Review

Responses of adipose-derived
stem cells during hypoxia:
enhanced skin-regenerative
1. Introduction potential
2. ASCs and skin regeneration
3. Cellular and molecular Hyung-Min Chung, Chong-Hyun Won & Jong-Hyuk Sung†
†
responses of ASCs during CHA University, Department of Biomedical Science, #606-16, Yeoksam-dong, Kangnam-gu,
hypoxia Seoul, 135-081, Republic of Korea

4. Functional enhancement by
Mesenchymal stem cells within the stromal–vascular fraction of subcutaneous
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hypoxia
adipose tissue (i.e., adipose-derived stem cells (ASCs)), have been used for
5. Expert opinion tissue engineering. In addition to serving a building-block function, ASCs are
reported to secrete growth factors that are essential for their function.
Increasing evidence indicates that ASCs play a significant role in skin regen-
eration, a function that is enhanced by hypoxia through upregulating secre-
tion of growth factors. Although the anatomical sites of ASCs in the body are
relatively oxygen-deficient, ASCs are usually cultured under normoxic condi-
tions (i.e., atmospheric oxygen levels). Culturing ASCs under physiologically
relevant low-oxygen-tension conditions may uniquely benefit the expansion,
differentiation, adhesion, growth factor secretion and regenerative potential
For personal use only.

of ASCs. Therefore, understanding the response and adaptation of ASCs to

hypoxia may be invaluable for developing novel cell- and cyto-therapy strat-
egies. This review highlights our current understanding of cellular and molec-
ular responses of ASCs to hypoxia, focusing on the enhancement of ASC
function and secretory activity by hypoxic culture conditions.

Keywords: adipose-derived stem cell, growth factor, hypoxia, skin regeneration

Expert Opin. Biol. Ther. (2009) 9(12):1499-1508

1. Introduction

Regenerative medicine uses the body’s own stem cells and growth factors as an
alternative therapeutic strategy for repairing damaged soft tissue such as skin.
Although such cell-based therapies are becoming more prominent, the limited
availability of human cells capable of self-renewal and differentiation into skin-type
cells are barriers to their expanded application. Adipose-derived stem cells (ASCs)
and their soluble factors, however, can be used to regenerate skin and thus offer a
potential workable solution to this dilemma [1-6]. ASCs can be easily obtained by
liposuction from human adipose tissue, they can be cultured on a large scale and
they can display multilineage developmental plasticity [7-9]. In addition to serving
a building-block function, ASCs secrete various growth factors that control and
regenerate damaged skin-type cells, a function that has been identified as essential
to the regenerative mechanisms of ASCs [1-4,10]. For example, ASCs and their solu-
ble factors accelerate wound healing, reduce wrinkling, improve pigmentation and
promote hair growth by activating neighboring cells in skin [1,3-5,10]. Recently, we
have demonstrated that hypoxia enhanced the regenerative function of ASCs by
increasing the secretion of growth factors [11]; however, the underlying mechanism
has not been fully characterized.
Oxygen deficiency may impair cell function. However, cellular responses to
hypoxic stress are highly dependent on cell type, maturity and environment. In

