucial role in neuroectoderm specification in amphibian and
chick embryos.
Antimycin A traditionally stimulates a shift in the metabolism from oxidative phosphorylation
to glycolysis (due to lower mitochondrial function) and promotes an increased superoxide
generation at complex III (due to a lack of normal electron flow to oxygen, and thus the
“leaking” of high-energy electrons from the transport chain). In order to determine if Antimycin
A treatment has the same effect in hESC, the levels of adenine nucleotides (ATP, ADP and
AMP) were measured by HPLC in Control and Antimycin A treated hESC. No significant
differences in the adenylate pool or adenylate charge were observed between the two conditions
(Fig.6 a-b). Lactate Dehydrogenase (LDH) is an enzyme that converts pyruvate, the final
product of glycolysis, to lactate when a decline in O2 availability or impaired ATP production
by mitochondria forces the cell into anaerobic metabolism, or when aerobic glycolysis is
favored, as is the case in some cancers. Antimycin A treated cells showed an increase of
approximately 40% in the rates of LDH activity (Fig.6 c). These results indicate that Antimycin
A induces a shift in the metabolism towards glycolysis similar to what is observed in other cell
types. However, hESC cultured in the presence of Antimycin A maintained ATP levels
consistent with untreated cells.
As mentioned above, Antimycin A increases the production of ROS in cells. To determine the
effect of Antimycin A on mitochondrial superoxide anion production in hESC, we used
MitoSox Red (Fig.7). Cells treated with 20 nM Antimycin A showed an increase of
approximately 61%±20.7% in the number of positive cells for MitoSox Red when compared
with control cells, as monitored by flow cytometry. This effect was dose dependent as treatment
of hESC with 2 μM of Antimycin A increased the number of positive cells approximately 250%
±16.3%. This effect was counteracted by the addition of MnTbap, a cell permeable Superoxide
Dismutase (SOD) mimetic which acts as a scavenger specifically targeting superoxide.
Treatment with 50μM MnTbap eliminated the effect of treatment with 20nM Antimycin A.
MnTbap was also able to reduce the number of positive hESC under control conditions (Fig.
7a, b).
Reactive oxygen species are important signaling molecules within the cell. To address whether
the effect of Antimycin A on pluripotency is mediated by ROS cells were maintained in
different combinations of Antimycin A and MnTbap and Real Time PCR for Nanog was
performed (Fig. 7c). MnTbap was able to partially abrogate the Antimycin A stimulated
increase in Nanog expression, although no significant differences were observed between
control and MnTbap treated cells. These results suggest that Superoxide anion generated at
complex III is at least partially responsible for the effect of Antimycin A on Nanog expression.
Discussion
Studies have demonstrated that hESC cultured under low oxygen tension (1.5-5%) are better
maintained in the undifferentiated state [12-36]. This suggests that a decrease in mitochondrial
oxidative phosphorylation and an increase in ROS signaling under these conditions might be
involved. We show here that this effect can be mimicked by directly inhibiting mitochondrial
function in a way that is at least partially dependent on ROS formation. hESC treated with 20
nM Antimycin A maintained pluripotency not only as evidenced by immunocytochemical
staining of the pluripotency markers but also as assayed by teratoma formation. Real Time
PCR and Western Blot analysis demonstrated that Antimycin A treated hESC had elevated
Varum et al.Page 8
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 9

levels of both Nanog mRNA and protein. While a similar increase was observed for TDGF1
mRNA expression, no significant differences were observed for Oct-4 protein or mRNA. These
results can best be explained by considering that, unlike the case for Oct-4, overexpression of
Nanog is beneficial for the maintenance of pluripotency, namely preventing neuroectoderm
differentiation induced by FGF signaling [33]. Others have shown that overexpression of
Nanog allows culture in the absence of feeders [37] and circumvents the need for both TGFb
and FGF signaling in the maintenance of pluripotency [38]. In contrast Oct- 4 levels must be
kept within defined ranges in order to maintain self-renewal of both mouse and human ESCs,
as overexpression results in upregulation of markers involved in endoderm and mesoderm
specification of both cell types [39-40] while, conversely, RNA interference targeting Oct-4
can trigger trophectoderm differentiation [41-43]. Additionally, Pan et al.[44] demonstrated
that a steady-state concentration of Oct-4 maintains Nanog expression, whereas an elevated
concentration of Oct-4 suppresses Nanog expression. Therefore, while upregulation of Nanog
is globally beneficial to the maintenance of pluripotency, upregulation of Oct-4 can be
detrimental and maintenance of the status quo is appropriate for Oct-4 expression. Taking into
account these findings it seems that Antimycin A does not increase Oct-4 expression above
the steady-state present in pluripotent hESC.
