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Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions


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Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3-5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.
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Hypoxia inducible factors regulate pluripotency and
proliferation in human embryonic stem cells cultured
at reduced oxygen tensions
Catherine E Forristal
, Kate L Wright
, Neil A Hanley
, Richard O C Oreffo
and Franchesca D Houghton
Centre for Human Development, Stem Cells and Regeneration,
Human Genetics Division and
Origins of Health & Disease Division, School of Medicine, University of Southampton, Southampton General
Hospital, Duthie Building (MP 808), Tremona Road, Southampton, SO16 6YD, UK
Correspondence should be addressed to F D Houghton; Email:
N A Hanley is now at Endocrine Sciences Research Group, Manchester Academic Health Science Centre, University of Manchester,
AV Hill Building, Oxford Road, Manchester, M13 9PT, UK
Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which
reside in a 3–5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-
dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic
responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced
expression of SOX2,NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein
was not expressed at 20% oxygen and displayed onlya transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed
a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and
were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both
hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was
concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the
reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a
highly proliferative, pluripotent population of hES cells. While HIF3Awas found to regulate the expression of both HIF1A and HIF2A, it is
HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.
Reproduction (2010) 139 85–97
Human embryonic stem (hES) cells, typically derived
from the inner cell mass (ICM) of the blastocyst,
proliferate by self renewal and have the potential to
differentiate into all cells of the body (Thomson et al.
1998). Thus, hES cells provide an excellent model to
investigate developmental mechanisms and are a
potentially unlimited source of cells for transplantation
in degenerative disease. However, hES cells are notor-
iously difficult to maintain in culture due to their
propensity for spontaneous differentiation, an effect
likely caused by a suboptimal culture environment.
Clues to improve the culture of hES cells may be gained
by investigating the environment from which these cells
are derived. In vivo, preimplantation embryos develop in
the secretions of the reproductive tract which are
characterised by low, 1.5–8% oxygen tensions (Fischer
& Bavister 1993), an environment which has been shown
to improve the in vitro embryo development of several
species including human (Dumoulin et al.1999,
Petersen et al. 2005,Kovacic & Vlaisavljevic 2008),
mouse (Orsi & Leese 2001) and bovine (Thompson et al.
1990,Olson & Seidel 2000). Moreover, mouse embryos
cultured at 5% oxygen rather than 20% oxygen
displayed a global gene expression pattern which more
closely resembled their in vivo counterparts (Rinaudo
et al. 2006). Harvey et al. (2004), also observed that
culture under 2% oxygen rather than atmospheric
oxygen significantly increased the proportion of ICM
q2010 Society for Reproduction and Fertility DOI: 10.1530/REP-09-0300
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via
This is an Open Access article distributed under the terms of the Society for Reproduction and Fertility’s Re-use Licence which permits unrestricted non-commercial use,
distribution, and reproduction in any medium, provided the original work is properly cited.
cells compared with trophectoderm cells, findings
consistent with pluripotent cells displaying increased
proliferation when cultured under reduced oxygen
tensions. Thus, mimicking the in vivo, physiological
oxygen concentration may be beneficial for the propa-
gation of hES cells in vitro. This is supported by data from
rat mesenchymal and neural crest stem cells, where
culture under physiological oxygen tensions showed an
increase in the rate of proliferation compared with those
maintained at 20% oxygen (Morrison et al. 2000,Lennon
et al. 2001). These combined data highlight the benefit
that culture under reduced oxygen tension has on the
ability to expand scarce stem/precursor cell populations.
In terms of hES cells, atmospheric oxygen remains
the culture environment of routine use. There is
emerging evidence to suggest that reducing the oxygen
concentration towards physiological levels is beneficial
for the in vitro maintenance of hES cells in terms of
decreasing the amount of spontaneous differentiation,
supporting self-renewal (Ezashi et al. 2005,Ludwig
et al. 2006,Westfall et al. 2008), and reducing spon-
taneous chromosomal aberrations (Forsyth et al. 2006).
Thus, while hES cells can be maintained under
atmospheric oxygen tensions, lowering the oxygen
tension to 2–5% appears beneficial for the propagation
of a highly proliferative, pluripotent population of
cells. However, controversy still remains as a recent
report suggests that there are no significant advantages
of culturing hES cells under reduced oxygen tension
(Chen et al. 2009).
Upon exposure to hypoxic conditions, cells mount a
physiological response to ensure sufficient levels for
oxygen-dependent processes. This response is regulated
by hypoxia inducible factors (HIFs) which regulate
the expression of over 200 genes including those
involved in erythropoiesis, apoptosis and proliferation
(Semenza 2000). HIFs are transcription factors consisting
of three oxygen-dependent asubunits: HIF1A, HIF2A
(also known as EPAS1) and HIF3A, and a constitutively
expressed bsubunit, HIF1B (also known as ARNT).
Under atmospheric oxygen tensions, HIF1A protein is
rapidly degraded due to hydroxylation by prolyl
hydroxylase proteins (PHDs). These hydroxylated
proteins are then recognised by the Von Hippel–Lindau
(VHL) protein which targets them for proteosomal
degradation. Under hypoxic conditions, PHDs are
unable to hydroxylate HIF1A which therefore cannot
be targeted for degradation by the VHL protein (Semenza
2003). Stabilised HIF1A subunits translocate from the
cytoplasm to the nucleus where they bind with HIF1B to
activate target genes (Wenger 2002). HIF1A, the first
HIF-asubunit described, is thought to be the global
regulator of the hypoxic response (Semenza & Wang
1992). However, the contribution of HIF2A and HIF3A,
which are believed to be regulated in a similar manner
(Ivan et al. 2001,Masson et al. 2001), remain to be fully
characterised. All three asubunits share many sequence
similarities (reviewed by Lee et al. (2004)); they all
possess the Per, Arnt/HIF1B and Sim domain essential for
binding to HIF1B and the oxygen-dependent
degradation domain, the target for degradation under
normoxic conditions. HIF3A differs from HIF1A and
HIF2A as it lacks the C-terminal activation domain
required for co-activator binding. Thus, HIF3A is unable
to recruit co-transcriptional regulators and basal tran-
scriptional machinery to gene targets. HIF-asubunits
have been found to activate genes that contain the
hypoxia response element sequence located in the
promoter region of hypoxia responsive genes (Wenger
2002). HIF2A can target this sequence independently of
HIF1A, suggesting that they have functionally diverse
roles. For example, HIF1A, originally thought to be the
main oxygen sensing subunit, predominantly regulates
glycolytic genes (Hu et al. 2003), whereas HIF2A is
the main regulator of hypoxia-induced erythropoietin
in tissues which express both HIF1A and HIF2A
(Warnecke et al. 2004,Rankin et al. 2007). HIF2A has
also been shown to promote cell cycle progression in
hypoxic renal clear cell carcinoma cells (Gordan et al.
2007). To date, little is known about which genes are
targeted by HIF3A.
The functional significance of HIF genes has been
highlighted using targeted gene inactivation. HIF1A
mice are non-viable, displaying developmental arrest by
E9.0 with significant mesenchymal cell death and
impaired vascular development (Kotch et al. 1999).
HIF1B null mice are embryonic lethal by E10.5,
displaying yolk sac and placental deficiencies and
decreased numbers of haematopoietic progenitors
(Nishi et al. 2004). HIF2A
mice develop severe
vascular defects and show developmental arrest
between E9.5 and E12.5 with variability depending on
the genetic background (Compernolle et al. 2002,Nishi
et al. 2004).
In mouse ES cells, both HIF1A and HIF2A are
expressed but HIF1A appears to be central to regulating
hypoxic responses, since it targets many oxygen-
dependent genes that are not regulated by HIF2A
(Hu et al. 2006). However, HIF2A has been found to
be a direct upstream regulator of POU5F1 (OCT4) in
mouse ES cells, suggesting that HIF2A is involved in the
regulation of stem cell maintanence (Covello et al.