10.1517/14712590903307362 © 2009 Informa UK Ltd ISSN 1471-2598 1499

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 Responses of adipose-derived stem cells during hypoxia
humans, oxygen concentrations vary significantly among tis-
sues. Oxygen concentrations range from 4 to 14% in highly
perfused organs (such as lung, liver, kidneys and heart) [12-15]
but are very low in bone marrow (0 – 4%) [16]. Importantly,
oxygen concentrations are typically less than 3% in adipose
tissue [17-20]; thus, ASCs reside in anatomical sites that are
relatively oxygen-deficient. Low oxygen tension is an impor-
tant component of the stem cell microenvironment (i.e.,
stem cell niche) and provides signals conducive to the main-
tenance of definitive stem cell properties [21-23]. Despite the
low oxygen concentration in the stem cell microenvironment,
stem cells are usually cultured under normoxic conditions cor-
responding to atmospheric oxygen (20 – 21%). Culturing ASCs
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under more physiologically relevant conditions of low-oxygen
tension may uniquely benefit the expansion, differentiation,
adhesion, growth factor secretion, and regenerative potential
of ASCs (Figure 1). Therefore, understanding the response
and adaptation of ASCs to hypoxia may be invaluable for
developing novel cell- and cyto-therapy strategies.
Despite a history of intensive research into the response
of stem cells to hypoxia, most previous literature has focused
on embryonic and bone marrow-derived mesenchymal stem
cells (BMSCs) [24-27]. Regarding ASCs, many studies have
examined the chondrogenic potentiality of ASCs during
For personal use only.
hypoxia, because cartilage is among the most hypoxic tissues
in the body [28,29]. In recent years, studies from our laboratory
and those of others have provided novel insights into the
cellular and molecular responses of ASCs to hypoxia in skin,
a subject that is briefly described in this review. The spe-
cific focus of this review is on the functional enhancement
of ASCs by hypoxia and the involvement of this enhanced
function in skin repair and regeneration.
2. ASCs and skin regeneration
Stem cell therapy as clinically applied is a safe and effective
method for repairing several types of tissue damage [6,30,31].
Recently, ASCs have been investigated as a substitute for
BMSCs, offering a potential alternative for skin repair and
regeneration [5,32]. ASCs differentiate into diverse skin cells
and secrete growth factors that exert their beneficial effects on
damaged skin via a paracrine mechanism.
2.1 Characteristics of ASCs
ASCs are obtained by liposuction from human adipose tissue
under physiological and pathological conditions [1,5]. Lipo-
aspirated tissues are digested with collagenase and, after
centrifugation, yield a high-density stromal–vascular fraction
that is subcultured to obtain pure ASCs. Freshly isolated ASCs
display surface markers that differ from those of cultured
ASCs, but both cell preparations are capable of multipoten-
tial differentiation. Recent studies have inferred that ASCs
may reside in a perivascular location where they appear to
co-express CD34 and smooth muscle actin (a-SMA) [33-35].
Paquet-Fifield et al. reported on the role of dermal pericytes