Very few growth factors have been identified as being necessary or sufficient for maintenance
of hESC pluripotency, though one leading candidate is bFGF [6-8]. As bFGF and Antimycin
A both promote pluripotency, we were interested in determining if Antimycin A could maintain
pluripotency in the absence of bFGF. Treatment with Antimycin A was able to alleviate the
requirement for exogenous bFGF as there was no difference in the number of totally positive
colonies between standard culture conditions and those including Antimycin A but lacking
bFGF. Furthermore, Antimycin A was able to sustain Nanog expression upon bFGF removal
in both WA07 and WA09 cell lines demonstrating that the effect is not ES line dependent.
Antimycin A treatment failed to maintain pluripotency upon the inhibition of the endogenous
bFGF pathway, indicating the requirement for the endogenous bFGF pathway in the
maintenance of pluripotency. The observation that Antimycin A increases Nanog expression
even when the endogenous pathway is suppressed suggests that Antimycin A works through
a pathway other than bFGF. Taken together, our results suggest that bFGF and Antimycin A
work through synergistic pathways to maintain pluripotency.
Cells cultured for several months in the presence of Antimycin A and absence of exogenous
bFGF remained pluripotent as assessed by ICC and Teratoma formation. Furthermore, Low
density Array analysis showed that Antimycin A treatment of hESC cultured in the absence of
bFGF resulted in an elevated expression of Nanog mRNA and maintained the expression of
differentiation genes at similar levels to those found in control cells.
Our findings can be explained in light of morphological and functional changes that
mitochondria undergo during early mammalian development. In the oocyte, mitochondria are
spherical with few christae, whereas between the zygote and the morula stage, mitochondria
become more elongated [45]. At the blastocyst stage two distinct forms of mitochondria are
present: mitochondria in the inner cell mass (ICM) are spherical and have low O2 consumption,
whereas those in the trophectoderm (TE) are elongated and have higher respiratory rates
[46-47]. As hESCs are derived from the ICM, one should expect that they share these metabolic
and morphological features. Indeed, it has been shown that undifferentiated hESC have few
mitochondria with an immature morphology, and a greater reliance on glycolysis [14-16]. As
hESC differentiate both the number of mitochondria with a mature morphology increases, as
well as ATP production by oxidative phosphorylation [16].
Finally, our results indicate that ROS produced at complex III of the mitochondrial electron
transfer chain are at least partially responsible for the Antimycin A effect on Nanog expression.
Varum et al.Page 9
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 10

Mitochondrial ROS constitute the major source of ROS in the cells and are produced as side
products of oxidative phosphorylation. The involvement of ROS in deleterious processes such
as DNA damage, changes in the native structure of proteins, and lipidic peroxidation of
membranes has long been recognized. More recently, however, it has been recognized that
ROS can modulate various intracellular signaling pathways through covalent modifications
(so called “redox signaling”) of target molecules, thereby inducing changes in cells that are
important in many physiological and pathophysiological processes [48]. In addition, there is
a growing body of evidence that there is an increase in ROS production following the addition
of a various peptide growth factors to cells in culture, and that these ROS are a crucial
component of downstream signaling. Our results demonstrate that Antimycin A increases
superoxide generation at complex III and that capture of superoxide anion by MnTbap partially
abrogates the effect of Antimycin A in Nanog expression, suggesting ROS as at least a partial
modulator of Antimycin A effect on pluripotency. Superoxide anion can rapidly convert to
other reactive species and therefore not be captured by MnTbap, thus continuing to signal,
which might also explain why MnTbap does not completely eliminate the Antimycin A effect.
