Hypoxia Promotes Osteogenesis but Suppresses

Adipogenesis of Human Mesenchymal Stromal Cells in a

Hypoxia-Inducible Factor-1 Dependent Manner
Markus Wagegg1,2,3., Timo Gaber1,2,3*., Ferenz L. Lohanatha1,2,3, Martin Hahne1,2,6, Cindy Strehl1,2,
Monique Fangradt1,2, Cam Loan Tran1,2, Kerstin Schonbeck1,2, Paula Hoff1,2, Andrea Ode3,4,
Carsten Perka5, Georg N. Duda3,4, Frank Buttgereit1,2,3
1 Department of Rheumatology and Clinical Immunology, Charite University Hospital, Berlin, Germany, 2 German Arthritis Research Center, Berlin, Germany, 3 BerlinBrandenburg Center of Regenerative Therapies, Charite University Hospital, Berlin, Germany, 4 Julius Wolff Institute and Center for Musculoskeletal Surgery, Charite
University Hospital, Berlin, Germany, 5 Orthopaedic Departments, Charite University Hospital, Berlin, Germany, 6 Berlin-Brandenburg School of Regenerative Therapies,
Charite University Hospital, Berlin, Germany

Abstract
Background: Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible
factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells
(hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs
towards adipogenic and osteogenic lineages.
Methodology/Principal Findings: Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app.
18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression
of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as
verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The
expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1a by lentiviral transduction was performed,
and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia
induced HIF-1a and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i)
suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and
associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1a enhanced adipogenesis under both
normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.
Conclusions/Significance: Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive
and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing,
which may potentially lead to the development of novel therapeutic approaches.
Citation: Wagegg M, Gaber T, Lohanatha FL, Hahne M, Strehl C, et al. (2012) Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human
Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner. PLoS ONE 7(9): e46483. doi:10.1371/journal.pone.0046483
Editor: Dimas Tadeu Covas, University of Sao Paulo - USP, Brazil
Received February 9, 2012; Accepted August 31, 2012; Published September 27, 2012
Copyright: 2012 Wagegg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported in part by the Bundesministerium fur Bildung und Forschung (BMBF) and the State of Berlin, BCRT-Grant II to FB and TG.
MW and TG have been supported in part by the project NMP4-LA-2009-228929 (Nanosciences, Nanotechnologies, Materials and new Production Technologies;
2/20101/2014) funded by the European Commission through the 7th Framework Programme for Research. MH has been supported by Deutsche
Forschungsgemeinschaft (DFG) funding through the Berlin-Brandenburg School for Regenerative Therapies GSC 203. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
. These authors contributed equally to this work.

Bone healing is characterized by a series of cellular and

molecular events that commence with hematoma formation and
an inflammatory cascade, finally leading to MSC recruitment and
terminal MSC differentiation. MSC recruitment is known to be
essential for successful fracture repair, and recent studies have
shown that migration of MSCs is strongly influenced by
mechanical stimulation equivalent to conditions of the early
bone-healing phase [4].
This process takes place under low O2 tensions so called
hypoxia which is mainly due to the disruption of supplying blood

Introduction
Mesenchymal stromal cells (MSCs) are a pluripotent cell
population capable of differentiating into a variety of cell types
including osteoblasts, chondrocytes, adipocytes, and myoblasts [1].
MSCs are essential for the repair and regeneration of damaged
tissues, and can be easily isolated from numerous tissues [2,3].
Therefore, cell therapy using MSCs represents a promising
approach to promote wound healing and tissue regeneration,
such as in repair of bone fractures.

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Differentiation of Human MSCs under Hypoxia

found no difference in cell morphology between MCSs maintained

under hypoxia and those incubated under normoxia for periods of
6 hours, 24 hours or 2 weeks (Fig. 2a). Subsequently, immunostaining analysis of surface markers of MSCs cultured under
normoxic or hypoxic conditions was performed. Under both
conditions, the surface markers CD13, CD44, CD73, CD90, and
CD105 were expressed whereas markers CD45, CD34, CD14,
and CD19 were absent without any detectable differences between
normoxic and hypoxic cell culture conditions (Fig. 2b, Tab. 2
and 3).