1500 Expert Opin. Biol. Ther. (2009) 9(12)
in promoting epithelial proliferation and differentiation, in
contrast to their well-known ability to regulate endothelial
cell function [36]. Consistent with the reported ability of
pericytes to act as mesenchymal stem cells (MSCs) in a variety
of tissues, the capacity of pericytes to differentiate into the
bone fat and cartilage lineage has been demonstrated [33,36].
Pericytes associated with blood vessels may account for the
presence of MSCs in tissues throughout the body [35]. In cul-
ture, ASCs display fibroblastic characteristics, with an abun-
dant endoplasmic reticulum and a large nuclear-to-cytoplasmic
volume ratio [5,7]. A comparative analysis of MSCs obtained
from bone marrow and adipose tissue clearly demonstrated
that ASCs are equivalent to BMSCs with regard to morphol-
ogy, cell surface receptor profile, and differentiation capac-
ity [37,38]. Both ASCs and BMSCs expressed embryonic stem
cell markers (Oct-4, Rex-1 and Sox-2) for at least 10 pas-
sages [39]. However, analyses of in vitro growth kinetics
revealed a shorter doubling time for ASCs. In addition,
ASCs underwent significantly more population doubling
than BMSCs [39]. Because ASCs are readily accessible, plen-
tiful and expandable, they offer diverse advantages for regen-
eration and replacement of damaged tissues. ASCs express
diverse surface markers, including adhesion molecules, recep-
tor molecules, surface enzymes, extracellular matrix proteins
and glycoproteins [7,9,40]. ASCs are not restricted to dif-
ferentiation along the adipocyte lineage; they can also differ-
entiate into chondrocytes, osteocytes, cardiomyocytes and
neurons in vitro [1,41,42].
2.2 Mechanism of action for skin regeneration
Although stem cell therapy has been safely and effectively used
to repair several types of tissue damage [30,31], the mechanism
underlying tissue regeneration is not well characterized.
Most research to date has focused primarily on stem cell
differentiation. It is generally believed that immature stem
cells migrate to the injured area, differentiate into the phe-
notype of the injured tissue, repopulate the diseased organ
with healthy cells and thereby repair the tissue. However, this
view cannot fully explain the mechanism of regeneration
because the survival of engrafted cells is too low (< 5%) to
be therapeutically relevant [43], and acute functional improve-
ment often occurs too rapidly – within days or even hours
after transplantation especially in skin [5,6,44,45]. In addition,
much of the functional improvement and attenuation of tis-
sue injury afforded by stem cell transplantation can be repro-
duced by treatment with cell-free conditioned medium from
stem cells [46]. This indicates that stem cells may exert their
beneficial effects via complex paracrine mechanisms in addition
to their proposed building-block function.
Altman et al. recently demonstrated that ASCs seeded on
a silk fibroin–chitosan scaffold (SFCS) differentiated into
fibrovascular, endothelial, and epithelial components of
restored tissue and enhanced wound healing [47]. Microvessel
density was significantly higher at wound sites, and engrafted
ASCs were positive for the fibroblastic markers, heat shock