This is in accordance with the reports of Carriere et al.[49] demonstrating that Antimycin A
inhibited murine preadipocyte differentiation towards the adipocyte phenotype by increasing
ROS formation at complex III. Indeed, several growth factor and cytokines such as bFGF and
TGFb are known to induce H2O2 generation in different cell types [50]. Alternatively, the effect
of Antimycin A in Nanog expression could be a synergistic action between ROS production
and a shift in oxidative metabolism towards glycolysis, which we have also demonstrated to
take place. This latter hypothesis is in accordance with findings of Chung et al.[51] who showed
that during the in vitro process of mESC differentiation, Antimycin A inhibits oxidative
phosphorylation and leads to a reduced appearance of beating cardiomyocytes. In addition, we
cannot rule out the hypothesis that the effect of Antimycin A on Nanog expression could be
partially mediated by changes in calcium homeostasis. Indeed, Spitkovsky et al.[52],
demonstrated that, while Antimycin A blocked cardiomyocyte differentiation by acting on
calcium signaling, and that the use of KCN (an inhibitor of complex IV of the mitochondrial
respiratory chain) did not. Further work is required to pinpoint the exact mechanism(s) involved
but our data provide the first evidence that modulation of mitochondrial function (probably
acting through a ROS-dependent pathway) can influence the pluripotent state of hESC.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We would like to acknowledge the invaluable help of several of our colleagues including: Gerald Schatten for critical
reading of the manuscript, discussion of the results and financial support. Carrie Redinger and Jody Mich-Basso for
hESC culture and RT PCR, Dave McFarland for help generating the teratomas and John Ozolek for analysis of
teratomas. Special thanks are due to Yuki Ohi and Miguel Ramalho-Santos (University of California, San Francisco)
for invaluable assistance with experiments involving the WA09 cell line. We would also like to thank Ana Sofia
Rodrigues, Andre Tartar, Dan Constantinescu and Charles Easley for critical reading of the manuscript. This work
was supported by a grant from the National Institute of Child Health and Human Development, 1PO1HD047675 (to
Gerald Schatten) and Fundacão para a Ciencia e Tecnologia (FCT) for scholarship support of S.V. J.-R.-S. was
supported by a Fulbright Fellowship.
References
[1]. Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T. STAT3 activation is
sufficient to maintain an undifferentiated state of mouse embryonic stem cells. Embo J 1999;18
(15):4261–4269. [PubMed: 10428964]
[2]. Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is
mediated via activation of STAT3. Genes Dev 1998;12(13):2048–2060. [PubMed: 9649508]
Varum et al. Page 10
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 11

[3]. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation
and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115(3):281–
292. [PubMed: 14636556]
[4]. Daheron L, Opitz SL, Zaehres H, Lensch MW, Andrews PW, Itskovitz-Eldor J, Daley GQ. LIF/
STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 2004;22
(5):770–778. [PubMed: 15342941]
[5]. Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA. BMP4 initiates
human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 2002;20(12):1261–1264.
[PubMed: 12426580]
[6]. Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson
JA. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative
potential for prolonged periods of culture. Dev Biol 2000;227(2):271–278. [PubMed: 11071754]
[7]. Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, Carpenter MK. Long-term culture of human
embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229(2):259–274. [PubMed:
14745951]
[8]. Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O’Sullivan C, Delavan-Boorsma K, Mok M,
Bronstein A, Carpenter MK. Basic fibroblast growth factor supports undifferentiated human
embryonic stem cell growth without conditioned medium. Stem Cells 2005;23(3):315–323.
[PubMed: 15749926]
[9]. Wang G, Zhang H, Zhao Y, Li J, Cai J, Wang P, Meng S, Feng J, Miao C, Ding M, Li D, Deng H.
Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the
absence of feeder layers. Biochem Biophys Res Commun 2005;330(3):934–942. [PubMed:
15809086]
[10]. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP
signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2005;2(3):185–
190. [PubMed: 15782187]
[11]. Vallier L, Alexander M, Pedersen RA. Activin/Nodal and FGF pathways cooperate to maintain
pluripotency of human embryonic stem cells. J Cell Sci 2005;118(Pt 19):4495–4509. [PubMed:
16179608]
[12]. Ezashi T, Das P, Roberts RM. Low O2 tensions and the prevention of differentiation of hES cells.