2006). In hES cells, the mechanism of hypoxic regulation
appears to differ since HIF1A protein is only transiently
expressed for w48 h following exposure to low oxygen
tension (Cameron et al. 2008).
While there has been controversy in the literature, this
study aims to prove that hES cell culture is improved
under physiological oxygen concentrations compared
with ambient, atmospheric oxygen tension. We investi-
gate the effect of oxygen tension on hES cell morphology,
pluripotency and proliferation and determine the
functional significance and potential hierarchy of HIFs
in regulating these hypoxic responses.
86 C E Forristal and others
Reproduction (2010) 139 85–97
Cell morphology
Under hypoxic conditions, morphology was altered
(Fig. 1a); colonies appeared more compact with clearly
defined borders. At 20% oxygen, the cells began to show
peripheral, spontaneous differentiation by day 4 post-
passage, characterised by enlarged, flattened cells with
translucent cytoplasm (Fig. 1aF). As a consequence, hES
cells cultured at 20% oxygen required more frequent
passaging to limit spontaneous differentiation. In con-
trast, hES cells maintained at 5% oxygen were passaged
due to space constraints, rather than to remove
differentiated areas. Thus, colony diameter was w50%
larger at 5% oxygen than those cultured under 20%
oxygen on each day post-passage (P!0.01–P!0.001;
Fig. 1b). In total, hES cells were maintained under both
5% and 20% oxygen for w10–12 months, or 36–40
passages and the same morphological differences were
maintained throughout this period. To further quantify
the increase in colony size, equal numbers of cells were
seeded on Matrigel coated plates on day 0 under both
oxygen tensions and total hES cell number determined
on each subsequent day post-passage. A significant
increase in cell number was apparent by 48 h post-
passage under 5% oxygen compared with 20% oxygen
and was maintained on days 3 and 4 (P!0.001; Fig. 1c).
Using Ki67 labelling of hES cells on Matrigel on day 3
post-passage, there was a dramatic decrease in the
number of proliferating cells at 20% oxygen compared
with 5% oxygen with w50% of hES cells cultured under
20% oxygen being Ki67 positive compared with virtually
Figure 1 (a) Phase contrast images of Hues 7 hES colonies cultured on MEFs under 5% (A, C and E) and 20% (B, D and F) oxygen on days 2 (A and B),
3 (C and D) and 4 (E and F) post-passage. Area of differentiation highlighted with arrow. Scale bar, 500 mm. (b) Average maximum hES cell colony
diameter at 5 and 20% oxygen on days 2–4 post-passage **P!0.01, ***P!0.001 significantly different from 5% oxygen (nZ6 for each day).
(c) Average hES cell number at 5% and 20% oxygen on days 1–4 post-passage. Cell numbers were normalised on day 0 by passaging equal numbers
of cells under both oxygen tensions. ***P!0.001 significantly different from 5% oxygen (nZ21 for each day). (d) Ki67 (green) and DAPI (blue)
labelling of Hues 7 hES cells at 5% (A–C) and 20% (D–F) oxygen on day 3 post-passage, phase contrast (C and F). Negative control, secondary
antibody only (G). Scale bar, 250 mm.
Hypoxic regulation of human embryonic stem cells 87 Reproduction (2010) 139 85–97
all cells at 5% oxygen (Fig. 1d). Similar results were
obtained for hES cells cultured on mouse embryonic
fibroblasts (MEFs; data not shown).
Pluripotency marker expression
To investigate the effect of oxygen tension on the
expression of pluripotency markers, hES cells cultured
under either 20% or 5% oxygen were obtained on day 3
post-passage, prior to the onset of any overt differentiation
observed in cells cultured at 20% oxygen. There was a
significant (P!0.05) reduction in POU5F1 (80G20%),
SOX2 (75G18%) and NANOG (60G10%) mRNA
expression in cells maintained at 20% oxygen compared
with those cultured at 5% oxygen (Fig. 2a). Although
immunocytochemistry detected similar immunoreactiv-
ity for POU5F1, SOX2, TRA-1-60 and TRA-1-81 (Fig. 2b),
using western blotting, POU5F1 was significantly
decreased by w40% in hES cells cultured at 20% oxygen
compared with 5% oxygen (P!0.01; Fig. 2c and d).
HIF expression
Real-time RT-PCR was used to investigate the effect
of environmental oxygen tension on the differential
expression of HIF1A,HIF1B,HIF2A and HIF3A
subunits. All HIFs were expressed in hES cells cultured
under both 20% and 5% oxygen, but there was no
significant difference in the mRNA expression of the
asubunits with respect to oxygen tension. Surprisingly,
HIF1B was significantly upregulated under hypoxic
conditions (Fig. 3a).
Western blotting was performed on day 3 post-passage
to quantify the level of HIF protein expression in hES
cells (Fig. 3b). HIF1A was not expressed following long-
term culture under 5% or 20% oxygen tension and there
was no significant difference in the expression of HIF1B.
However, HIF2A and HIF3A were significantly upregu-
lated under 5% oxygen compared with 20% oxygen
(P!0.05; Fig. 3c).
Using immunocytochemistry, HIF1A was found to be
only transiently expressed being present in the nucleus of
hES cells cultured under 5% oxygen for 48 h but absent
after long-term culture under hypoxic conditions
(Fig. 3d). HIF1A was also not expressed in hES cells
cultured under 20% oxygen. HIF2A and HIF3A both
displayed a largely cytoplasmic expression following
48 h of culture at 5% oxygen. However, following
long-term hypoxic culture they both displayed a
predominantly nuclear localisation. Under 20% oxygen,
HIF2A and HIF3A remained cytoplasmic. HIF1B, which
is known to be constitutively expressed, was present in
the nuclei of hES cells when cultured under both 20%
and 5% oxygen tensions. It was therefore not necessary
to investigate the localisation of HIF1B in hES cells
cultured in 5% oxygen for 48 h.
Figure 2 (a) mRNA expression of POU5F1,SOX2 and NANOG cultured under 5% and 20% oxygen using relative quantification real-time RT-PCR.
All data have been normalised to UBC and to 1 for 5% oxygen **P!0.01 (nZ6). (b) Immunocytochemistry of POU5F1 (A and E), SOX2 (B and F),
TRA-1-60 (C and H) and TRA-1-81 (D and G) of hES cells cultured under 5% (A–D) and 20% (E–H) oxygen. Scale barZ100 mm. (c) Representative
western blot of POU5F1 expression in hES cells cultured under 5% and 20% oxygen. (d) Quantification of POU5F1 western blots. Data were
normalised to b-actin and to 1 for 5% oxygen. **P!0.01 significantly different to 5% oxygen (nZ3).
88 C E Forristal and others
Reproduction (2010) 139 85–97
Figure 3 (a) mRNA expression of HIFs in hES cells at 5% and 20% oxygen. All data have been normalised to UBC and to 1 for 20% oxygen. *P!0.05
significantly different to 20% oxygen (nZ3). (b) Representative western blots of HIFs after long-term culture at 5% or 20% oxygen (C) represents
positive control protein. (c) Quantification of HIF western blots. Data were normalised to b-actin and to 1 for 20% oxygen. *P!0.05 significantly
different to 20% oxygen (nZ3). (d) Protein expression of HIFs by immunocytochemistry in hES cells cultured under 5% oxygen for 48 h (5% 48 h;
A and B, G and H, M and N), 5% oxygen for long-term (5% LTZmore than three passages; C and D, I and J, O and P, S and T) and 20% oxygen
(E and F, K and L, Q and R, U and V). HIF1A (green; A–F), HIF2A (green; G–L), HIF3 (red; M–R) and HIF1A (green; S–V). DAPI (blue). Negative
controlsZFITC secondary only (W) and Texas Red secondary only (X). Scale barZ40 mm.