Hypoxia
Survival and proliferation ↑
C
AS
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HIF-1α stabilization
Upregulation of specific growth factors
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Angiogenesis ↑
Figure 1. Responses of ASCs during hypoxia.
protein 47, smooth muscle actin, and von Willebrand factor.
Green fluorescent protein (GFP)-positive stem cells were also
found to differentiate into epidermal epithelial cells. However,
transdifferentiation of ASCs into multiple skin cell types has
not been reported in vitro. Trottier et al. have reconstituted
a skin substitute that produced a more complete tri-layered
skin structure consisting of the epidermis, the dermis and
the adipocyte-containing hypodermis using ASCs in place
of dermal fibroblasts [48], indicating the potential of ASCs
in skin reconstruction.
A number of reports have described a paracrine mechanism
for ASCs in skin. ASCs secrete various growth factors that
control and manage skin-type cells, a process that is essential
to the regenerative function of ASCs [1,2,10]. Our group
examined the paracrine effect of ASCs in various systems
using ASC-conditioned medium (ASC-CM), showing, for
example, that ASC-CM stimulated both collagen synthesis
and migration of primary cultured dermal fibroblasts. In
addition, ASC-CM protected dermal fibroblasts from oxida-
tive stress induced by chemicals or UVB irradiation [1,2], and
treatment of B16 melanoma cells with ASC-CM inhibited
tyrosinase activity and melanin synthesis [10]. In animal stud-
ies, ASC-CM treatment reduced wrinkling and accelerated
wound healing [2,5]. Collectively, these results indicate that
skin regeneration is mediated by factors secreted by ASCs.
Expert Opin. Biol. Ther. (2009) 9(12)
Chung, Won & Sung
Differentiation into adipocyte ↓
Adhesion and migration ↑
Secretion ↑
Wound-healing ↑ Anti-apoptosis ↑
2.3 Proteomic analysis of ASC-CM
Using a two-dimensional electrophoretic (2-DE) analysis,
Roche et al. confirmed that the intracellular proteins of
BMSCs and ASCs were very similar, arguing that these two
cell types are interchangeable with respect to use in cell-based
therapy [49]. Zvonic et al. detected approximately 300 features
in ASC-CM by 2-DE, and showed that secreted proteins are
upregulated and downregulated by induction of adipogen-
esis [50]. Although both intracellular and secretory proteins
of ASCs have been analyzed by several groups, the techniques
used have failed to identify those proteins secreted by ASCs
that are responsible for skin regeneration. This may reflect the
fact that these techniques are limited to the analysis of highly
abundant proteins.
We have also analyzed ASC-CM using the liquid
chromatography–tandem mass spectrometry (LC-MS/MS)
technique, and detected approximately 100 proteins by map-
ping peptides from tryptic digests. However, only five iden-
tified proteins (IGF-binding protein 4 (IGFBP4), IGFBP7,
metalloproteinase inhibitor 1 precursor, IL-6 and IL-8) are
cytokines or growth factors [1]. Recently, we analyzed the
growth factors in ASC-CM using a membrane-based anti-
body array against 41 growth factors, and epithelial growth
factor (EGF), EGF fibroblast growth factor 4 (FGF 4),
IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, G-CSF,
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 Responses of adipose-derived stem cells during hypoxia
GM-CSF, IGF-III, macrophage colony-stimulating factor
(MCSF), platelet-derived growth factor (PDGF), TGF-b
and VEGF [4]. Although this method is limited to growth
factors for which antibodies are commercially available, it has
advantages over the 2-DE-based method in terms of assay
sensitivity. New proteomic analysis techniques in conjunction
with other state-of-the-art analytical tools and follow-up
functional studies are needed to clarify which proteins in
ASC-CM are active in skin repair and regeneration.
3. Cellular and molecular responses of ASCs
during hypoxia
ASCs are typically harvested from the stromal–vascular frac-
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tion of lipo-aspirated tissues. However, the architectural rela-
tionship of ASCs to other cell types and structures in vivo is
still unknown. Despite the fact that adipose tissue is highly
vascularized, in vivo measurements of oxygen concentrations
have shown that the oxygen concentration in adipose tissues is
approximately 3% [18]. Therefore, hypoxic cultures may more
accurately reflect the actual in vivo environment and may
prove beneficial to ASCs during in vitro expansion.
3.1 Proliferation
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Sotiropoulou et al. attempted to establish an optimal proto-
col for the large-scale production of MSCs and identified