Proc Natl Acad Sci U S A 2005;102(13):4783–4788. [PubMed: 15772165]
[13]. Fischer B, Bavister BD. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and
rabbits. J Reprod Fertil 1993;99(2):673–679. [PubMed: 8107053]
[14]. St John JC, Ramalho-Santos J, Gray HL, Petrosko P, Rawe VY, Navara CS, Simerly CR, Schatten
GP. The expression of mitochondrial DNA transcription factors during early cardiomyocyte in vitro
differentiation from human embryonic stem cells. Cloning Stem Cells 2005;7(3):141–153.
[PubMed: 16176124]
[15]. Oh SK, Kim HS, Ahn HJ, Seol HW, Kim YY, Park YB, Yoon CJ, Kim DW, Kim SH, Moon SY.
Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2,
and SNUhES3. Stem Cells 2005;23(2):211–219. [PubMed: 15671144]
[16]. Cho YM, Kwon S, Pak YK, Seol HW, Choi YM, Park do J, Park KS, Lee HK. Dynamic changes
in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of
human embryonic stem cells. Biochem Biophys Res Commun 2006;348(4):1472–1478. [PubMed:
16920071]
[17]. Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker
PT. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible
factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem 2000;275(33):25130–
25138. [PubMed: 10833514]
[18]. Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, Simon MC, Hammerling U,
Schumacker PT. Mitochondrial complex III is required for hypoxia-induced ROS production and
cellular oxygen sensing. Cell Metab 2005;1(6):401–408. [PubMed: 16054089]
[19]. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth
of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19(10):971–974. [PubMed:
11581665]
Varum et al.Page 11
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 12

[20]. Mohammadi M, Froum S, Hamby JM, Schroeder MC, Panek RL, Lu GH, Eliseenkova AV, Green
D, Schlessinger J, Hubbard SR. Crystal structure of an angiogenesis inhibitor bound to the FGF
receptor tyrosine kinase domain. EMBO J 1998;17(20):5896–5904. [PubMed: 9774334]
[21]. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. The ground
state of embryonic stem cell self-renewal. Nature 2008;453(7194):519–523. [PubMed: 18497825]
[22]. Navara CS, Redinger C, Mich-Basso J, Oliver S, Ben-Yehudah A, Castro C, Simerly C. Derivation
and characterization of nonhuman primate embryonic stem cells. Curr Protoc Stem Cell Biol. 2007
Chapter 1:Unit 1A 1.
[23]. Navara CS, Redinger C, Mich-Basso J, Oliver S, Ben-Yehudah A, Castro C, Simerly C. Derivation
and characterization of nonhuman primate embryonic stem cells. Curr Protoc Stem Cell Biol. 2007a
Chapter 1: Unit 1A 1.
[24]. Navara, CS.; Redinger, C.; Mich-Basso, J.; Oliver, S.; Ben-Yehudah, A.; Castro, C.; Simerly, CR.
Derivation and Characterization of Non-human Primate Embryonic Stem Cells. John Wiley and
Sons; 2007.
[25]. Amaral S, Moreno AJ, Santos MS, Seica R, Ramalho-Santos J. Effects of hyperglycemia on sperm
and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes.
Theriogenology 2006;66(9):2056–2067. [PubMed: 16860381]
[26]. Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P. Comparative study of mouse
and human feeder cells for human embryonic stem cells. Int J Dev Biol 2008:353–363. [PubMed:
18415935]
[27]. Dvorak P, Hampl A. Basic fibroblast growth factor and its receptors in human embryonic stem cells.
Folia Histochem Cytobiol 2005;43(4):203–208. [PubMed: 16382885]
[28]. Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A. Expression
and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells.