Hypoxic regulation of human embryonic stem cells 89 Reproduction (2010) 139 85–97
HIF knockdown
Following siRNA real-time RT-PCR confirmed an 83%,
86% and 90% knockdown of HIF1A,HIF2A and HIF3A
respectively compared with transfection control siRNA
(Fig. 4a, c and e). Initial studies examined the effect of
knocking down individual HIF-asubunits for 48 h.
When HIF2A was silenced HIF1A mRNA expression
was not affected (Fig. 4a). However, the knockdown of
HIF3A significantly upregulated HIF1A mRNA
expression (P!0.05, Fig. 4a). As expected, HIF1A
protein was not expressed in hES cells maintained at
5% oxygen and was not induced by silencing HIF2A.
Interestingly, HIF1A protein was induced when
HIF3A was silenced (Fig. 4b). When both HIF2A and
HIF3A were silenced, HIF1A was expressed but at a
significantly reduced level compared with when HIF3A
alone was knocked down (P!0.001, Fig. 4b).
When HIF1A was silenced, HIF2A mRNA expression
were unaffected. However, there was a significant
reduction of HIF2A mRNA (P!0.01) and protein
(P!0.05) expression observed when HIF3A was
silenced (Fig. 4c and d).
HIF3A mRNA (Fig. 4e) and protein (Fig. 4f) expression
were found to be significantly upregulated when HIF1A
and HIF2A were knocked down independently.
Effect of HIFs on pluripotency marker expression
Using real-time RT-PCR there was a significant reduction
in POU5F1 (Fig. 5a; P!0.001), SOX2 (Fig 5c; P!0.001,
P!0.05) and NANOG (Fig. 5e; P!0.001) when HIF2A
and HIF3A were silenced independently. As expected,
silencing of HIF1A did not alter the mRNA expression of
POU5F1,SOX2 and NANOG compared with transfection
control siRNA (Fig. 5a, c and e). At the protein level,
POU5F1, SOX2 and NANOG were significantly reduced
when HIF2A (P!0.001) and HIF3A (P!0.05) were
silenced (Fig. 5b, d and f).
Effect of HIF expression on hES cells morphology
Silencing HIF1A or HIF3A did not affect hES cell
morphology (Fig. 6a) and following knockdown these
cells could be maintained in culture, remaining TRA-1-
60 and POU5F1 positive 48 h (Fig. 6b) and two passages
(Fig. 6c) post transfection. Moreover, HIF1A and HIF3A
silenced colonies contained similar levels of SSEA1
expression as the transfection controls (Fig. 6b).
However, when HIF2A expression was knocked down
colonies appeared to have less clearly defined borders
and large areas of differentiation (Fig. 6a). These cells
failed to maintain pluripotency, being SSEA1 positive
Figure 4 mRNA expression levels of (a) HIF1A,
(c) HIF2A and (e) HIF3A when each HIF-aisoform
was silenced in hES cells cultured under 5% oxygen
for 48 h. All data has been normalised to UBC and
to 1 for the transfection control. *P!0.05,
**P!0.01, ***P!0.001 significantly different to
transfection control (nZ3). (b) Representative
HIF1A protein expression and quantification when
HIF2A and HIF3A were silenced separately and
simultaneously. *** P!0.001 significantly different
to HIF3A siRNA. Representative (d) HIF2A and
(f) HIF3A protein expression and quantification
when each HIF-aisoform was silenced *P!0.05,
**P!0.01, *P!0.001 significantly different to
transfection control (nZ3). Protein was collected
for all samples 48 h post-transfection.
90 C E Forristal and others
Reproduction (2010) 139 85–97
and displaying large areas that were TRA-1-60 and
POU5F1 negative (Fig. 6b) and failed to proliferate
during culture. Consequently, HIF2A silenced hES cells
that were cultured for two passages post transfection
were negative for TRA-1-60 and POU5F1 (Fig. 6c).
Double knockdowns combining HIF1A and either
HIF2A or HIF3A showed significant areas of differen-
tiation and the cells failed to form colonies (Fig. 6a).
When HIF2A and HIF3A were both silenced simul-
taneously, hES cells were capable of forming colonies
but possessed large areas of differentiation. In contrast,
triple HIF-aknockdown hES cells did not form colonies
and did not survive in culture.
Effect of HIF expression on hES cells proliferation
At 48 h post-transfection, hES cell number (Fig. 7a) and
colony size (Fig. 7b) were significantly (P!0.001)
reduced by w30% when HIF2A expression was knocked
down and displayed a further decrease (P!0.01) when
HIF2A and HIF3A were silenced simultaneously. There
was no additional increase in the diameter of HIF2A
silenced cells 72 h post-transfection (data not shown).
Colony size and cell number were unaffected by HIF1A
or HIF3A knockdown. It was not possible to measure the
colony size of double knockdown combinations of
HIF1A and HIF2A or HIF1A and HIF3A or the triple
knockdown due to the absence of colony formation
occurring in these populations. Virtually all cells were
positive for Ki67 when either HIF1A or HIF3A expression
was silenced whereas Ki67 expression decreased to
w85% when HIF2A was knocked down (P!0.001,
Fig. 7c and d).
There is emerging evidence to suggest that culture under
more physiological oxygen conditions (2–5% oxygen)
decreases the amount of spontaneous hES cell differen-
tiation (Ezashi et al. 2005,Westfall et al. 2008). Similarly,
we found that spontaneous differentiation was apparent
by day 4 post-passage in hES cells cultured under
atmospheric conditions, whereas cells cultured at 5%
oxygen maintained pluripotency. As a result of this, hES
cells cultured under 20% oxygen required more frequent
passaging prior to confluency to remove differentiated
areas, whereas hES cells maintained at 5% oxygen were
only passaged due to space constraints on the plate.
These findings suggest that environmental oxygen
tension has a critical role in the maintenance of hES
Figure 5 mRNA expression levels of (a) POU5F1,
(c) SOX2 and (e) NANOG when HIF-asubunits
were silenced in hES cells cultured under 5%
oxygen for 48 h. Data normalised to UBC and to 1
for the transfection control. *P!0.05, ***P!0.001
significantly different to transfection control (nZ3).
Representative (b) POU5F1, (d) SOX2 and
(f) NANOG protein expression and quantification
by western blotting when HIF-asubunits were
silenced. *P!0.05, ***P!0.001 significantly
different to transfection control (nZ3). Protein was
collected for all samples 48 h
Hypoxic regulation of human embryonic stem cells 91 Reproduction (2010) 139 85–97
cell pluripotency. To investigate differences between the
two oxygen concentrations, results were obtained on day
3 post-passage, prior to overt cell differentiation.
Culture at 5% oxygen, whether on MEFs or feeder-free
on Matrigel, increased the rate of cell proliferation which
agrees with data from Ludwig et al. (2006), producing
significantly larger hES cell colonies. This may be due to
the observed increase in NANOG expression levels,
since it has recently been shown that NANOG regulates
S-phase entry in hES cells and that overexpression of
NANOG significantly increases proliferation by binding
to regulatory regions of CDK6 and CDC25A, two
important cell cycle regulators (Neganova et al. 2009,
Zhang et al. 2009). Similar increased rates of prolifer-
ation have also been observed in many other primitive
populations including mesenchymal stem cells and
neural progenitor cells when cultured under low oxygen
tensions (Grayson et al. 2007,Zhao et al. 2008). Data
from mesenchymal stem cells suggest the involvement of
HIFs, specifically HIF2A (Grayson et al. 2007). This is
supported in hypoxic carcinoma cells where HIF2A has
been shown to promote cell cycle progression (Gordan
et al. 2007).