several culture conditions that influenced the proliferation of
MSCs [51]. In addition to the quality of the fetal calf serum
used, other culture parameters shown to affect proliferation
included basal medium type, glucose concentration, stable
glutamine, cell-plating density and plastic surface quality [51].
It is also well established that BMSC proliferation is signif-
icantly increased by hypoxia [52]. Similarly, our study dem-
onstrated that the survival and proliferation of ASCs was
influenced by culture medium, plastic surface quality and
oxygen content [11]. Clearly, ASC proliferation was greater
under hypoxic versus normoxic conditions in vitro [21,29,53,54].
An oxygen tension of 1 – 5% during culture is sufficient to
increase the proliferation and function of ASCs, while main-
taining their morphology and multi-lineage capacity. In addi-
tion, ASCs demonstrated the capacity to proliferate in vivo in
response to a hypoxic insult remote from their resident niche,
and showed increasing proliferation in vitro with greater
degrees of hypoxia [55]. Collectively, these results support the
idea that hypoxic culture conditions are favorable for ASCs.
3.2 Differentiation
As described above, studies of the differentiation of ASCs into
multiple skin-cell types are at an early stage, and the effects
of hypoxia on differentiation have not been investigated.
However, there are interesting reports regarding adipogenic
differentiation of ASCs and the effects of hypoxia [56-58].
Adipocytes in human adipose tissue originate by differentia-
tion from resident progenitor cells in the stromal–vascular
fraction of adipose tissue, and oxygen tension may affect ASC
1502 Expert Opin. Biol. Ther. (2009) 9(12)
differentiation. For example, Carriere et al. (2004), reported
that hypoxia (1% oxygen) strongly increased the generation
of reactive oxygen species, up-regulated hypoxia-inducible
factor-1a (HIF-1a), and inhibited adipocyte differentiation
in 3T3 F442A pre-adipocytes [56]. Inhibition of adipogene-
sis at low oxygen tension is associated with activation of
HIF-1a, a transcription factor essential for cellular responses
to decreased oxygen levels [57]. Lin et al. also demonstrated
that hypoxia reversibly arrests ASCs in an undifferentiated
state and maintains the expression of pre-adipocyte factor 1
(Pref-1), which negatively regulates adipogenic differen-
tiation [58]. However, there are controversial reports that
HIF-1a expression of ASCs is stabilized transiently during
the adipogenic process, and hypoxia exerted a potent lipo-
genic effect on MSCs [59-61]. Hypoxia may not simply
reflect an ‘on/off’ process of adipogenesis of ASCs but a
physiological continuum that operates flexibly in response
to microenvironmental conditions.
3.3 Adhesion and migration
It is well known that ASCs have the inherent ability to
preferentially localize to inflamed and injured tissues, where
they aid tissue regeneration [55,62-64]. ASCs adhere to extra-
cellular matrix (ECM) proteins, an interaction that can be
manipulated in vitro to augment this migration. Amos et al.
evaluated the ability of ASCs to bind ECM proteins under
physiological flow conditions [64], and showed that culturing
under hypoxic conditions significantly increased the ability
of ASCs to adhere to vascular cell adhesion molecule-1
(VCAM-1) and endothelial intercellular adhesion molecule-1
(ICAM-1). ASCs also adhere to inflammatory-mediating
adhesion molecules. Importantly, the functional adhesion
capabilities of ASCs are affected by oxygenation levels in the
culture environment. For example, ASCs migrate towards stro-
mal cell-derived factor-1 (SDF-1), a chemokine expressed by
ischemic tissue; in culture, hypoxia augments ASC expression
of SDF-1 receptor and potentiates ASC migration [55]. These
observations highlight the importance of expanding ASCs
under hypoxic conditions, which increases the attachment
and migration/homing potential of transplanted ASCs.
3.4 Intracellular response
The most prominent response of ASCs to hypoxia is a change
in protein expression profile (Table 1). The levels of HIF-1a
are regulated mainly by the available oxygen; under normoxic
conditions, HIF-1a degradation is rapid. As shown in
Figure 2, HIF-1a expression in ASCs was clearly detected
under hypoxic culture conditions, but was scarcely present
under normoxic conditions. Under low oxygen tension (2%),
HIF-1a stabilization has been shown to enhance the expres-
sion of target genes, including those related to angiogenesis,
cell proliferation, and energy metabolism [65-69]. Consistent
with this, our group showed, using quantitative reverse tran-
scriptase-polymerase chain reaction (RT-PCR), that cellular
levels of VEGF, bFGF and IGF were significantly increased
 Chung, Won & Sung

Table 1. Hypoxia-induced changes in protein expression proliferation, angiogenesis and apoptosis [70]. This highlights
in ASCs. the importance of further studies on the use of oxygen-
tension regulation as a tool for preparing ASCs in a way that
Oxygen Protein level Ref. allows the full potential of these cells to be exploited.
concentration

5% Collagen type II protein, total [79] 3.5 Alteration of secretion by hypoxia

collagen synthesis In a preliminary study, we measured total protein con-
5% Collagen type II, IX, XI, SOX6, [29] centration in ASC-CM using the Bradford method, and
SOX5, SOX9, HIF2a found that there was no difference in the level of total
5% Collagen type II, SOX9, collagen X [80] secreted proteins between normoxic (1.02 ± 0.04 µg/ml)
1% Leptin [70] and hypoxic (1.01 ± 0.01 µg/ml) conditions. Because
1% VEGF [77] intracellular levels of some growth factors are increased
5%, 1% VEGF, SDF-1, SDF-1 receptor [55]
in ASCs by hypoxia ( Table 1), we measured secreted
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(CXCR4) proteins in ASC-CM using ELISA. Hypoxia significantly

Not described IGF-1, nuclear b-catenin [81] upregulated some proteins in the conditioned medium,
Not described VEGF, IGF-1 [82]
including VEGF (2.8-fold) and bFGF (1.8-fold). IGF was
upregulated intracellularly but was not detected among the
Not described c-kit [83]
secreted proteins [11] . Using a commercially available
Not described HIF-1a [84]
membrane-based antibody array for 41 growth factors,
5% VEGF, HIF-1a, leptin, matrix [85]
we recently determined which growth factors were upre-
metalloproteinases 2 and 9
gulated in conditioned medium by hypoxia. Most growth
factors were upregulated in conditioned medium harvested
from hypoxic cultures (hypoCM) with some exceptions.
Normoxia Hypoxia The signal intensities of IGFBP1, IGFBP2, macrophage
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colony stimulating factor (M-CSF), M-CSF receptor

HIF-1α (120 kDa)
α-tubulin (55 kDa)
Figure 2. Protein expression of hypoxia inducible factor
(HIF)-1a in ASCs. HIF-1a was clearly detected after culture under
hypoxic conditions (2% oxygen) for 48 h, while that was not
detected under normoxic condition (our unpublished data).
(over 1.5-fold) by 2% hypoxia, but mRNAs for other
growth factors, including TGF-b, hepatocyte growth factor
(HGF), keratinocyte growth factor (KGF) and PDGF-A,
were not induced by low oxygen tension. Rubina et al. also
evaluated the expression of angiogenesis-related growth fac-
tor genes in ASCs by quantitative PCR, showing that expres-
sion of VEGF, bFGF, HGF, c-Met and PDGF-B mRNA
were significantly increased by hypoxia (1% oxygen) [67].
Martin-Rendon et al. reported that ASCs respond to hyp-
oxic stimuli by upregulating the genes for angiogenic and
anti-apoptotic factors, primarily through stabilization of
HIF-1a [65]. Activation of HIF-1a-mediated signaling is
complex and is regulated by a cascade of molecular events.
HIF-1a accumulates, dimerizes with HIF-1b, recruits the
co-activators CBP/p300, and activates the transcription of
genes, including VEGF and angiopoietins [66].
Recently, Pilgaard et al. used microarray analyses to
measure genetic changes in ASCs during hypoxia. Exposure
to 1% oxygen produced a specific expression profile: 2581
genes were differentially expressed, and there was a substan-
tial enrichment in genes involved in cell growth and
(MCSF R), PDGF receptor-b, and VEGF were signifi-
cantly greater in hypoCM than in conditioned medium
harvested under normoxic conditions (norCM)
(p < 0.05, Figure 3 ). By contrast, EGF was reduced
by almost 70% in hypoCM relative to norCM.
4. Functional enhancement by hypoxia
As described above, culturing ASCs under the more physio-
logically relevant conditions of low oxygen tension during
expansion may manifest as beneficial effects on proliferation,
adhesion, migration and specific gene expression and secre-
tion. Altered characteristics acquired during hypoxia may
increase the regenerative potential of ASCs, as evidenced by
the enhanced angiogenic, anti-apoptotic and wound healing
potential in ASCs cultured in hypoxia.
4.1 Angiogenesis
Recent reports describing the ability of ASCs to differentiate
into vascular endothelial cells have inspired a number of
researchers to investigate the use of ASCs to enhance neo-
vascularization in the treatment of ischemic disorders [71-73].
These studies have demonstrated that human and murine
adipose-derived stromal cells can incorporate into blood
vessels by differentiating into endothelial cells, and shown
that the indirect effects of angiogenic growth factor released
from ASCs may enhance the recovery from perfusion in a
murine model of hind-limb ischemia. Lu et al. also reported
that ASCs have the potential to enhance the blood supply in
random-pattern skin flaps [74].

Expert Opin. Biol. Ther. (2009) 9(12) 1503

 Responses of adipose-derived stem cells during hypoxia
norCM
Pos Neg
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Figure 3. Growth factor antibody array. Secretory factors in conditioned medium harvested from normoxic cultures (norCM) and
hypoxic cultures (hypoCM) were concentrated and incubated with a membrane containing antibodies against 41 growth factors. With the
exception of EGF (highlighted in blue), most growth factors were highly represented in hypoCM. Growth factors significantly increased by
hypoxia (p < 0.05) are marked in red (our unpublished data).
1.2
Fold fibroblast viability
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1 structures [67]. The pro-neovascular activity of ASCs may be
* upregulated via a paracrine mechanism in response to hypoxia,
0.8
0.6
0.4
0.2
0
No treatment Control norCM
tbOOH treatment
Figure 4. Anti-apoptotic effect of conditioned medium
harvested under normoxia (norCM) or hypoxia (hypoCM).
Tert-butyl hydroperoxide (tbOOH) treatment reduced the survival
of dermal fibroblasts, an effect that was reversed by ASC-CM.
HypoCM had a more potent anti-apoptotic effect than did norCM
(our unpublished data).
Hypoxia reportedly enhances the neoangiogenesis of ASCs
via a paracrine mechanism that involves increased secretion
of certain growth factors [71,75,76]. For example, hypoCM
significantly increased endothelial cell growth and reduced
endothelial cell apoptosis. One growth factor that played an
essential role in the cellular response to hypoxia was VEGF,
which was secreted at fivefold higher levels in hypoCM and
induced angiogenesis and anti-apoptosis [71]. The a-subunit
of HIF-1 is stabilized under low oxygen tension and regu-
lates the expression of VEGF, which promotes the adoption
of functional characteristics necessary for new blood vessel
growth and maturation [77]. Rubina et al. showed that ASCs
stimulate vessel-like structure formation and stabilization in a
1504
hypoCM
Pos Neg
EGF
IGFBP-1 IGFBP-2
M-CSF M-CSF R PDGF R β
VEGF
Matrigel implant model, and demonstrated that hypoxia-
cultured ASCs showed enhanced formation of capillary-like
and function cooperatively with the existing vasculature to
promote angiogenesis.
4.2 Anti-apoptotic effect
Emerging evidence indicates that ASCs can be used for the
hypoCM treatment of skin diseases related to oxidative stress [1-3]. Both
chronologic and extrinsic aging are associated with excessive
production of free radicals, molecules containing one or more
unpaired electrons. When allowed to remain in their highly
reactive state, these compounds can damage cell membranes,
proteins and DNA. However, the components of the antiox-
idant defense system in human skin are regulated and pro-
tected in a complex manner. One such mechanism by which
ASCs protect dermal fibroblasts from oxidative damage is by
reducing apoptosis [1-3]. The anti-apoptotic effect of ASCs was
demonstrated by incubating primary cultured fibroblasts in
ASC-CM. Morphological changes and cell survival assays
revealed that incubation with ASC-CM protected dermal
fibroblasts from free radicals induced by tert-butyl hydro-
peroxide (tbOOH) and UVB [1,2]. In a cell-cycle analysis,
ASC-CM decreased the fraction of sub-G1 cells relative to
controls and reversed chemical- and UVB-induced apoptotic
cell death.
Modulation of the anti-apoptotic effect of ASCs by
hypoxia was further studied in the same experimental sys-
tem using hypoCM and norCM. tbOOH treatment reduced
the survival of dermal fibroblasts; however, this effect was
reversed by ASC-CM. The anti-apoptotic effect was greater
in hypoCM than in norCM (Figure 4). Secretion of VEGF
Expert Opin. Biol. Ther. (2009) 9(12)

and IGFBPs was significantly increased by hypoxia (Figure 3),
indicating a possible mechanism for the protection against
free radicals.
4.3 Wound healing
Under physiological conditions, ASCs may be frequently
found within the stromal–vascular fraction of subcutaneous
adipose tissue where they have ready access to oxygen.
However, capillary injury during wound healing generates
a hypoxic environment. In general, ASCs are involved in
wound-healing processes and ASC-mediated wound regener-
ation is accelerated by hypoxia [11]. In this study, wound-
healing potential was determined by comparing collagen
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synthesis and migration of dermal fibroblasts following
exposure to hypoCM and norCM. HypoCM had a greater
stimulatory effect on type I collagen secretion and migration
of dermal fibroblast than did norCM. In animal studies,
hypoCM treatment significantly reduced wound area size
and depth compared with norCM.
Because hypoxia significantly increased the secretion of
VEGF and bFGF, we suggested that these factors are respon-
sible for the hypoxia-induced enhancement of wound-healing
function. To test this, we used neutralizing anti-VEGF and
anti-bFGF antibodies. The migration of dermal fibroblasts
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was significantly decreased by the addition of neutralizing
antibodies against VEGF and bFGF [11]. Consistent with these
in vitro experiments, the wound-healing effect of ASC-CM was
significantly reduced in an animal model by the addition of
antibodies to both VEGF and bFGF [11]. Thus, the hypoxia-
induced enhancement of ASC-mediated wound healing may
be due, at least in part, to secreted VEGF and bFGF.
5. Expert opinion
Typically, ASCs are cultured under ambient, or normoxic,
conditions. However, the oxygen tension in the physiological
Expert Opin. Biol. Ther. (2009) 9(12)
Chung, Won & Sung
niches occupied by ASCs is much lower. Thus, hypoxic
culture conditions can function as a ‘prophylactic’ signal,
increasing proliferation, adhesion and migration of ASCs.
In addition, HIF-1a expression is significantly increased
by hypoxia, and angiogenic and anti-apoptotic growth fac-
tors are upregulated. Collectively, the cellular and molecular
changes induced by hypoxia enhance the skin-regenerative
potential of ASCs through effects on functions such as
angiogenesis, anti-apoptosis and wound healing. Thus,
when used as a therapeutic tool to repair skin injuries,
ASCs must be cultured under low-oxygen conditions. How-
ever, given the potential cancer-inducing risks, the clinical
application of cultured ASCs in human skin can be hazard-
ous [78]. In addition, industrial-scale handling and commer-
cialization of ASCs are problematic. Instead, ASC secretory
factors can be used and provide a number of advantages over
cell-based therapies. Because secretory factors can be stored
long-term in a stable form and are relatively devoid of safety
issues, they are suitable for commercialization. Harvesting
conditioned medium for skin regeneration (i.e., to produce
growth factors for cosmetic raw materials) would simply
require predetermination of optimal oxygen tension to max-
imize the content of regenerative proteins in the conditioned
medium. In short, hypoxia induces cellular and molecular
changes in ASCs and improves their skin-regenerative
potential.
Acknowledgments
Figure 1 and Table 1 were kindly edited by JA Yang.
Declaration of interest
This study was supported by a grant from the Korea
Healthcare Technology R&D project, Ministry for Health,
Welfare & Family Affairs (A0851360).
1505
 Responses of adipose-derived stem cells during hypoxia
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Affiliation
Hyung-Min Chung1,2,3, Chong-Hyun Won4 &
Jong-Hyuk Sung†1,2
†
Author for correspondence
1
CHA Stem Cell Institute,
Stem Cell Research Laboratory,
Seoul, Republic of Korea
2
CHA University,
Department of Biomedical Science,
#606-16, Yeoksam-dong,
Kangnam-gu, Seoul, 135-081, Republic of Korea
Tel: +82 2 3468 3491; Fax: +82 2 3468 3373;
E-mail: brian99@empal.com
3
CHA bio and Diostech Co., Ltd.,
Seoul, Republic of Korea
4
University of Ulsan College of Medicine,
Asan Medical Center,
Department of Dermatology,
Seoul, Republic of Korea