vessels [5]. When comparing with atmospheric oxygen levels (21%

O2) or in vitro culture condition (,18% O2), in vivo physiological O2
tension of peripheral tissues, e.g. in the stem cell niches such as
adipose tissue or bone marrow is much lower (17% O2) (as
reviewed in [6]).
One key event in the cellular adaptation towards a hypoxic
environment is the induction/stabilization of the transcription
factor hypoxia-inducible factor (HIF)-1, which is composed of an
oxygen-sensitive a-subunit and a constitutively expressed bsubunit. In the presence of higher oxygen levels (.5%), HIF-1a
protein is subjected to proteosomal degradation almost as soon as
it is translated. Under hypoxic conditions, HIF-1a protein is
stabilized, dimerizes with HIF-1b and transactivates a number of
genes whose products participate in a variety of cellular processes
involved in adaptation to hypoxia, such as glycolysis, erythropoiesis, and angiogenesis [7,8].
Several studies have been carried out in order to analyze the
effects of hypoxia on MSCs, but the results were either inconsistent
or yielded conflicting results. For example, some reports demonstrated that human bone marrow-derived MSCs cultured under
hypoxia showed a diminished capacity to differentiate into
adipocytes and osteocytes, supporting the notion that low oxygen
tension promotes an undifferentiated state [9,10,11]. In contrast,
other reports demonstrated that MSCs expanded under reduced
oxygen tension to be primed for chondrogenic differentiation
[12,13]. Moreover, Tsai et al recently demonstrated that (i)
hypoxic culture conditions promote chondrogenic, osteogenic, and
adipogenic differentiation and (ii) hypoxic cells show an increased
bone repairing ability in vivo [14].
In summary, although the observations are highly variable
depending on the experimental conditions and specimens used, it
is clear that hypoxia (and hence HIF-1a) affects the differentiation
characteristics of MSCs. However, much less attention has been
paid to the role of oxygen as a signalling molecule that influences
human stromal cell survival, proliferation and differentiation in
culture. Only limited and inconsistent information is available on
the impact of hypoxia and HIF-1a on the differentiation potential
of primary human multipotent MSCs. Information on these
crucial issues are, however, highly clinically relevant. Therefore,
we investigated the effect of hypoxia on the differentiation
potential of primary human MSCs.

Hypoxia induces HIF-1a and HIF-1 target gene expression

Next, we analyzed in MSC the influence of hypoxia on the
transcription of HIF-1a. We found hypoxia resulted in a
significant time-dependent increase in the expression of HIF-1a
mRNA (HIF1A) in MSCs after 72 hours (p,0.05) and 2 weeks
(p,0.001) of incubation when compared to cells under normoxia
(Fig. 3a). HIF-1a is known to be primarily regulated at the
protein/post-translational level. Therefore, we also investigated
the protein expression of HIF-1a and found a time-dependent
increase in HIF-1a protein expression (Fig. 2b). Furthermore,
typical HIF target genes such as VEGF, GLUT1, LDHA, PGK1
were up-regulated after 72 hours under hypoxic conditions
(Fig. 3c). The observed increase of HIF-1a and HIF-1-targetgene expression in primary human MSCs suggest that HIF-1a in
MSCs is a key regulator for adaption to hypoxia.

Hypoxia suppresses adipogenic and promotes

osteogenic differentiation of human MSCs
From the results above and the reports that hypoxia promotes
chondrogenesis [13], we suspected hypoxia may also influence
adipogenesis and osteogenesis. In order to test this hypothesis, we
induced differentiation of human MSCs into adipocytes and
osteoblasts under normoxic and hypoxic conditions, respectively.
We found adipogenic differentiation to be suppressed under
hypoxia, but osteogenic differentiation to be clearly promoted
(Fig. 4a). We also analyzed mRNA expression of HIF1A and
VEGFA during adipogenesis and osteogenesis (Fig. 4b and c). In
the case of adipogenesis and osteogenesis, HIF1A mRNA is
differentially expressed in MSCs incubated under normoxic and
hypoxic conditions (Fig. 4d). In adipogenesis, the expression
significantly decreases after 2 weeks under hypoxia compared to
normoxia (p,0.05). In contrast, during osteogenesis there is an
increase of expressed HIF1A mRNA under hypoxic conditions
compared to normoxia (p,0.05). Surprisingly, expression of
VEGFA transcription is up to 20 fold higher under hypoxic
conditions during osteogenesis than during adipogenesis. It
increases with time and is higher under hypoxia than under
normoxia (p,0.05). In contrast, VEGFA mRNA expression is
decreased during adipogenesis (Fig. 4c). Furthermore, we
analysed the expression of PPARG (a key marker for the adipogenic
switch), and RUNX2 (a key marker for the osteogenic switch). In
adipogenesis the expression of PPARG is significantly higher after 2
weeks under normoxia compared to hypoxia (p,0.001; Fig. 4d).
In the case of RUNX2, we observed a significant up-regulation of
gene expression after 2 weeks under hypoxia compared to
normoxia which is much more pronounced during osteogenic
differentiation (p,0.01) than during adipogenic differentiation
(p,0.05; Fig. 4e).
We found that osteogenesis of human MSC was facilitated by
chemical inducers of HIF-1a, such as the iron chelating agent
desferrioxamine mesilate (DFX) or the dimethyloxallyl glycine
(DMOG), even under normoxic conditions, but to a lesser extent
than under hypoxic conditions (Fig. 4f).

Results
Bone marrow derived human MSCs express typical
surface markers and are able to differentiate into
adipogenic, osteogenic and chondrogenic lineages
Isolated bone marrow derived human MSCs were characterized
via their surface marker expression according to the declaration of
the Mesenchymal and Tissue Stem Cell Committee of the International Society
for Cellular Therapy, being positive for the expression of CD13,
CD44, CD73, CD90, and CD105 and negative for the expression
of CD45, CD34, CD14, and CD19 (Fig. 1a, Tab. 1). The MSC
phenotype was confirmed by showing differentiation into different
mesenchymal lineages such as adipogenic, chondrogenic and
osteogenic (Fig. 1bd).

Hypoxia does not alter MSC phenotype and surface

marker expression
In order to analyze the impact of hypoxia on human MSC
phenotype and surface marker expression, isolated bone marrow
derived cells were cultured in DMEM without growth or
differentiation factors supplemented with 10% FCS. MSCs were
cultured for 2 weeks under normoxic and hypoxic conditions. We
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Differentiation of Human MSCs under Hypoxia

Figure 1. Characterization of human MSCs by their surface marker expression and their ability to differentiate into mesenchymal
lineages. (a) Bone marrow derived human MSCs express CD13, CD44, CD73, CD90, and CD105 but do not express CD45, CD34, CD14, and CD19;
they differentiate into different mesenchymal lineages such as (b) adipogenic, (c) osteogenic and (d) chondrogenic lineages (scale indicated on the
figures; n = 8).
doi:10.1371/journal.pone.0046483.g001

(sh1 and sh2) and one non-silencing shRNA (scr) as control.

Efficiency of lentiviral transduction was up to 93% (data not
shown). Knockdown of HIF-1a was verified at the protein level
(Fig. 5a).
In HIF-1a knockdown cells, adipogenesis and osteogenesis were
induced for 4 weeks. In the case of adipogenesis (Fig. 5b), control
cells (scr-treated cells) behaved as expected, i.e. adipogenesis was
suppressed under hypoxia. However, adipogenic differentiation of
transduced MSCs was increased compared with controls, under
both normoxic and hypoxic conditions.

Reduction of HIF-1a expression in human MSCs (i)

enhances adipogenesis under normoxic conditions, (ii)
partially restores hypoxia-induced attenuation of
adipogenesis and (iii) suppresses hypoxia-enhanced
osteogenesis
From the results above, we considered that HIF-1a could play a
key role in regulating adipogenesis and osteogenesis. In order to
investigate the function of HIF-1a in greater detail, a shRNA
mediated knockdown of HIF-1a was performed using lentiviral
transduction. To this end, we used two HIF-1a-silencing shRNAs
Table 1. Percentage of CD-positive cell populations.

Patient#

CD13

CD14

CD19

CD34

CD44

CD45

CD73

CD90

CD105

99.5

0.1

0.5

2.1

95.1

0.1

100.0

95.0

87.5

99.9

0.2

0.8

1.2

96.2

0.2

100.0

93.2

86.1

99.8

0.5

0.4

0.4

91.8

0.0

100.0

94.1

89.9

99.2

1.2

0.7

0.6

96.5

0.2

100.0

93.2

93.4

98.7

0.3

0.1

3.5

99.0

0.1

100.0

91.1

85.2

98.2

0.6

0.7

2.0

90.1

0.3

100.0

92.1

92.2

99.5

0.4

0.5

4.1

92.4

0.1

100.0

94.3

96.1

99.3

1.1

0.2

0.6

91.6

0.1

100.0

95.2

81.7

mean

99.1

0.7

0.4

1.9

93.6

0.1

100.0

93.3

89.8

SD

0.6

0.4

0.3

1.6

3.4

0.1

0.0

1.5

5.4

doi:10.1371/journal.pone.0046483.t001

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Differentiation of Human MSCs under Hypoxia

Figure 2. Hypoxia does not alter MSC phenotype and surface marker expression. No difference between MCSs maintained under
normoxia and those incubated under hypoxia were observed in terms of (a) cell morphology (after 6 h, 24 h, and 2w of incubation; scale indicated on
the figures; n = 3) and (b) surface marker expression of the positive-markers CD13, CD44, CD73, CD90, CD105 and the negative-markers CD45, CD34,
CD14, and CD19 (normoxia = red; hypoxia = green; n = 6).
doi:10.1371/journal.pone.0046483.g002

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Differentiation of Human MSCs under Hypoxia

Table 2. Percentage of CD-positive cell populations after 2 week incubation under normoxia.

Patient#

CD13

CD14

CD19

CD34

CD44

CD45

CD73

CD90

CD105

98.7

1.2

0.2

2.3

97.5

0.0

100.0

99.8

81.8

91.2

1.1

1.3

1.4

98.2

1.2

100.0

99.2

89.4

99.3

0.3

0.5

2.2

99.8

0.3

100.0

97.2

90.8

92.4

0.6

1.2

1.0

98.7

0.2

100.0

99.9

89.0

92.3

0.4

0.4

1.3

99.5

0.0

100.0

99.0

95.1

98.1

1.1

0.9

1.2

98.5

1.1

100.0

99.3

95.2

mean

95.3

0.8

0.8

1.6

98.7

0.5

100.0

99.3

90.2

SD

3.7

0.4

0.5

0.5

0.8

0.5

0.0

1.1

4.9

doi:10.1371/journal.pone.0046483.t002

Interestingly, osteogenic differentiation showed the converse

(Fig. 5c), with promotion of osteogenesis under hypoxic conditions. In the case of osteogenic differentiation of MSCs with HIF1a knockdown, we found suppression of hypoxia-enhanced
osteogenesis.

the stem cell niche where the physiological oxygen tension is

known to be as low as 12% [3].
Moreover, MSCs also face pathophysiologic hypoxia. For
example, low oxygen levels are strongly associated with injury
and/or inflammation. Under these circumstances the microenvironment is also characterized by cell accumulation/infiltration,
and high levels of humoral factors such as cytokines and growth
factors.
An interesting and typical example is bone fracture repair
[16,17,18]. Here, a series of cellular and molecular events is
initiated leading to structural reconstitution and tissue regeneration [19]. The bone healing process has been characterized by four
phases: an inflammatory phase, a soft callus phase, a hard callus
phase, and a remodelling phase [20,21]. MSCs are thought to be
recruited to the fracture site in the initial stages of fracture healing
(day 1) and to proliferate by day 3 after fracture [5,22]. During
these stages, MSCs that are utilized to regenerate bone tissue do
not only face hypoxic conditions but also high levels of proinflammatory mediators [23,24]. Both factors are considered
capable of modifying behaviour and functionality of MSCs.
Given this background, we investigated the differentiation
potential of human MSCs towards adipogenic and osteogenic
lineages. We found that adipogenesis although induced with
adipogenic conditioned medium was suppressed by hypoxia,
whereas osteogenesis was enhanced, which directly correlated to
the gene expression of PPARG and RUNX2, respectively but also
HIF-1a. Furthermore, the enhancement of osteogenesis under
hypoxia could be mimicked under normoxic conditions by so
called chemical hypoxia as induced by DFX or DMOG (Fig. 4).
We also demonstrate here that the hypoxia-mediated increase in

Discussion
Mesenchymal stromal cells (MSCs) are pluripotent cells, capable
of differentiating into a variety of cell types and being essential in
the repair and regeneration of damaged tissues [1]. To this end,
MSCs are known to be recruited to sites of injury and
inflammation for repair and regeneration of damaged tissue such
as injured muscles, tendons and bones where they face conditions
of reduced oxygen availability [6,15]. Hypoxia, and hence HIF1a, affects (i) MSC maintenance and self-renewal in the stem cell
niche under physiological hypoxia and (ii) MSC differentiation in
the process of tissue regeneration after injury under pathophysiological hypoxia [3]. Therefore, we investigated the effect of
hypoxia on both maintenance and differentiation potential of
primary human MSCs.
To this end, we first confirmed the immunophenotype and
validated the function of the MSCs isolated in our experiments
(Fig. 1). We then showed that incubation under hypoxia for up to
2 weeks did not alter the morphology or immunophenotype of
human MSCs (Fig. 2). Furthermore, we demonstrated the
capability of human MSCs to induce HIF-1a and HIF-1 target
gene expression under hypoxic conditions (Fig. 3). The data
demonstrate both the maintenance of the stem cell capabilities,
indicated by morphology and immunophenotype, and the switch
from aerobic to anaerobic cell metabolism such as postulated for

Table 3. Percentage of CD-positive cell populations after 2 week incubation under hypoxia.

Patient#

CD13

CD14

CD19

CD34

CD44

CD45

CD73

CD90

CD105

99.3

1.6

0.9

1.9

99.3

0.2

100.0

99.5

82.5

90.9

0.2

2.3

1.9

96.2

0.4

100.0

98.7

88.4

98.7

0.0

0.7

1.4

98.2

1.3

100.0

94.5

87.6

99.3

1.4

0.2

2.1

99.0

0.9

100.0

98.7

92.4

94.8

1.3

0.0

2.3

97.6

0.0

100.0

99.3

93.5

97.5

0.8

1.7

1.6

98.8

2.3

100.0

98.2

96.8

mean

96.8

0.9

1.0

1.9

98.2

0.9

100.0

98.2

90.2

SD

3.3

0.7

0.9

0.3

1.1

0.9

0.0

1.8

5.1

doi:10.1371/journal.pone.0046483.t003

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Differentiation of Human MSCs under Hypoxia

MSCs towards osteoblasts, thereby inhibiting adipogenesis, in

order to guarantee the initiation of proper bone regeneration in a
HIF-1 dependent manner.
This competitive effect of hypoxia on human MSCs has not
been reported previously. However, the investigations on the effect
of hypoxia/HIF on human MSC maintenance and differentiation
published by other authors show conflicting results.
Focusing on the effect of hypoxia alone, Lavrentieva et al.
(2010) reported that hypoxia facilitates a switch from aerobic to
anaerobic metabolism that we could confirm by analyzing the
gene expression of the glycolytic genes glucose transporter 1
(GLUT1), lactate dehydrogenase A (LDHA), and phosphoglycerate
kinase 1(PGK1) [25]. Moreover, Holzwarth et al. (2010) also
showed in accordance with our data that low oxygen levels
stabilize the immunophenotype of human MSCs [11]. Furthermore, Tamama et al. (2011) and Tsai et al. (2011) very recently
demonstrated that hypoxia enhances the proliferation and colony
formation thus increasing expansion efficiency of human MSCs
[14,26]. Summarizing the data it has been postulated that oxygen
tension is an important regulator in the determination of cell fate
and for the maintenance of MSCs in an undifferentiated state,
which is supported by our findings [6].
When focusing on the differentiation capability of human MSCs
under pathophysiologic conditions, in the case of osteogenic
differentiation, hypoxic conditions were shown to reduce the
capability of MSCs to differentiate into osteoblasts and into bone
marrow-isolated adult multilineage inducible (MIAMI) cells
[9,10,26,27,28]. On the other hand, hypoxia and/or HIF has
been reported to promote osteogenesis [29,30,31]. The latter data
support in part our findings.
With regard to adipogenesis our data suggest that hypoxic
condition decreases adipogenic differentiation in a HIF-1-dependent manner. These data were in contrast to the findings of Tsai et
al. (2011) as given above [14] but were at least in part supported
by Holzwarth et al. (2010) and Tamama et al. (2011) [11,26]. In
the latter report it was shown that hypoxic condition promotes
MSC self-renewal through preserving colony forming early
progenitors and maintaining undifferentiated phenotypes [26].
Therefore, these authors demonstrated that hypoxic conditions
reversibly decreased adipogenic differentiation dependent on the
activation of unfolded protein response (UPR), but not on HIFs
[26].
We assume that possible reasons for these different results
include the variety of sources of MSCs (e.g. umbilical cord-derived
[25]), the different culture conditions [14], and differences in the
experimental design (use of immortalized human MSCs [26]).
Consequently, it is difficult to make broad conclusions regarding
the role of hypoxia on the biology of MSCs [32]. However, with
regard to our defined experimental conditions we can clearly
conclude that hypoxia suppresses adipogenesis and promotes
osteogenesis of human MSCs in a competitive and HIF-1dependent manner. We therefore conclude that the hypoxic
nature of the fracture hematoma guides the differentiation of
MSCs towards osteoblasts, thereby inhibiting adipogenesis, which
is crucial for effective bone healing. During this process, the master
regulator of the hypoxic cellular response, HIF-1, balances the
differentiation decision between osteogenesis and adipogenesis.
Consequently, stem cell therapy in combination with chemical
induced hypoxia/HIF-1 could be a new approach to improve
fracture healing.

Figure 3. Hypoxia induces HIF-1a and HIF-1-target-gene

expression. (a) HIF1A gene expression of human MSCs obtained by
real-time PCR (n = 6; unpaired t-test). (b) HIF-1alpha and beta-actin
protein expression obtained by immunoblot. (c) Hypoxia-induced HIF1-target-gene expression of VEGFA, LDHA, GLUT1, and PGK1 (n = 6; 2weeks data; one sample t-test; dotted-line indicates normalization to
gene expression of normoxic cells; *** p,0.001; ** p,0.01; * p,0.05).
doi:10.1371/journal.pone.0046483.g003

osteogenic differentiation and decrease in adipogenic differentiation could be reversed by shRNA against HIF-1a (Fig. 5). Of
note, HIF-1a in MSCs and other cells mediates the cellular
adaption to hypoxia in order to ensure the function of the cells in
hypoxic areas through the readjustment of the cellular bioenergetics [7,8]. Therefore, we conclude from our data that the
hypoxic nature of the fracture site facilitates the differentiation of

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Differentiation of Human MSCs under Hypoxia

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Differentiation of Human MSCs under Hypoxia

Figure 4. Hypoxia suppresses adipogenic and promotes osteogenic differentiation of human MSCs. (a) Oil-Red-O stain for the analysis
of adipogenesis and von-Kossa stain for the analysis of osteogenesis of MSCs incubated under normoxia (<18% pO2) or hypoxia (1% pO2) for 4 weeks
using either osteogenic or adipogenic differentiation medium (scale indicated on the figures; n = 6). (b) HIF1A, (c) VEGFA, (d) PPARG and (e) RUNX2
gene expression of MSCs incubated under normoxia (<18% pO2) or hypoxia (1% pO2) for 2 weeks using either osteogenic or adipogenic
differentiation medium as obtained by real-time PCR (n = 6; unpaired t-test; dotted-line indicates normalisation to gene expression of undifferentiated
cells; * p,0.05). (f) Analysis of osteogenesis by von-Kossa stain of MSCs incubated under normoxia (<18% pO2) without treatment, or with either
250 mM DFX and 100 mM DMOG, respectively (scale indicated on the figures; n = 3).
doi:10.1371/journal.pone.0046483.g004

DMOG. Both substances were added to the incubation medium

once weekly for a period of 4 weeks.

Materials and Methods

Antibodies
Antibodies for flow cytometric analysis were purchased at
Immunotools (CD13, CD34, CD45, all APC labelled), NatuTec/
eBiosience (CD44, CD90, CD105, all APC labelled) and BD
Biosience, Heidelberg, Germany (CD73 labelled at DRFZ with
Cy5). CD14 and CD19 (labelled with Cy5) were obtained from
DRFZ.
For immunoblot, rabbit whole serum anti-human HIF-1a and
mouse anti-human b-actin antibody were bought from Abcam plc
(Cambridge, UK) and Sigma-Aldrich Chemie GmbH (Munich,
Germany), respectively.

Osteogenic, adipogenic and chondrogenic

differentiation of human MSCs (transduced and nontransduced)
Human MSCs (transduced and non-transduced) were plated in
6-wells at a density of 10000 cells/cm2 for osteogenic and
adipogenic differentiation, or at a density of 25000 cells in a pellet
culture in a 15 ml polypropylene conical tube for chondrogenic
differentiation in a micromass culture. After 24 h, medium was
changed to conditioned medium (CM) in order to induce
differentiation. In the case of differentiation in the osteogenic
linage, DMEM was supplemented with 10 mM b-glycerophosphate, 10 nM dexamethasone and 0.1 mM L-ascorbic acid-2phosphate. For differentiation of MSCs to adipocytes, 10 mg/ml
insulin, 0.2 mM indomethacine, 1 mM dexamethasone and
0.5 mM 3-isobutyl-1-methyl-xanthine was used to supplement
DMEM. For chondrogenesis, NH Chondrodiff Medium (Miltenyi
Biotec) was used. Undifferentiated MSCs were maintained in
control medium, consisting of DMEM supplemented with 10%
FCS, penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Medium was replaced every 4 days (chondrogenic) or weekly
(osteogenic and adipogenic). Differentiation was terminated after
28 days and assessed by specific staining as described below.

Isolation and expansion of human MSCs

Primary human mesenchymal stromal cells (MSCs) were
isolated from bone marrow obtained from human donors
undergoing total endoprosthesis (TEP) of the hip joint. The donor
population was reasonably homogenous (age range 49 to 68 years,
equal numbers of males and females, and exclusively hip
osteoarthritis as the reason for TEP). All patients were more or
less continuously treated with non-steroidal anti-inflammatory
drugs (NSAIDS) (for patient information see Tab. 4). Written
informed consent was obtained from each donor. All experiments
were conducted according to the protocols approved by the
Charite University Hospital ethics committee and according to the
Helsinki Declaration. MSCs were isolated with a density gradient
and re-suspended in complete culture medium. Human MSCs
were cultured in Dulbeccos modified Eagles medium-high
glucose (DMEM) containing 10% fetal calf serum (FCS), 50 mM
2-ME (Sigma-Aldrich), 100 units/ml penicillin, and 0.1 mg/ml
streptomycin at 37uC in 5% CO2 atmosphere. After 3 days, nonadherent cells were removed by medium change. MSCs were
expanded as adherent cells. Cells from each donor were cultured
separately. Cells were passaged at 7080% confluence and
passages 38 were used for experiments.

Oil-Red-O staining of differentiated human MSCs

Cells undergoing adipogenic differentiation were stained with
Oil-Red-O dye to visualize lipid droplet accumulation. Medium
was aspirated and cells were washed with PBS followed by fixation
with 2% neutral buffered formalin for 20 minutes. Then cells were
first washed with distilled water and again with 60% isopropanol.
A fresh 60% Oil-Red-O working solution was prepared from a
stock solution (0.5 g Oil-Red-O in 100 mL isopropanol), left for
10 minutes and filtered through a 0.25 mm syringe filter. The
working solution is not stable and has to be used within one day.
Cells were stained with the working solution for 15 min, washed
with 60% isopropanol and as second with distilled water. Imaging
was performed at room temperature by microscopy.

Characterization of human MSCs (transduced and nontransduced)

Flow cytometric analysis was used for the characterization of
surface protein expression pattern shown by the adherent cells.
Typical surface marker CD13, CD44, CD73, CD90, CD105,
CD14, CD19, CD34 and CD45 were measured according to
protocols previously published [33]. Furthermore, cells were tested
for their capacity to differentiate into the adipogenic, osteogenic
and chondrogenic lineages in vitro (for details see below).

Von-Kossa staining of differentiated human MSCs

Cells undergoing osteogenic differentiation were stained with
von-Kossa dye to visualize calcium deposits, which were replaced
by silver. Medium was aspirated and cells were washed with PBS,
followed by two washes using distilled water. Cells were fixed with
2% neutral buffered formalin for 20 minutes, and then incubated
with 1% silver nitrate solution (w/v) for 60 min under a bright
light bulb. Subsequently cells were washed twice with distilled
water and then incubated with 5% sodium thiosulfate solution (w/
v) for 5 minutes to remove non-reacted silver. Afterwards the cells
were washed with distilled water. Imaging was performed at room
temperature by microscopy.

Induction of hypoxia and chemical hypoxia

Human MSCs (transduced and non-transduced) were incubated
in a hypoxic chamber (Binder) at 5% CO2 level and less than 2%
O2, balanced with N2. Normoxic controls were incubated at 5%
CO2 in a humidified atmosphere with ,18% O2. When inducing
chemical hypoxia, human MSCs were incubated under
normoxia in the presence of either 250 mM DFX or 100 mM

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Differentiation of Human MSCs under Hypoxia

Figure 5. Reduction of HIF-1a of human MSCs (i) enhances adipogenesis under normoxic conditions, (ii) partially restores hypoxiainduced attenuation of adipogenesis and (iii) suppresses hypoxia-enhanced osteogenesis. (a) Transduction of anti HIF-1a-shRNAconstructs efficiently reduced HIF-1a protein expression as shown by immunoblot (2-weeks data). (b) Oil-Red-O stain for the analysis of adipogenesis
of shRNA-construct transduced MSCs and (c) von-Kossa stain for the analysis of osteogenesis of shRNA-construct transduced MSCs. Cells were
maintained under normoxia (<18% pO2) or hypoxia (1% pO2) for 4 weeks using either osteogenic or adipogenic differentiation medium (scale
indicated on the figure; n = 3).
doi:10.1371/journal.pone.0046483.g005

sections were covered with liquid mounting medium and a

coverslip. The tissue sections were either stored at 220uC or
assessed directly by microscopy.

Alzian blue staining of differentiated human MSCs

After chondrogenic differentiation, the medium was removed
and the micromass culture pellet was washed with PBS. The pellet
was embedded in cryomedium and deep frozen at 280uC. After 2
days, tissue sections were generated and transferred onto
microscope slides and either stored at 220uC or directly stained.
For the staining, the tissue sections were fixed with 2% neutral
buffered formalin for 20 minutes, washed and stained with alzian
blue solution for 30 minutes. Staining was followed by washing
with tap water and rinsing with distilled water. The stained tissue

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Imaging of differentiated human MSCs

Imaging was performed at room temperature with a microscope
(LEITZ DM IL, Leica) equipped with Zeiss A-Plan 106 (NA 0.25)
and LD A-Plan 326 (0.40) objectives. Images were captured by a
Nikon E4500 camera. Photoshop software (Adobe Systems Inc.)
was used to adjust levels and colour balance.

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Differentiation of Human MSCs under Hypoxia

Table 4. Patient information and experiments conducted.

Patient#

Age

Gender

Medication

Disease

Type of experiment

normoxia (FACS, morphology, adipogenic & osteogenic & chondrogenic

differentiation)

56

NSAID*

hip OA

68

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic & chondrogenic

differentiation)

66

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic differentiation); hypoxia

(FACS, morphology, adipogenic & osteogenic differentiation)

49

NSAID*

hip OA#

normoxia (FACS, morphology, WB1); hypoxia (FACS, morphology, WB1)

65

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR, WB1);

hypoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR, WB1)

59

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR, WB1);

hypoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR, WB1)

58

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR);

hypoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR)

62

NSAID*

hip OA#

normoxia (adipogenic & osteogenic differentiation, qPCR); hypoxia (adipogenic &

osteogenic differentiation, qPCR)

65

NSAID*

hip OA#

normoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR);

hypoxia (FACS, morphology, adipogenic & osteogenic differentiation, qPCR)

10

54

NSAID*

hip OA#

normoxia (adipogenic & osteogenic differentiation); hypoxia (adipogenic &

osteogenic differentiation)

11

57

NSAID*

hip OA#

normoxia (adipogenic & osteogenic differentiation, qPCR); hypoxia (adipogenic &

osteogenic differentiation, qPCR)

12

59

NSAID*

hip OA#

HIF-knock-down (WB1, adipogenic & osteogenic differentiation)

NSAID*

hip OA

HIF-knock-down (WB1, adipogenic & osteogenic differentiation)

hip OA

HIF-knock-down (adipogenic & osteogenic differentiation)

13
14

60
54

m
m

NSAID*

*nonsteroidal anti-inflammatory drugs,

#
hip osteoarthritis,
1
Western-Blot.
doi:10.1371/journal.pone.0046483.t004

Quantitative PCR of selected genes of expanded and/or

differentiated human MCSs

Immunoblot of HIF-1a and ACTB

Human MSC were lysed after 0 h, 24 h, 72 h, and 2 weeks of
incubation under normoxia or hypoxia. Nuclei were prepared
using the Nuclear Extract Kit from Active Motif, according to the
manufacturers instructions. For immunodetection of proteins,
10 mg of nuclear fractions were separated by SDS-PAGE and
blotted onto PVDF membranes (Millipore). Blotted proteins were
analyzed as indicated and visualized by enzymatic chemiluminescence (Amersham Biosciences).

The cDNAs were synthesized by reverse transcription using

SensiscriptH Reverse Transcription Reagents (Qiagen, Darmstadt,
Germany). Quantitative PCR (qPCR) was carried out using the
LightCyclerH Fast Start DNA Master SYBRH Green I Kit
(ROCHE Diagnostics - Applied Science, Mannheim, Germany).
Data were normalized to the expression of b-actin (ACTB). All
primers used were obtained from TIB Molbiol (Berlin, Germany;
Tab. 5).
Table 5. Primersets used.

Gene symbol

Gene name

Gene function

Primerset

ACTB

beta-actin

structural houskeeper

gACAggATgCAgAAggAgATCACT;
TgATCCACATCTgCTggAAggT

GLUT1

glucose transporter 1

glucose transport

ACgCTCTgATCCCTCTCAgT; gCAgTACACACCgATgATgAAg

HIF1A

hypoxia-inducible factor 1, alpha subunit

transcription factor

CCATTAgAAAgCAgTTCCgC; TgggTAggAgATggAgATgC;

LDHA

lactate dehydrogenase A

glycolysis enzyme

ACCCAgTTTCCACCATgATT; CCCAAAATgCAAggAACACT;

PGK1

phosphoglycerate kinase 1

glycolysis enzyme

ATggATgAggTggTgAAAgC; CAgTgCTCACATggCTgACT;

VEGFA

vascular endothelial growth factor A

pro-angiogenic factor

AgCCTTgCCTTgCTgCTCTA; gTgCTggCCTTggTgAgg.

PPARG

Peroxisome proliferator-activated receptor

gamma

transcription factor (marker of

adipogenesis)

CCCAggTTTgCTgAATgTgAAg;
gAAgggAAATgTTggCAgTgg

RUNX2

Runt-related transcription factor 2

transcription factor (marker of

osteogenesis)

AggTACCAgATgggACTgTg; TCgTTgAACCTTgCTACTTgg

doi:10.1371/journal.pone.0046483.t005

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Differentiation of Human MSCs under Hypoxia

Lentiviral based shRNA mediated knockdown of HIF-1a

Statistical analysis

Based on the pLentiLox 3.7 (Addgene plasmid 11795), shRNA

constructs were generated by subcloning short hairpin oligo
nucleotides (pLL-scr 59-T gCTATCgAgAAgATCAgCC TTCAAgAgA
ggCTgATCTTCTTTAgC TTTTTTC-39; pLL-HIFsh1 59-T
CCgCTggAgACACAATCATAT TTCAAgAgA ATATgATTgTgTCTCCAgCgg TTTTTTC-39; pLL-HIFsh2 59-T CCAgTTATgATTgTgAAgTTA TTCAAgAgA TAACTTCACAATCATAACTgg TTTTTTC39) as previously described [34]. Lentiviral stocks were obtained by
calcium phosphate co-transfection of HEK293 cells (ATCC) with
the lentiviral packaging plasmids pVSVG and pPAX2. The
medium was replaced after 4 h and viral supernatants were
collected 4872 h later. Human MSCs were infected by 90 min
centrifugation at 700 g at 37uC with viral supernatants and 8 mg/
ml polybrene (Sigma-Aldrich), followed by replacement of the viral
supernatant with fresh full supplemented culture medium after
4 h. shRNA construct containing cells were analyzed for coexpressed GFP using flow cytometry. Transfected MSC with a
frequency of .90% GFP positive cells were used for osteogenic
and adipogenic differentiation.

Data shown are reported as the mean 6 SD of at least three

independently performed experiments. Differences between normally distributed groups were compared using the Students t test.
Statistical significance was considered when p,0.05 (*** p,0.001;
** p,0.01; * p,0.05).

Acknowledgments
We would like to thank Manuela Jakstadt for expert technical assistance.
We also acknowledge Luk Van Parijs for providing the plasmid pLL3.7.

Author Contributions
Conceived and designed the experiments: MW TG PH CP GND FB.
Performed the experiments: MW TG FLL MH CS MF PH AO CLT KS.
Analyzed the data: MW TG FLL MH CS MF PH CLT KS. Contributed
reagents/materials/analysis tools: TG AO CP GND FB. Wrote the paper:
TG MW CP GND FB.

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