In agreement with Forsyth et al. (2008) and Westfall
et al. (2008), there was no significant increase in the
mRNA expression of the HIF-asubunits under hypoxia
suggesting that altered protein levels are due to post-
transcriptional regulation as found in other cell types
(Huang et al. 1998,Lang et al. 2002). However, in
contrast to Westfall et al. (2008),HIF1B mRNA
expression was upregulated under hypoxia which may
reflect differences in both the methodology and cell lines
used. To date, most research has focused on the HIF1A
subunit which is thought to be the master regulator of the
Figure 6 (a) Representative phase contrast images
of hES cells cultured under 5% oxygen 48 h after
transfection with HIF-asiRNA. Controls contain
the same volume and concentration of transfection
reagent and AllStars control siRNA as each of the
knockdowns. Scale barZ100 mm. (b) Protein
expression of pluripotency markers TRA-1-60
(A, G, M and S), merged with DAPI (B, H, N and T),
POU5F1 (C, I, O and U), merged with DAPI (D, J, P
and V) and differentiation marker SSEA1 (E, K, Q
and W), merged with DAPI (F, L, R and X) in hES
cells 48 h following siRNA transfection of HIF-a
subunits. Scale barZ100 mm. (c) Phase contrast
and protein expression of POU5F1 and TRA-1-60
in hES cells two passages following HIF-agene
silencing. Scale barZ100 mm.
92 C E Forristal and others
Reproduction (2010) 139 85–97
hypoxic response (Mazure et al. 2004). However, our
data suggests this is not the case, at least in hES cells
since HIF1A was only transiently expressed in the
nucleus for w48 h following exposure to hypoxia.
These findings are in agreement with recent data from
Cameron et al. (2008) using a 2% environmental oxygen
tension, although contrary to those of Westfall et al.
(2008), who found HIF1A protein to be expressed in hES
cells after 10 days of culture under both 5 and 20%
oxygen. The reason for this discrepancy is unknown
although HIF1A would be expected to be rapidly
degraded by PHDs under atmospheric conditions.
Moreover, the HIF1A staining observed by the latter
investigators was cytoplasmic and therefore would not
be expected to regulate the expression of hypoxia
regulated genes. Our study suggests that HIF1A may
play a role in the initial adaptation of hES cells to hypoxia
but some other factor(s) must be responsible for
maintaining the hypoxic response. We propose that
HIF2A or HIF3A, both of which are translocated from the
cytoplasm to the nucleus and are upregulated following
culture under hypoxic conditions, may take over the
initial, transient role of HIF1A. This is the first time that
HIF3A and HIF1B have been demonstrated in hES cells.
hES cell colonies cultured at 5% or 20% oxygen were
both positive for pluripotency markers albeit at a
significantly decreased level at 20% oxygen. These
results were not apparent using immunocytochemistry
and highlight the importance of performing quantitative
techniques when assessing pluripotency. The current
results suggest that although hES cells are capable of
being maintained under atmospheric conditions plur-
ipotency is reduced before the appearance of morpho-
logical differentiation. This shows that the manual
removal of differentiated cells at the time of passage, a
method often used during routine culture at 20% oxygen
is not effective in ensuring the maintenance of a highly
pluripotent population. However, culturing hES cells at
5% oxygen does not require the routine removal of
differentiated areas and can therefore be considered a
much more effective method of maintaining pluripo-
tency during long term culture. Interestingly, our data
Figure 7 Effect of silencing HIF-asubunits in hES
cells cultured under 5% oxygen 48 h following
siRNA transfection on average cell number (nZ3).
Cell numbers were normalised on day 0 by
passaging an equal number of cells. (b) Effect of
HIF-asilencing 48 h following siRNA transfection
on average maximum colony diameter (nZ6).
***P!0.001 significantly different from
transfection control. Bars with the same superscript
are significantly different;
P!0.01. (c) Ki67
(green) and DAPI (blue) labelling of HIF-asilenced
hES cells and transfection control. Scale
barZ100 mm. (d) Percentage of HIF-asilenced
Ki67 positive hES cells. ***P!0.001 significantly
different from transfection control.
Hypoxic regulation of human embryonic stem cells 93 Reproduction (2010) 139 85–97
shows that hES cells cultured at 5% oxygen have an
increased expression of SOX2, NANOG and POU5F1
compared with cells cultured under 20% oxygen even
on day 3 post-passage, before these cells appear to be
morphologically differentiated. These data are in agree-
ment with similar POU5F1 results obtained by Ludwig
et al. (2006). However, they are contrary to those of
Forsyth et al. (2008) and Westfall et al. (2008), who both
observed no difference in the expression SOX2,POU5F1
and NANOG between hES cells cultured at 2 and 4%
oxygen respectively compared with atmospheric oxygen
tensions. However, the latter investigators did show that
POU5F1-regulated genes such as left-right determina-
tion factor 2 and fibroblast growth factor receptor 2
(FGFR2) were down-regulated under atmospheric oxy-
gen tensions, suggesting that although mRNA expression
of pluripotency markers was not reduced under 20%
oxygen, their downstream targets display decreased
Taken together, our data suggest that either low oxygen
tension is beneficial for the maintenance of pluripotency
or that cultures at 5% oxygen contain a greater
proportion of undifferentiated cells (or both). In favour
of the former, an increase in POU5F1 mRNA and protein
has also been observed in mouse ES cells when HIF2A
expression was increased using a genetic knock-in
strategy (Covello et al. 2006). It is tempting to speculate
that HIF2A regulates the long-term response of hES cells
to reduced oxygen but this should be interpreted with
caution as the little studied HIF3A is also differentially
regulated and may also be fundamental to the hypoxic
Our concept is illustrated in Fig. 8, which summarises
our data on HIF expression and localisation in hES cells.
We suggest that HIF1A and HIF3A are able to regulate
each other’s expression because when HIF1A is knocked
down HIF3A is upregulated and when HIF3A is silenced
HIF1A expression increases. Thus, HIF1A may be key in
the initial adaptation of hES to the hypoxic environment,
but when this expression is lost after w48 h in hypoxic
conditions, HIF3A is upregulated, translocates to the
nucleus where it is transcriptionally active, and takes
over from HIF1A in maintaining the long-term response
to hypoxia. In addition, HIF3A appears to be an
upstream regulator of HIF2A since when HIF3A
expression is lost HIF2A expression significantly
decreases. Thus, it appears that HIF3A regulates the
expression of the other HIF-aisoforms in hES cells. The
precise role of HIF3A has previously been unknown but
data from the human kidney suggests that it may be a
negative regulator of HIF-agene expression (Hara et al.
2001) and therefore might have a role in the down-
regulation of HIF1A in hES cells exposed to long-term
hypoxia. This is supported by data from mouse ES cells
where hypoxic expression of HIF1A was shown to
suppress LIF–STAT signalling leading to the inhibition
of self renewal and the promotion of cell differentiation
(Jeong et al. 2007).
HIF3A is also able to regulate downstream targets
through the upregulation of HIF2A in hES cells. Our data
show that HIF2A is an upstream regulator of POU5F1
expression in hES cells, which agrees with data from the
mouse (Covello et al. 2006). Moreover, we believe this is
the first description of HIF2A regulating the expression of
SOX2 and NANOG and highlights the importance of
HIF2A in the maintenance of hES cells under hypoxic
conditions. Since HIF3A positively regulates HIF2A, a
decrease in HIF3A expression results in a decrease in
HIF2A expression. Thus, the decrease in POU5F1, SOX2
and NANOG mRNA and protein expression observed in
HIF3A knockdowns may be a result of loss of HIF2A, not
HIF3A. HIF1A does not appear to affect hES cell
pluripotency or morphology.
As well as maintaining pluripotency, HIF2A was found
to regulate proliferation in hES cells with a significant
decrease in both cell number and colony size occurring
when HIF2A expression was silenced. This may be due
to the reduced NANOG expression observed when
HIF2A was silenced since a decrease in NANOG has
been found to delay entry into S-phase of the cell cycle,
thus decreasing proliferation (Zhang et al. 2009).
Colony morphology was severely affected when two
HIF-asubunits were silenced simultaneously, with a
combination of HIF1A and either HIF2A or HIF3A
being non-viable in culture. This may be due to HIF2A
being instrumental in controlling pluripotency and
HIF3A in regulating the expression of HIF2A. Not
surprisingly, therefore, the simultaneous loss of HIF1A,
HIF2A and HIF3A results in these cells being unable to
survive hypoxia. hES cells in which both HIF2A and
HIF3A were silenced formed viable colonies but
contained large areas of differentiation. The survival
of these cells may be due to compensation by HIF1A,
which was switched back on at the protein level. This
highlights the importance of HIF1A in maintaining the
hypoxic response when the other asubunits are not
Figure 8 A schematic representation of the HIF-asubunit localisation,
regulation and effect on proliferation in hES cells cultured under
atmospheric and hypoxic oxygen tensions.
94 C E Forristal and others
Reproduction (2010) 139 85–97
active as observed in the initial adaptation of hES cells
to hypoxia.
In conclusion, these studies demonstrate that the
culture of hES cells is preferential under a reduced
oxygen environment. In contrast to other cell types,
HIF1A is only responsible for the initial adaptation of
cells to hypoxia, whereas HIF2A regulates the long-term
hypoxic response by controlling hES cell pluripotency
and proliferation. HIF3A acts to regulate the expression
level of both HIF1A and HIF2A. These data provide a
greater understanding of the mechanisms which regulate
hES cell function and the pluripotent state.
Materials and Methods
Culture of hES cells
Hues-7 hES cells (D Melton, Howard Hughes Medical
Institute/Harvard University) were maintained on g-irradiated
MEFs in knockout (KO) DMEM (Invitrogen) supplemented with
10% KO serum replacement (Invitrogen), 1 mM L-glutamine,
50 mM b-mercaptoethanol, 1% non-essential amino acids,
10 ng/ml basic FGF factor (Peprotech Ltd, London, UK) and
100 mg/ml penicillin/streptomycin before being transferred on
to plates pre-coated with Matrigel (BD Biosciences, San Diego,
CA, USA), in medium that had been cultured over-night on
g-irradiated MEFs. hES cells were initially cultured under
atmospheric (w20%) oxygen before half were transferred into
5% O
and balanced nitrogen. Unless stated, cells
were cultured at 5% oxygen for a minimum of three passages
prior to use.
Quantitative real-time RT-PCR
mRNA was isolated from hES cells cultured on Matrigel
on day 3 post-passage using TriReagent (Sigma) and RNA
(2 mg) was reverse transcribed into cDNA using Moloney
murine leukaemia virus reverse transcriptase (Promega). The
standard curve method of relative quantification real-time
PCR was performed using Applied Biosystems reagents
in 20 ml reactions containing 4 mgofcDNA,10ml2!
Taqman Universal PCR Master Mix, 1 ml Probes and Primer
Mix (HIF1A: Hs00936368_m1; HIF1B: Hs00231048_m1;
HIF2A: Hs01026142_m1; HIF3A: Hs00541709_m1; POU5F1:
Hs01895061_u1; NANOG: Hs02387400_g1; SOX2: Hs0060
2736_s1; ubiquitin C (UBC): Hs00824723_m1) using a 7500
Real-Time PCR System. The following conditions were used;
50 8C for 2 min, 95 8C for 10 min, 45 cycles at 95 8C for 15 s
and 60 8C for 1 min. Placental cDNA (0–10 ng) was used to
create a standard curve for each gene of interest as well as
for UBC, used as an endogenous control. All target transcripts
were analysed in duplicate and normalised to UBC.
Western blotting
hES cells cultured on Matrigel were rinsed with ice-cold PBS
and treated with ice-cold radio immunoprecipitation assay
lysis buffer (50 mM Tris–HCl (pH 7.4), 1% nonyl
phenoxylpolyethoxylethanol, 0.25% Na-deoxycholate and
complete protease inhibitors (Roche) supplemented with
1 mM phenylmethylsulphonyl fluoride, 1 mM Na
1 mM NaF) for 30 min with gentle agitation. Protein concen-
tration was quantified using the Bradford assay (Bradford 1976)
and lysates (75 mg) were resolved on an 8% SDS bisacrylamide
gel. Positive controls were run for each protein of interest;
hypoxic colorectal carcinoma cell protein for HIF1A, foetal
heart (obtained following ethical approval from the South-
ampton & South West Hampshire Local Research Ethics
Committee, under guidelines issued by the Polkinghorne
Committee) for HIF1B and NT2 cells treated with 130 mM
cobalt chloride for 4 h for HIF2A and HIF3A. The protein was
electro-transferred to a nitrocellulose membrane and blocked
in PBS containing 5% non-fat powdered milk and 0.1% Tween-
20 for 1 h at room temperature. Primary antibodies were
diluted in blocking buffer and incubated with the membrane
overnight at 4 8C; HIF1A (BD Biosciences) 1:250, HIF1B (Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA) 1:250, HIF2A
(Novus Biologicals, Cambridge, UK) 1:500, HIF3A (Santa Cruz)
1:1000, POU5F1 (Santa Cruz) 1:1000, SOX2 (Millipore,
Billerica, MA, USA) 1:000 and NANOG (Abcam, Cambridge,
UK) 1:1000. Membranes were washed and incubated with
either peroxidase labelled anti-mouse antibody (Amersham)
1:50 000, or anti-goat antibody (Sigma) 1:200 000, in blocking
buffer for 1 h at room temperature. Protein expression was
quantified relative to b-actin expression which was detected
with mouse anti-b-actin peroxidase-conjugated antibody (1:50
000; Sigma). Membranes were developed using ECL advanced
western blotting detection kit (Amersham).
hES cells cultured on Matrigel were fixed in 4% paraformalde-
hyde for 20 min. Non-specific antibody binding was blocked
with 3% donkey serum and where necessary cells were
permeabilised with 0.1% tritonX-100 for 1 h before the
addition of primary antibodies diluted in PBS and 3% donkey
serum. Primary antibodies used were TRA-1-60 (Santa Cruz)
1:100, TRA-1-81 (gift from P Andrews) 1:50, SSEA1 (Santa
Cruz) 1:100, POU5F1 1:100, SOX2 (Chemicon) 1:150, HIF1A
1:250, HIF1B 1:100, HIF2A 1:100, HIF3A 1:50, Ki67
(Novocastra Laboratories, Newcastle upon Tyne, UK) 1:100
and incubated overnight in a humidified chamber at 4 8C.
Secondary antibody staining was performed with anti-mouse
IgG conjugated-FITC 1:100, anti-mouse IgM conjugated-FITC
1:200 (Sigma) or anti-goat IgG Alexa Fluor 594 1:100 (Sigma)
for 1 h in a humidified chamber. Nuclei were labelled with
DAPI (Vecta Laboratories, Peterborough, UK).
siRNA experiments were carried out on hES cells cultured on
Matrigel under 5% oxygen for a minimum of three passages.
hES cells cultured under 5% oxygen were passaged and
incubated overnight. For each transfection 50 nM siRNA
HsHIF3A_1_HP; Qiagen) along with 12 ml HiPerfect transfec-
tion reagent (Qiagen) were mixed into 200 ml of KO-DMEM
Hypoxic regulation of human embryonic stem cells 95 Reproduction (2010) 139 85–97
and added in a dropwise manner to 1-well of a 6-well plate.
At 48 h after transfection, cells were harvested and extracts
were prepared for mRNA and protein. For HIF3A, siRNA
targeting exon 5 was used which is present in all the major
known HIF3A isoforms (Maynard et al.2003). AllStars
Negative Control (Transfection control, Qiagen) siRNA that
has no homology to any known mammalian gene was used
as a negative control for each transfection. For double and
triple knockdowns 50 nM siRNA and 12 ml HiPerfect
transfection reagent were added in 600 ml of KO-DMEM.
Twice or three times the volume of Allstars Negative control
was added to controls for double and triple knockdowns
respectively. hES cells were analysed for knockdown using
relative quantification real-time RT-PCR.
Statistical analysis
All data were analysed to determine whether they were
normally distributed, using the Anderson–Darling normality
test. Differences between oxygen tension and maximum
colony diameter and cell number were analysed using a
Student’s t-test. Differences in relative gene and protein
expression between cells cultured at 5 and 20% oxygen were
analysed using a 1-sample t-test. Differences between negative
control and knockdown results were analysed using a Student’s
t-test. All data represent at least three independent experi-
ments. A value of P!0.05 was considered significant. Data are
presented as meanGS.E.M.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
This work was funded by the Gerald Kerkut Charitable Trust, a
Wellome Trust Research Career Development Fellowship
(WT066492MA), the MRC (G0701153), the Wellcome Trust
in partnership with JDRF (WT074320MA). N A H receives
support from Manchester NIHR BRC.
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Received 16 July 2009
First decision 18 August 2009
Revised manuscript received 8 September 2009
Accepted 14 September 2009
Hypoxic regulation of human embryonic stem cells 97 Reproduction (2010) 139 85–97
... Global comparison of gene expression profiles of short-term (2 days) and long-term (13 days) NSC cultures derived from fetal midbrain or cortical tissue showed that initial adaptation to O 2 deficiency is distinct from maintenance of the response to physioxic culture conditions as suggested in previous reports [48,49]. ...
... Consecutively, the concordance of genes between short-term and long-term physioxia within the same culture (mesencephalon or cortex) was less than 33%, and hypoxia response genes such as Vegf, glucose transporter, or Pdk1 are less or absent in NSCs cultured in long-term physioxia. This results in a switch from affected metabolic pathways in short-term physioxic cultures to signaling pathways such as Notch signaling or Wnt signaling, which is consistent with human studies showing that the response to short-term hypoxia/physioxia differs substantially from adaptation to long-term hypoxia/ physioxia [48,49]. Intriguingly, the affected signaling pathways Notch and Wnt (only in midbrain NSCs) are key Figure 5: Comparison of differentiation-related genes in short-versus long-term physioxia of mesencephalic and cortical NSC cultures. ...
Full-text available
Oxygen is an essential factor in the cellular microenvironment with pivotal effects on neural development with a particular sensitivity of midbrain neural stem cells (NSCs) to high atmospheric oxygen tension. However, most experiments are still performed at atmospheric O2 levels (21%, normoxia), whereas mammalian brain tissue is physiologically exposed to substantially lower O2 tensions around 3% (physioxia). We here performed serial Affymetrix gene array analyses to detect expression changes in mouse fetal NSCs from both midbrain and cortical tissues when kept at physioxia compared to normoxia. We identified more than 400 O2-regulated genes involved in cellular metabolism, cell proliferation/differentiation, and various signaling pathways. NSCs from both regions showed a low number but high conformity of regulated genes (9 genes in midbrain vs. 34 in cortical NSCs; 8 concordant expression changes) after short-term physioxia (2 days) with metabolic processes and cellular processes being the most prominent GO categories pointing to cellular adaption to lower oxygen levels. Gene expression profiles changed dramatically after long-term physioxia (13 days) with a higher number of regulated genes and more diverse expression patterns when comparing the two NSC types (338 genes in midbrain vs. 121 in cortical NSCs; 75 concordant changes). Most prominently, we observed a reduction of hits in metabolic processes but an increase in biological regulation and signaling pointing to a switch towards signaling processes and stem cell maintenance. Our data may serve as a basis for identifying potential signaling pathways that maintain stem cell characteristics in cortical versus midbrain physioxic stem cell niches.
... We further calculated the ratio of the protein expression levels of BAX to MCL-1 and compared them between normoxia and different time points upon hypoxia (1% O 2 ) induction. We found that BAX/MCL-1 ratio was increased by hypoxia (H9: 1.79 ± 0.11, 1.59 ± 0. 18 (Fig. 5a). These findings suggest that BAX/MCL-1 ratio may serve as a rheostat to determine the susceptibility of hPSCs to undergo apoptosis upon hypoxia (1% O 2 ) exposure. ...
... Moreover, it has become evident that embryonic stem cells frequently occupy hypoxic niches and low O 2 levels (generally < 5% O 2 ) regulate their differentiation 4 . In this regard, several reports indicate that the culture of hPSCs at 2-5% O 2 is advantageous for their maintenance in terms of reduced spontaneous differentiation, improved proliferation, and increased expression of crucial pluripotent markers 8,18,[26][27][28][29] . Contrarily, in the absence of O 2 or severe hypoxia, most cell types undergo cell death. ...
Full-text available
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (hPSCs) that can differentiate into a wide range of specialized cells. Although moderate hypoxia (5% O2) improves hPSC self-renewal, pluripotency, and cell survival, the effect of acute severe hypoxia (1% O2) on hPSC viability is still not fully elucidated. In this sense, we explore the consequences of acute hypoxia on hPSC survival by culturing them under acute (maximum of 24 h) physical severe hypoxia (1% O2). After 24 h of hypoxia, we observed HIF-1α stabilization concomitant with a decrease in cell viability. We also observed an increase in the apoptotic rate (western blot analysis revealed activation of CASPASE-9, CASPASE-3, and PARP cleavage after hypoxia induction). Besides, siRNA-mediated downregulation of HIF-1α and P53 did not significantly alter hPSC apoptosis induced by hypoxia. Finally, the analysis of BCL-2 family protein expression levels disclosed a shift in the balance between pro- and anti-apoptotic proteins (evidenced by an increase in BAX/MCL-1 ratio) caused by hypoxia. We demonstrated that acute physical hypoxia reduced hPSC survival and triggered apoptosis by a HIF-1α and P53 independent mechanism.
... Therefore, injury results in the disturbance of the cell microenvironment at the wound site, creating a hypoxic niche that embryonic stem cells (ESCs) and adult stem cells rely on for self-renewal (28). Previous studies have shown that hypoxic conditions can promote the self-renewal and maintenance of pluripotency in embryonic and other types of stem cells (29)(30)(31). Under hypoxia, human ESCs control HIF2α through glycolytic flux, thereby upregulating the expression of C-terminal binding proteins 1 and 2 to sustain self-renewal (29). ...
... Under hypoxia, human ESCs control HIF2α through glycolytic flux, thereby upregulating the expression of C-terminal binding proteins 1 and 2 to sustain self-renewal (29). In addition, HIF2α is closely associated with the pluripotency regulatory network of genes, such that HIF2α knockdown leads to the downregulation of octamer-binding transcription factors 3 and 4 (OCT3/4), sex determining region Y-box 2 (SOX2) and NANOG (30). In vitro reprogramming experiments have also revealed that hypoxia can promote the expression of pluripotent factors, such as OCT3, OCT4, SOX2, NANOG and Krüppel-like factor 4 (KLF4), to increase the efficiency of reprogramming, and can reduce the number of transcription factors required for reprogramming (31). ...
Accumulating evidence has shown that cell dedifferentiation or reprogramming is a pivotal procedure for animals to deal with injury and promote endogenous tissue repair. Tissue damage is a critical factor that triggers cell dedifferentiation or reprogramming in vivo. By contrast, microenvironmental changes, including the loss of stem cells, hypoxia, cell senescence, inflammation and immunity, caused by tissue damage can return cells to an unstable state. If the wound persists in the long‑term due to chronic damage, then dedifferentiation or reprogramming of the surrounding cells may lead to carcinogenesis. In recent years, extensive research has been performed investigating cell dedifferentiation or reprogramming in vivo, which can have significant implications for wound repair, treatment and prevention of cancer in the future. The current review summarizes the molecular events that are known to drive cell dedifferentiation directly following tissue injury and the effects of epigenetic modification on dedifferentiation or reprogramming in vivo. In addition, the present review explores the intracellular mechanism of endogenous tissue repair and its relationship with cancer, which is essential for balancing the risk between tissue repair and malignant transformation after injury.
... The defined xeno-free cGMP culture media, freezing media and serum replacements are also available on the market. These defined cGMP products, together with the hypoxia that supports the pluripotent state of hESCs, are the basis of a high-quality clinical-grade hESC culture [17,28,29]. ...
... Derivation and culture conditions are undeniably the main factors that have an impact on the quality of hESCs. We chose a hypoxic environment (5% O2) for both derivation and culture, because it has been proven that hypoxia supports the pluripotency of hESCs [29]. Moreover, the embryos that were used for the derivation were cultured under hypoxia as the ESHRE guidelines also recommend a low oxygen concentration for blastocyst culture [35]. ...
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Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.
... Physiological oxygen tensions vary across organs and tissue components and typically range from 1-14 % in vivo for most tissues depending on the distance away from the vascular system [30]- [32]. The physiological normoxia (physoxia) environment for ESCs typically ranges from 2 to 5% [33]. Oxygen affects epigenetic modifications, and epigenetics is essential for initiation of hypoxic response pathways [34]. ...
Telomere repeats at the ends of human chromosomes protect chromosomes from degradation, and telomerase has a prominent role in telomere maintenance. Telomerase also affects cell proliferation, DNA replication, differentiation, and tumourigenesis. TERT (telomerase reverse transcriptase enzyme) is the catalytic subunit of telomerase and is critical for enzyme activity. TERT promoter mutations and promoter methylation are strongly associated with increased telomerase activation in cancer cells. Notably, TERT and telomerase are downregulated in stem cells during their differentiation. Therefore, the link between differentiation and telomerase provides a valuable tool for studying the epigenetic regulation of TERT enzyme. Oxygen tension affects several cellular behaviours including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen tension in driving promoter modifications of the TERT gene in embryonic stem cells (ESCs) is poorly understood either in vitro or in vivo. We adopted a monolayer ESCs differentiation model to explore the role of low, physiological, oxygen (physoxia) in the epigenetic regulation of telomerase and associated genes, including TERT, DNMTs, and HDACs. Cells were cultured in either air, a 2% O2 incubator, or a 2% O2 oxygen workstation to provide a fully defined 2% O2 environment. Pre-gassed media (pre-conditioned to 2% O2 in a HypoxyCool unit) was used in all 2% O2 experimentation. As anticipated, physoxia culture increased the proliferation rate and stemness of ESCs and a slower onset of differentiation in physoxia was evident. Further, downregulated TERT expression was correlated to reduced telomerase activity during differentiation. TERT expression and telomerase activity remained significantly elevated in physoxia during differentiation. A substantial increase in TERT promoter methylation levels was noted during differentiation. Chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B CHiP demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B binding to the promoter. In conclusion, we have demonstrated that DNMT3B can directly bind TERT promoter, change its methylation levels, and contribute to regulation of telomerase activity.
... It participates in the regulation of cell survival, apoptosis, energy metabolism, oxygen homeostasis, and other cellular functions by affecting the expression of more than 200 genes. HIF-1α is an early oxygen regulator that is more sensitive to hypoxia, and it is stimulated by hypoxia or other cytokines [27][28][29]. However, HIF-1α is easily degraded via ubiquitin pathway in the cornea under normoxic state, and thereby its expression level is low. ...
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The hyperbaric oxygen therapy is often used in the management of acid and base burns of the eyes. However, oxygen is rarely supplied locally through goggles or face mask in ophthalmology. Therefore, in this study, we aim to investigate how oxygen delivery affects eye recovery after injury. We used a rabbit model with corneal epithelial injury to examine the effects of local oxygen supply via goggles or face mask on the recovery of cornea. A total of 75 healthy New Zealand white rabbits were randomly divided into three groups, A, B, and C, with 25 rabbits in each group. Then, on each rabbit eye (150 eyes in total), a circle of corneal epithelium with 5 mm in diameter was scraped off from the center of the cornea with a corneal epithelial scraper. Group A was given oxygen goggles every day (the oxygen flow rate was 3 L/min, once a day, 2 hours each time); group B was given nasal inhalation of oxygen every day (the oxygen flow rate was 3 L/min, once a day, 2 hours each time); and group C did not receive any treatment and was healed naturally. We found that the group A, which received oxygen supply via goggles, showed the best eye recovery. Transmission electron microscopy showed that the cornea with local oxygen supply via goggles or face mask exhibited intact capillary structure and obvious desmosome/hemidesmosome connections between cells. Moreover, the protein and RNA levels of hypoxia-related genes were lower in group A and B, suggesting that the hypoxia factor is a sensitive and early regulator in the low oxygen environment.
... Numerous studies on mouse and human embryonic stem cells (ESCs) revealed that hypoxia promotes ESC differentiation, especially towards endodermal lineages (Burr et al., 2017;Forristal et al., 2010;Houghton, 2021;Kusuma et al., 2014;Pimton et al., 2015). Hypoxia is also implemented in some protocols that model mammalian embryo development in a dish (Sozen et al., 2021;Aguilera-Castrejon et al., 2021), yet the mechanisms through which hypoxia exerts its beneficial effects are not clear. ...
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The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.
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The heart harbors a population of resident cardiac stem cells (CSC) that maintain tissue homeostasis. These cells also find application in cell-based therapy, where cardiac stem cells are transplanted as cardiospheres or cardiosphere-derived cells (CDCs) for myocardial regeneration. Cardiac stem cells are expanded in culture to obtain sufficient numbers for transplantation. On transplantation, cells home into the site of injury and mediate myocardial regeneration. Conventionally, cell cultures are carried out in ambient O 2 (21% O 2 ). Stem cells reside in niches where the O 2 level is close to 3%. Assuming that maintenance of cells in a milieu that simulates the in-situ conditions will help in the preservation of stem cell attributes, CDCs isolated from adult Wistar rats was cultured in 3% O 2 (hypoxia) and compared with CDCs maintained in 20% O 2 (normoxia). Expression of c-kit and Nkx 2.5 was higher and cardiac troponin lower in hypoxia, indicating the maintenance of an undifferentiated state. Expression of von Willebrand factor and angiogenic differentiation was higher in cultures maintained in 3% O 2 suggesting enhanced vasculogenic potential. Cellular senescence and intracellular reactive oxygen generation (ROS) were lower, and the proliferation rate was higher in CSCs maintained in 3% O 2 . Enhanced migration, proliferation, and preservation of undifferentiated state lead to the inference that, culture in low oxygen tension can increase the yield of healthy cardiac stem cells for myocardial regeneration.
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Over the past 2 decades, mesenchymal stem cells (MSCs) have attracted a lot of interest as a unique therapeutic approach for a variety of diseases. MSCs are capable of self-renewal and multilineage differentiation capacity, immunomodulatory, and anti-inflammatory properties allowing it to play a role in regenerative medicine. Furthermore, MSCs are low in tumorigenicity and immune privileged, which permits the use of allogeneic MSCs for therapies that eliminate the need to collect MSCs directly from patients. Induced pluripotent stem cells (iPSCs) can be generated from adult cells through gene reprogramming with ectopic expression of specific pluripotency factors. Advancement in iPS technology avoids the destruction of embryos to make pluripotent cells, making it free of ethical concerns. iPSCs can self-renew and develop into a plethora of specialized cells making it a useful resource for regenerative medicine as they may be created from any human source. MSCs have also been used to treat individuals infected with the SARS-CoV-2 virus. MSCs have undergone more clinical trials than iPSCs due to high tumorigenicity, which can trigger oncogenic transformation. In this review, we discussed the overview of mesenchymal stem cells and induced pluripotent stem cells. We briefly present therapeutic approaches and COVID-19-related diseases using MSCs and iPSCs.
Objective: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. Methods: EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. Results: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). Conclusion: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
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During mammalian embryogenesis, the early embryo grows in a relatively hypoxic environment due to a restricted supply of oxygen. The molecular mechanisms underlying modulation of self-renewal and differentiation of mouse embryonic stem cells (mESCs) under such hypoxic conditions remain to be established. Here, we show that hypoxia inhibits mESC self-renewal and induces early differentiation in vitro, even in the presence of leukemia inhibitory factor (LIF). These effects are mediated by down-regulation of the LIF-STAT3 signaling pathway. Under conditions of hypoxia, hypoxia-inducible factor-1α (HIF-1α) suppresses transcription of LIF-specific receptor (LIFR) by directly binding to the reverse hypoxia-responsive element located in the LIFR promoter. Ectopic expression and small interference RNA knockdown of HIF-1α verified the inhibitory effect on LIFR transcription. Our findings collectively suggest that hypoxia-induced in vitro differentiation of mESCs is triggered, at least in part, by the HIF-1α-mediated suppression of LIF-STAT3 signaling.
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Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1alpha and/or Hif-2alpha (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel-Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1alpha. Here we show that conditional inactivation of Hif-2alpha in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2alpha but not Hif-1alpha and that the hypoxic induction of liver Epo in anemic adults was Hif-2alpha dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1alpha dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.
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Respiratory distress syndrome (RDS) due to insufficient production of surfactant is a common and severe complication of preterm delivery. Here, we report that loss of the hypoxia-inducible transcription factor-2α (HIF-2α) caused fatal RDS in neonatal mice due to insufficient surfactant production by alveolar type 2 cells. VEGF, a target of HIF-2α, regulates fetal lung maturation: because VEGF levels in alveolar cells were reduced in HIF-2α-deficient fetuses; mice with a deficiency of the VEGF164 and VEGF188 isoforms or of the HIF-binding site in the VEGF promotor died of RDS; intrauterine delivery of anti-VEGF-receptor-2 antibodies caused RDS and VEGF stimulated production of surfactant proteins by cultured type 2 pneumocytes. Intrauterine delivery or postnatal intratracheal instillation of VEGF stimulated conversion of glycogen to surfactant and protected preterm mice against RDS. The pneumotrophic effect of VEGF may have therapeutic potential for lung maturation in preterm infants.
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In this study, we show that NANOG, a master transcription factor, regulates S-phase entry in human embryonic stem cells (hESCs) via transcriptional regulation of cell cycle regulatory components. Chromatin immunoprecipitation combined with reporter-based transfection assays show that the C-terminal region of NANOG binds to the regulatory regions of CDK6 and CDC25A genes under normal physiological conditions. Decreased CDK6 and CDC25A expression in hESCs suggest that both CDK6 and CDC25A are involved in S-phase regulation. The effects of NANOG overexpression on S-phase regulation are mitigated by the down-regulation of CDK6 or CDC25A alone. Overexpression of CDK6 or CDC25A alone can rescue the impact of NANOG down-regulation on S-phase entry, suggesting that CDK6 and CDC25A are downstream cell cycle effectors of NANOG during the G1 to S transition.
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Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O2) using microarray technology. Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O2, then bifurcated into 21% O2 and 2% O2, and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% O(2) (p0p10) with 2% O2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 under-represented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% O2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O2 cultured cells. The gene changes associated with 2% O2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity.
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Human embryos grow naturally in vivo in lower oxygen (O(2)) tension environments than atmospheric O(2) tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O(2) tension. This study aimed to compare the behavior of hESC under reduced O(2) tension (5%) versus normoxia (21%). hESC lines were cultured in different O(2) tensions and then examined for morphology, apoptosis and gene expression profiles. hESC grown in 5% O(2) tension were not morphologically different from hESC grown in normoxia on day 7 of the first and fourth passages. However, after prolonged culture without splitting (10-14 days), hESC colonies were thinner and looked better morphologically in 5% O(2), but the cells proliferated more slowly and their sizes were larger. At most time points, the gene expression profiles in both O(2) tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, alpha-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1alpha and VEGF). A lower level of cyclin-D1 mRNA (suggestive of less Wnt pathway signaling on day 7 of the fourth passage) and a higher level of Desmin (suggestive of more differentiation to mesoderm, at day 7 of the first passage) were detected in 5% O(2). This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O(2) tension (5% O(2)) probably does not provide significant advantages over the standard 21% O(2) tension for the maintenance of an undifferentiated state by the criteria used in this study.
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To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O(2) atmosphere (approximately 4% O(2)), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O(2) conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O(2). Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O(2). Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O(2), while a few genes implicated in lineage specification were up-regulated. Although the differences between O(2) conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O(2) favors the molecular mechanisms required for the maintenance of pluripotency.
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One of the characteristic features of human embryonic stem cells (hESCs) is the competence for self-renewal and pluripotency. To date, little is known about cell cycle regulation in these cells and how the cell cycle machinery influences hESCs properties. A common feature of human, murine and primate ESCs is the presence of a short G1 phase, which has been viewed as a time window during which stem cells are exposed to differentiation signals. We used the hESCs differentiation model and comparisons to human embryonic carcinoma (EC) cells to study the key regulators of G1 to S transition in hESCs. Our studies show that hESCs express all G1-specific CYCLINs (D1, D2, D3 and E) and cyclin-dependent kinases (CDK) (CDK2, CDK4 and CDK6) at variable levels. In contrast to murine ESCs, most of the cell cycle regulators in hESCs show cell cycle-dependent expression, thus revealing important differences in the expression of cell cycle regulatory components between these two embryonic cell types. Knockdown of CDK2 using RNA interference resulted in hESCs arrest at G1 phase of the cell cycle and differentiation to extraembryonic lineages. This suggests an important role for CDK2 in cell cycle regulation in hESCs that are likely to bear significant impacts on the maintenance of their pluripotent phenotype.
Isolated neural crest stem cells (NCSCs) differentiate to autonomic neurons in response to bone morphogenetic protein 2 (BMP2) in clonal cultures, but these neurons do not express sympathoadrenal (SA) lineage markers. Whether this reflects a developmental restriction in NCSCs or simply inappropriate culture conditions was not clear. We tested the growth and differentiation potential of NCSCs at ∼5% O_2, which more closely approximates physiological oxygen levels. Eighty-three percent of p75^+P_0 ^− cells isolated from embryonic day 14.5 sciatic nerve behaved as stem cells under these conditions, suggesting that this is a nearly pure population. Furthermore, addition of BMP2 plus forskolin in decreased oxygen cultures elicited differentiation of thousands of cells expressing tyrosine hydroxylase, dopamine-β-hydroxylase, and the SA lineage marker SA-1 in nearly all colonies. Such cells also synthesized and released dopamine and norepinephrine. These data demonstrate that isolated mammalian NCSCs uniformly possess SA lineage capacity and further suggest that oxygen levels can influence cell fate. Parallel results indicating that reduced oxygen levels can also promote the survival, proliferation, and catecholaminergic differentiation of CNS stem cells (Studer et al., 2000) suggests that neural stem cells may exhibit a conserved response to reduced oxygen levels.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.