Stem Cells 2005;23(8):1200–1211. [PubMed: 15955829]
[29]. Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, Bello PA,
Benvenisty N, Berry LS, Bevan S, Blum B, Brooking J, Chen KG, Choo AB, Churchill GA, Corbel
M, Damjanov I, Draper JS, Dvorak P, Emanuelsson K, Fleck RA, Ford A, Gertow K, Gertsenstein
M, Gokhale PJ, Hamilton RS, Hampl A, Healy LE, Hovatta O, Hyllner J, Imreh MP, Itskovitz-
Eldor J, Jackson J, Johnson JL, Jones M, Kee K, King BL, Knowles BB, Lako M, Lebrin F, Mallon
BS, Manning D, Mayshar Y, McKay RD, Michalska AE, Mikkola M, Mileikovsky M, Minger SL,
Moore HD, Mummery CL, Nagy A, Nakatsuji N, O’Brien CM, Oh SK, Olsson C, Otonkoski T,
Park KY, Passier R, Patel H, Patel M, Pedersen R, Pera MF, Piekarczyk MS, Pera RA, Reubinoff
BE, Robins AJ, Rossant J, Rugg-Gunn P, Schulz TC, Semb H, Sherrer ES, Siemen H, Stacey GN,
Stojkovic M, Suemori H, Szatkiewicz J, Turetsky T, Tuuri T, van den Brink S, Vintersten K,
Vuoristo S, Ward D, Weaver TA, Young LA, Zhang W. Characterization of human embryonic stem
cell lines by the International Stem Cell Initiative. Nat Biotechnol 2007;25(7):803–816. [PubMed:
17572666]
[30]. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression
cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113(5):643–
655. [PubMed: 12787505]
[31]. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M,
Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse
epiblast and ES cells. Cell 2003;113(5):631–642. [PubMed: 12787504]
[32]. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith
A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription
factor Oct4. Cell 1998;95(3):379–391. [PubMed: 9814708]
[33]. Baldassarre G, Bianco C, Tortora G, Ruggiero A, Moasser M, Dmitrovsky E, Bianco AR, Ciardiello
F. Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and
inhibits transformation in a human embryonal carcinoma cell line. Int J Cancer 1996;66(4):538–
543. [PubMed: 8635871]
[34]. Vallier L, Mendjan S, Brown S, Chng Z, Teo A, Smithers LE, Trotter MW, Cho CH, Martinez A,
Rugg-Gunn P, Brons G, Pedersen RA. Activin/Nodal signalling maintains pluripotency by
controlling Nanog expression. Development 2009;136(8):1339–1349. [PubMed: 19279133]
Varum et al.Page 12
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 13

[35]. Stern CD. Neural induction: old problem, new findings, yet more questions. Development 2005;132
(9):2007–2021. [PubMed: 15829523]
[36]. Westfall SD, Sachdev S, Das P, Hearne LB, Hannink M, Roberts RM, Ezashi T. Identification of
oxygen-sensitive transcriptional programs in human embryonic stem cells. Stem Cells Dev 2008;17
(5):869–881. [PubMed: 18811242]
[37]. Darr H, Mayshar Y, Benvenisty N. Overexpression of NANOG in human ES cells enables feeder-
free growth while inducing primitive ectoderm features. Development 2006;133(6):1193–1201.
[PubMed: 16501172]
[38]. Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, Yu J, Antosiewicz-Bourget J, Tian
S, Stewart R, Thomson JA. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling
in human ESCs. Cell Stem Cell 2008;3(2):196–206. [PubMed: 18682241]
[39]. Rodriguez RT, Velkey JM, Lutzko C, Seerke R, Kohn DB, O’Shea KS, Firpo MT. Manipulation
of OCT4 levels in human embryonic stem cells results in induction of differential cell types. Exp
Biol Med (Maywood) 2007;232(10):1368–1380. [PubMed: 17959850]
[40]. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation,
dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24(4):372–376. [PubMed: 10742100]
[41]. Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm
and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells
2004;22(2):225–235. [PubMed: 14990861]
[42]. Velkey JM, O’Shea KS. Oct4 RNA interference induces trophectoderm differentiation in mouse
embryonic stem cells. Genesis 2003;37(1):18–24. [PubMed: 14502573]
[43]. Matin MM, Walsh JR, Gokhale PJ, Draper JS, Bahrami AR, Morton I, Moore HD, Andrews PW.
Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human
embryonic stem cells and embryonic carcinoma cells. Stem Cells 2004;22(5):659–668. [PubMed:
15342930]
[44]. Pan G, Li J, Zhou Y, Zheng H, Pei D. A negative feedback loop of transcription factors that controls
stem cell pluripotency and self-renewal. FASEB J 2006;20(10):1730–1732. [PubMed: 16790525]
[45]. Ramalho-Santos J, Varum S, Amaral S, Mota PC, Sousa AP, Amaral A. Mitochondrial functionality
in reproduction: from gonads and gametes to embryos and embryonic stem cells. Hum Reprod
Update. 2009
[46]. Stern S, Biggers JD, Anderson E. Mitochondria and early development of the mouse. J Exp Zool
1971;176(2):179–191. [PubMed: 5559227]
[47]. Houghton FD. Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst.
Differentiation 2006;74(1):11–18. [PubMed: 16466396]
[48]. Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol 2003;15(2):247–254. [PubMed:
12648682]
[49]. Carriere A, Carmona MC, Fernandez Y, Rigoulet M, Wenger RH, Penicaud L, Casteilla L.
Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and
adipocyte differentiation: a mechanism for hypoxia-dependent effect. J Biol Chem 2004;279(39):
40462–40469. [PubMed: 15265861]
[50]. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell
Mol Physiol 2000;279(6):L1005–1028. [PubMed: 11076791]
[51]. Chung S, Dzeja PP, Faustino RS, Perez-Terzic C, Behfar A, Terzic A. Mitochondrial oxidative
metabolism is required for the cardiac differentiation of stem cells. Nat Clin Pract Cardiovasc Med
2007;4(Suppl 1):S60–67. [PubMed: 17230217]
[52]. Spitkovsky D, Sasse P, Kolossov E, Bottinger C, Fleischmann BK, Hescheler J, Wiesner RJ. Activity
of complex III of the mitochondrial electron transport chain is essential for early heart muscle cell
differentiation. Faseb J 2004;18(11):1300–1302. [PubMed: 15180963]
[53]. Cauffman G, Van de Velde H, Liebaers I I, Van Steirteghem A. Oct-4 mRNA and protein expression
during human preimplantation development. Mol Hum Reprod 2005;11(3):173–181. [PubMed:
15695770]
Varum et al. Page 13
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 14

Figure 1. Analysis of Pluripotency after Antimycin A Treatment
(a) Real Time PCR for the pluripotency markers Nanog and Oct-4 in hESC treated with
Antimycin A for 5 days or 4-8 passages. Statistical significant differences were determined by
paired t-test; n=6 and 3 respectively. **P<0.01; *P<0.05. (b-c) Western blot analysis for Nanog
and Oct-4 protein levels in hESCs treated with Antimycin A for 4-8 passages. (d) hESC
morphology and pluripotency marker pattern expression after treatment with Antimycin A for
4-8 passages, examined by phase contrast microscopy, ICC and confocal laser scanning
microscopy. (e) H&E staining of teratoma sections from hESC treated with Antimycin A for
9 passages. Abbreviations: Ant A, Antimycin A; Error bars, SEM. Scale bar, 50μm.
Varum et al. Page 14
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Page 15

Figure 2. Analysis of Apoptosis/ Necrosis and Proliferation Rates after Antimycin A Treatment
(a-b) Representative dot plots of flow cytometry for Annexin V/PI of control and Antimycin
A treated hESC. Four populations were identified: viable cells (negative for both Annexin V
and PI); early apoptotic cells (positive for Annexin V and negative for PI); late apoptoic cells
(positive for both Annexin V and PI) and necrotic cells (only positive for PI). (c) Percentage
of the different cell populations in both control and Antimycin A treated cells (d) Brdu
incorporation in both control and Antimycin A treated hESCs, examined by Confocal
microscopy. (e) Percentage of Brdu positive cells. Error bars, SEM. Scale bar, 50μM
Varum et al. Page 15
Stem Cell Res. Author manuscript; available in PMC 2010 September 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript