Mitochondrion 19 (2014) 105–112

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Mesenchymal stem cells and hypoxia: Where are we? L.B. Buravkova a,b, E.R. Andreeva a, V. Gogvadze b,c, B. Zhivotovsky b,c,⁎ a b c

Institute of Biomedical Problems, Russian Academy of Sciences, Moscow, Russia Faculty of Basic Medicine, MV Lomonosov Moscow State University, 119991 Moscow, Russia Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177 Stockholm, Sweden

a r t i c l e

i n f o

Available online 15 July 2014 Keywords: Mesenchymal stem cells Hypoxia Mitochondria Cell death

a b s t r a c t Multipotent mesenchymal stromal cells (MSCs) are involved in the organization and maintenance of tissue integrity. MSCs have also attracted attention as a promising tool for cell therapy and regenerative medicine. However, their usage is limited due to cell impairment induced by an extremely harsh microenvironment during transplantation ex vivo. The microenvironment of MSCs in tissue depots is characterized by rather low oxygen consumption, demonstrating that MSCs might be quite resistant to oxygen limitation. However, accumulated data revealed that the response of MSCs to hypoxic conditions is rather controversial, demonstrating both damaging and ameliorating effects. Here, we make an attempt to summarize recent knowledge on the survival of MSCs under low oxygen conditions of varying duration and severity and to elucidate the mechanisms of MSC resistance/ sensitivity to hypoxic impact. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Multipotent mesenchymal stromal cells (MSCs) are a subject of increasing scientific interest due to their key role in the organization and maintenance of tissue integrity and their involvement in physiological/ pathological tissue repair. These cells play an important role in hematopoiesis as a part of the hematopoietic stem cell microenvironment, and also constitute a self-renewing cell population with the ability to differentiate into distinctive end-stage cell types that produce specific mesenchymal tissues including bone, cartilage, muscle, bone marrow stroma, tendon/ligament, fat, dermis, and other connective tissues (Barry and Murphy, 2004; Bruder et al., 1998; Caplan, 2007; Friedenstein et al., 1968). As a key and constitutive element of tissue homeostasis MSCs are subjected to different stimuli that may cause their damage and even death, especially at the site of tissue injury. Recently, MSCs have also attracted attention as a promising tool for cell therapy and regenerative medicine. However, their usage is limited due to cell impairment induced by an extremely harsh microenvironment during transplantation ex vivo. It is believed that ischemic conditions, i.e. a lack of both nutrients and an oxygen supply, can cause MSC death and therapeutic failure (Noort et al., 2010). On the other hand, the microenvironment of MSCs in tissue depots is characterized by a rather low O2 partial pressure, demonstrating that MSCs might be quite resistant to oxygen limitation. It is generally recognized that hypoxia activates many stress and ⁎ Corresponding author at: Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden. Tel.: + 46 8 524 87588; fax: + 46 8 329041. E-mail address: [email protected] (B. Zhivotovsky).

http://dx.doi.org/10.1016/j.mito.2014.07.005 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

survival pathways. In the case of MSCs, the data on their response to hypoxic conditions are rather controversial, demonstrating both damaging and ameliorating effects. Here, we make an attempt to summarize recent knowledge on the survival of MSCs under low oxygen conditions of varying duration and severity and to elucidate the mechanisms of MSC resistance/sensitivity to hypoxic impact. 2. Cellular response to hypoxia During the past few decades, significant progress has been made in understanding cell responses to decreased oxygen concentrations and the mechanisms underlying this response. Convincing evidence now exists that every cell is sensitive to changes in the oxygen concentration in the surrounding medium. Alterations in the oxygen concentration represent a physiological stimulus, which triggers certain intracellular mechanisms responsible either for cell death or for cell adaptation to new environmental conditions. Oxygen deprivation and hypoxic conditions can be deleterious to cells because of hypoxia-mediated, p53dependent cell death (Graeber et al., 1996). However, some cells, in particular, tumor cells, can survive under hypoxic conditions because of mutations in p53 or its low expression (Levine, 1989). The key adaptive response to hypoxic conditions is the stabilization of hypoxia inducible factor (HIF)-1 (Wang and Semenza, 1993). HIF-1 is a heterodimer that consists of a constitutively expressed HIF1β subunit and a HIF-1α subunit; the expression of the latter is highly regulated and is determined by the relative rates of its synthesis and degradation. Synthesis of HIF-1α is regulated via oxygen-independent mechanisms, whereas its degradation is oxygen-dependent (Semenza, 2003). Specifically, in the presence of oxygen, HIF-1α chains are

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polyhydroxylated on conserved prolyl and arginyl residues by oxygendependent prolyl and arginyl hydroxylases. Once hydroxylated, HIF-1α protein binds to von Hippel–Lindau (VHL) tumor suppressor protein — the recognition component of E3 ubiquitin-protein ligases. Ubiquitinated HIF-1α is rapidly degraded by the proteasome. Under conditions of low oxygen tension (hypoxia or ischemia), hydroxylation is suppressed, which causes the stabilization of HIF-1α and its accumulation in the nucleus, where it forms a complex with the constitutively expressed HIF-1β (Semenza, 2004). In addition, HIF-1 alters mitochondrial function suppressing mitochondrial respiration (Papandreou et al., 2006). This suggests that HIF-1 might play the role of a switch between glycolysis and oxidative phosphorylation. Several mechanisms may be involved in such regulation. The fate of pyruvate, the end-product of glycolysis, depends on the relative activities of two enzymes, pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH). PDH converts pyruvate into acetyl CoA, which enters the Krebs cycle where it undergoes stepwise transformation. The activity of PDH is controlled by pyruvate dehydrogenase kinase 1 (PDK1), the enzyme that phosphorylates and inactivates PDH. HIF-1 has been shown to upregulate PDK1 thereby inactivating PDH (Kim et al., 2006). Under these circumstances, instead of supplying reduced equivalents to mitochondria, pyruvate undergoes conversion into lactate, reoxidizing cytosolic NADH which facilitates glycolysis. In addition HIF-1 stimulates expression of the LDH-A (Semenza et al., 1996), whose product converts pyruvate into lactate. Taken together, these factors suppress the delivery of acetyl-CoA to the Krebs cycle and mitochondrial respiration. According to the latest data, HIF-1 induction in response to hypoxia occurs almost immediately. Experiments on the HeLa S3 cell line have shown that under hypoxic (0.02–5% O2) or anoxic (~0% O2) conditions, HIF-1α can be detected in the cell nucleus within 2 min after exposure. The HIF content in the nucleus increases rapidly within 30 min, whereupon its accumulation rate decreases; the maximal level of HIF is observed after 60 min of exposure (Jewell et al., 2001). Using perfused and ventilated lung preparations subjected to hypoxia/reoxygenation it was demonstrated that the half-life of HIF does not exceed 1 min, which is the shortest half-life among proteins (Wenger, 2002). This means that HIF accumulation in the cell nucleus under hypoxia occurs almost immediately and represents a crucial mechanism of cell survival. It also means that the detection and verification of HIF-1 are rather intricate tasks. HIF plays an important role in MSC physiology under hypoxic conditions. It is the main regulator controlling the metabolic fate and multipotency of MSCs. The stabilization of HIF-1α exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation without affecting the proliferation of the MSC population. Moreover, HIF-1α stabilization in MSCs leads to the induction of pluripotent genes (Oct-4 and klf-4) and the inhibition of terminal differentiation into osteogenic and adipogenic lineages (Park et al., 2013). Experiments performed with human MSCs isolated from three origins, i.e. adult and pediatric bone marrow and umbilical cord blood (UCB), revealed that pediatric bone marrow and UCB MSCs show stabilization of HIF-1α under normoxic conditions, when it should be degraded completely (Palomaki et al., 2013). This was explained by a high expression level of HIF-1α mRNA rather than compromised proteasomal degradation of the HIF-1α protein. In all three MSC populations high normoxic HIF levels led to increased expression of various glycolytic HIF target genes and stimulated glycolysis. Hypoxia has been shown to enhance mouse MSC radioresistance in vitro, increase the MSC proliferation rate and colony size, increase long-term survival after irradiation, and improve MSC recovery from radiation-induced cell cycle arrest. HIF-1α has been found to contribute to the enhanced double strand base repair by regulating the expression of DNA ligase IV and DNA-PK, and promote Rad51 foci formation in response to DNA double strand break in hypoxic MSCs (Sugrue et al., 2013). Thus, stabilization of HIF under hypoxic conditions represents a key event in MSC adaptation to hypoxia.

3. The role of hypoxia in MSC biology The effects of a low oxygen environment on MSC behavior have been intensively studied in vitro in the last 20 years. Despite the variety of experimental protocols, the accumulated data can be divided in two main pools according to the duration of exposure to hypoxia: a) MSCs grown at ambient oxygen (20% O2) then exposed to acute short-term hypoxia (up to 72 h) and b) MSCs permanently cultured at a low oxygen level. 3.1. MSCs after short-term hypoxic exposure To discuss the currently available experimental data on short-term hypoxia, it is necessary to specify the duration of such a temporary impact and the severity of hypoxic exposure. The exposure time usually varies from 0 to 72 h with an oxygen concentration from 0 to 5% (Fig. 1). Such significant variations in experimental design lead to certain difficulties in the comparative analysis of the data. Analyzing the available data on the “oxygen drop”, one can speculate that the earliest MSC response to short-term hypoxia is the impairment of cell functions and even stimulation of cell death. Thus, an increase in the number of apoptotic cells after 3–24 h of hypoxic treatment (0.5–5% O2) has been detected in MSCs derived from rats (Zhu et al., 2006). This was the first report on the proapoptotic effect of hypoxia on MSCs, although it should be mentioned that in these experiments cells were subjected not only to hypoxia, but also to serum deprivation. Therefore, it is hard to draw any conclusion on the impact of the hypoxic conditions alone. Similar results were later described by others groups (Chacko et al., 2010; Peterson et al., 2011). When hypoxia was combined with serum deprivation (ischemic condition), the number of apoptotic cells increased to 50% (Nie et al., 2011; Zhang et al., 2009). Twenty four hours of severe hypoxia (~0% O2) caused cell death by both apoptosis and necrosis in up to 70% of rat MSCs (Chang et al., 2009). Inhibition of proliferation in human bone marrow (BM) MSCs and murine adiposederived MSCs was observed after 48 h of incubation at 1% O2 (Efimenko et al., 2011; Hung et al., 2007). Surprisingly, a slight increase in the duration of hypoxia evoked the opposite effects, as an increase in the proliferation rate was observed after 3–4 days in 1% O2 in human umbilical cord and BM-MSCs (Lavrentieva et al., 2010). Additionally, despite the inhibition of MSC proliferation, the migratory activity of MSCs both in wound healing and in Boyden chamber assays was enhanced after short-term (6–20 h) hypoxic treatment (Busletta et al., 2011; Hung et al., 2007; Lee et al., 2010). The results mentioned above clearly demonstrate that short-term hypoxia is a stress factor to MSCs (Fig. 1). It also appears that MSCs possess compensatory mechanisms that are rapidly evoked under stress conditions, such as oxygen limitation. It has been demonstrated that temporary exposure of MSCs to a hypoxic environment induces a rapid (within 1 h) translocation of HIF-1α to the nucleus (Kanichai et al., 2008). Nuclear localization of HIF-1 was also demonstrated after 6–12 h (Lee et al., 2010), 24 h (Chacko et al., 2010; Deschepper et al., 2011; Hu et al., 2008; Peterson et al., 2011) and even 72 h (Lavrentieva et al., 2010) after exposure to hypoxia. Stabilization of HIF-1 induces various signaling and metabolic pathways in MSCs, providing a cellular response to the hypoxic trigger. Thus, after 24 h in 1% O2, a decrease was observed in the level of survivin, a negative regulator of apoptosis (Busletta et al., 2011); however, Chacko et al. (2010) under the same experimental conditions showed an increase in the level of this antiapoptotic protein. Hypoxic conditions also cause a mild increase in the pAkt/Akt (Chacko et al., 2010; Peterson et al., 2011) and PI3K/PTEN ratios (Peterson et al., 2011; Xu et al., 2008). Temporary hypoxia also decreases the ratio of phospho/non-phospho-ERK 1–2 (Crisostomo et al., 2008; Peterson et al., 2011) and causes a slight although significant elevation in the Bcl-2/Bax ratio (Peterson et al., 2011). Data on the regulation of HIF-dependent kinases in MSCs are rather controversial. Hypoxia-induced phosphorylation of p38 mitogenactivated protein kinase (MAPK) has been described (Lee et al., 2010),

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HYPOXIC IMPACT

MSCs

Short-term exposure

0,5-10% O2 Duration, weeks

0-5% O2 Duration, hours 72

48

, = viability ,

24 ,

migration

,

HIF1a ,

1

p38 MAPK

proliferation

“stemness” genes Oct-4, Rex-1, Nanog, SSEA-4 etc

survivin

Bcl-2/Bax

CFU-F

chondrodifferentiation

NF-kB

pERK 1-2/ERK 1-2ratio

3…

osteo- and adipodifferentiation

SAPK/JNK

pAkt/Akt, PI3K/PTEN ratio

2

, = viability

ROS

proliferation

Permanent exposure

Angiogenic activity Anaerobic glycolysis ATP production

VEGF, FGF2, HGF, and IGF-1

Fig. 1. Schematic representation of short- and long-term hypoxic effects on MSCs. The cellular response to short-term exposure is to a certain extent controversial, displaying both hampering and stimulating outcomes. Permanent expansion under hypoxia provides maintenance of low differentiated state of MSCs. In filled ellipse the common for both impacts phenomena are highlighted.

but downregulation of p38 MAPK has also been demonstrated (Peterson et al., 2011). Hypoxia induces stress-activated protein kinase/c-Jun NH2terminal kinase (SAPK/JNK) (Busletta et al., 2011; Lee et al., 2010), but others have demonstrated the inhibition of this enzyme (Chang et al., 2009; Zhang et al., 2009). Hence, the cellular response to an acute oxygen drop is a very dynamic process with a very sensitive balance of pro/ antiapoptotic pathways. Another critical factor associated with oxygen deprivation is increased oxidative stress, which can lead to mitochondrial dysfunction. The level of reactive oxygen species (ROS) may increase under hypoxic conditions, when electron transport complexes are in the reduced state (Guzy and Schumacker, 2006). The main source of ROS under these circumstances is Complex II of the mitochondrial respiratory chain (Guzy et al., 2005). Additionally, an enhancement in ROS formation (Busletta et al., 2011; Lee et al., 2010; Peterson et al., 2011; Zhang et al., 2009) can result from increased expression of the oxidative stress enzyme NAD(P)H oxidase under hypoxic conditions (Peterson et al., 2011). An additional factor contributing to the increased ROS content is a decrease in the expression of the anti-oxidant enzyme catalase, a major metabolizer of ROS, while the levels of SOD and its subunits are unaltered by hypoxic challenge. A decrease in the endogenous scavenger enzyme catalase will result in intracellular accumulation of hydrogen peroxide, with deleterious effects on the cell (Peterson et al., 2011). Short-term hypoxia evokes a significant decrease in MSC mitochondrial transmembrane potential (Nie et al., 2011; Zhang et al., 2009; Zhu et al., 2006) and causes cytochrome c release (Zhang et al., 2009; Zhu et al., 2006). The level of intracellular ATP decreases (Chang et al., 2009; Deschepper et al., 2011), and energy metabolism is switched to anaerobic glycolysis with significantly enhanced glucose consumption and corresponding lactate production (Deschepper et al., 2011; Lavrentieva

et al., 2010). Elevated expression of energy metabolism-associated genes including GLUT-1, LDH, and PDK1 has been demonstrated (Lavrentieva et al., 2010). Another factor which might be involved in the MSC response to acute hypoxia is nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), a protein complex that controls the transcription of DNA (Fig. 1). NF-κB is found in almost all animal cells and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized low-density lipoprotein, and bacterial or viral antigens. Short-term hypoxic exposure leads to NF-κB activation in MSCs and a subsequent increase in vascular endothelial growth factor (VEGF), FGF2, HGF, and IGF-1 expression (Crisostomo et al., 2008). Thus, short-term oxygen limitation activates various intracellular mechanisms responsible for the development of the adaptive response including changes in gene expression. It is clear that hypoxia switches on various signaling pathways, which can be interrelated and interdependent, and this determines the overall cell response. The switching on of all the above described intracellular mechanisms modifies the MSC population, inducing alterations in MSC functional properties in a constant low oxygen microenvironment. 3.2. Permanent expansion at hypoxia An important component of the stem and progenitor cell microenvironment is low oxygen tension which provides signals that are conducive to the maintenance of stem-cell functions (Buravkova et al., 2009). However, in many studies, cells are usually cultured under normoxic conditions equivalent to atmospheric oxygen (20%). Permanent cell maintenance under hypoxia should reflect the physiological conditions more adequately.

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Numerous studies have demonstrated that MSCs constantly propagated at low oxygen (1–5%) generally display enhanced proliferative potential (Basciano et al., 2011; D'Ippolito et al., 2006; Dos Santos et al., 2010; Fehrer et al., 2007; Grayson et al., 2007; Grinakovskaya et al., 2009; Iida et al., 2010; Lopez et al., 2013; Malladi et al., 2006; Nekanti et al., 2010; Valorani et al., 2010; Volkmer et al., 2010; Zhambalova et al., 2009; Zhao et al., 2010; Zscharnack et al., 2009) and greater colony-forming activity as compared to cells cultivated under normoxic conditions (20% O2) (Dos Santos et al., 2010; Fehrer et al., 2007; Iida et al., 2010; Lopez et al., 2013; Zscharnack et al., 2009). However, these effects can be driven by specific circumstances. It has been demonstrated that at passage 0, the colony formation capacity of MSCs is markedly decreased in hypoxia: the colonies were fewer and smaller than the colonies that formed under normoxic conditions (Basciano et al., 2011). In addition, hypoxic cells contained fewer mitochondria as compared to normoxic cells, pointing to a prominent alteration in energy metabolic pathways caused by hypoxia. However after 2–3 passages under hypoxic conditions, the numbers of colonies increased. The attenuation of cell growth was observed when MSCs were induced to undergo chondrogenic differentiation under low O2 (Khan et al., 2007) or when proliferation rate was measured in the passage directly after cell explantation (Basciano et al., 2011). The viability of MSCs at low oxygen (5%) was the same (Buravkova et al., 2009; Grinakovskaya et al., 2009) or even higher than that under normoxic conditions (Buravkova and Anokhina, 2007; Valorani et al., 2010). The capacity for multilineage differentiation is one hallmark of MSCs. It is well established that under specific conditions, MSCs can differentiate into osteoblasts, adipocytes, and chondroblasts. However, incubation at a low oxygen level (2%) reduces the osteogenic differentiation of murine AT-MSCs (Malladi et al., 2006) and attenuates the osteogenic and adipogenic differentiation of human AT-MSCs (Lee and Kemp, 2006). Significant attenuation of osteogenic and adipogenic differentiation was demonstrated at the gene expression and corresponding product level. Alkaline phosphatase activity (D'Ippolito et al., 2006; Fehrer et al., 2007) and calcium matrix formation (D'Ippolito et al., 2006; Fehrer et al., 2007; Grinakovskaya et al., 2009; Iida et al., 2010; Malladi et al., 2006; Valorani et al., 2012; Volkmer et al., 2010) were diminished as well as the expression of the corresponding genes ALP (Iida et al., 2010; Volkmer et al., 2010), OPN (Volkmer et al., 2010), RUNX2 (D'Ippolito et al., 2006; Merceron et al., 2010; Yang et al., 2011; Zscharnack et al., 2009), and IBSP (Dos Santos et al., 2010; Fehrer et al., 2007). Adipocyte formation, identified as intracellular lipid droplet accumulation, was also significantly reduced. The mRNA expression of adipogenesis-related genes LPL and FABP4 (Dos Santos et al., 2010; Fehrer et al., 2007; Valorani et al., 2010) was downregulated. Moreover, decreased differentiation activity was revealed when MSCs were first cultivated in 20% O2 for 1–2 weeks, then subjected to differentiation at low O2 (Volkmer et al., 2010). In contrast to the abovementioned differentiation routes, the chondrogenic potential of hypoxic MSCs is strongly elevated. It has been demonstrated that low oxygen favors the chondrogenic differentiation of MSCs, as evidenced by increased production of glycosaminoglycan, chondroitin-4-sulfate (Lee and Kemp, 2006), aggrecan, and collagen types II, IX, and XI (Annabi et al., 2003; Lopez et al., 2013) and the significantly upregulated expression of the chondrogenic markers SOX6, SOX5, SOX9 (Buravkova et al., 2013; Estrada et al., 2012; Lee and Kemp, 2006; Lopez et al., 2013), COL2A1, and AGC1 (Buravkova et al., 2013; Rylova and Buravkova, 2014) (Fig. 1). Under reduced oxygen, MSCs also display angiogenic activity. At 1% O2, murine bone marrow MSCs rapidly migrate and form threedimensional capillary-like structures in Matrigel, synthesize more VEGF, and downregulate matrix metalloproteinase (MMP)-2 mRNA expression and protein secretion, while the expression of membrane-type (MT)1-MMP is strongly induced by hypoxia (Annabi et al., 2003). Capillary-like structures have also been demonstrated in hypoxic (5%)

cultures of human bone marrow MSCs (Zhambalova et al., 2009) and adipose tissue MSCs. However, several recent studies of hypoxia-propagated MSCs have presented results that seem to be a bit out of the “mainstream” discussed above (Fig. 1). These data should also be considered to obtain a complete idea concerning in vitro capacities of MSCs. Thus, a decrease in MSC viability at 1% O2 has been demonstrated for sheep MSCs after 12 days without medium replacement (Deschepper et al., 2011) and for rat MSCs after 7 days in culture (Zhang et al., 2009). There are data describing the opposite effects of long-term hypoxia on MSC differentiation capacity. In particular, incubation of human MSCs under hypoxic conditions results in an enhanced ability to differentiate into adipocytes and osteocytes (Valorani et al., 2012). It has been demonstrated that MSCs produce more osteomatrix and intracellular lipid droplets, and the expression of differentiation-related genes RUNX2, ALP, PPARgamma and LPL is up-regulated (Basciano et al., 2011; Grayson et al., 2007). Equal MSC osteo- and adipo-potential has been demonstrated under both hypoxic and normoxic conditions (Dos Santos et al., 2010). Incubation at low oxygen levels (2%) has also been shown to inhibit the chondrogenic differentiation of murine AT-MSCs (Malladi et al., 2006). The analysis of the metabolic activities of MSCs under permanent hypoxia has revealed an increase in the consumption of glucose and glutamine and production of lactate as a consequence of switching cell metabolism from oxidative phosphorylation to anaerobic glycolysis (Dos Santos et al., 2010). The metabolic shift to glycolysis is accompanied by reduced mitochondrial transmembrane potential (Buravkova et al., 2013). The importance of various metabolic pathways in generating ATP for MSC function and survival is still poorly understood. It is thought that the activation of glycolysis in MSCs at low oxygen tension may improve their growth and genetic stability, while at 20% O2, stem cell physiology is disturbed due to abnormally increased oxidative stress (Estrada et al., 2012). An increased proliferative activity and attenuated differentiating potential beyond doubt support the assumption that hypoxia may modulate MSCs, favoring self-renewal and multipotency. RT-PCR data have revealed the up-regulation of “stemness” gene expression, e.g. Oct-4, Rex-1 (D'Ippolito et al., 2006; Zhao et al., 2010), STRO-1 (Iida et al., 2010), Sca-1 + and Sca-1 +/CD44 + (Valorani et al., 2010), Sox2 (Valorani et al., 2010; Zhao et al., 2010), Nanog (Fehrer et al., 2007; Valorani et al., 2010), and SSEA-4 (D'Ippolito et al., 2006). Enhanced expression of the gene group involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, and LAMA) and epithelial development (DMKN) assigned by the Gene Ontology program to “plasticity” has also been described (Basciano et al., 2011) (Fig. 1). Using the Human Stem Cell Pluripotency Array, it was demonstrated that early mesodermal/endothelial (CD34, ACTC), stemness (CRABP2, DNMT3B, GRB7, IFITM1, KIT, LIN28, IMP2 and IL6ST), and mesoderm lineage (NOGGIN, DES, COL1A, and DESMIN) genes were upregulated with low oxygen levels, suggesting that MSCs under hypoxic conditions possess the properties of true stem cells (Nekanti et al., 2010). Thus, it may be likely that long-term in vitro hypoxia enhances a genetic program that maintains the MSCs in an undifferentiated state and in parallel stimulates the expression of genes involved in the development of various, mesodermal and non-mesodermal, cell lineages. In this respect, hypoxia may increase self-renewal, multipotency and the transdifferentiation potential of MSCs. Considerable data clearly demonstrate that long-term hypoxia significantly modulates MSC properties. However, the question arises as to whether these changes are temporary or permanent. In order to clarify this issue, MSCs expanded under hypoxia were switched to normoxia (20% O2). On the next passage after such shifting, MSCs showed restored osteogenic (Fehrer et al., 2007; Grinakovskaya et al., 2009) and adipogenic (Fehrer et al., 2007; Valorani et al., 2010) potential based on the level of gene expression and calcium and triglyceride deposition. We have also demonstrated attenuated proliferation and

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colony-formation under hypoxic conditions (Rylova and Buravkova, 2014). It is interesting that when adipo-differentiation induced in hypoxic MSCs is switched to normoxia, the up-regulation of the adiporelated genes PPAR-gamma, LPL and FABP4 is more pronounced than that in normoxia (Valorani et al., 2010). Thus, low O2 in the MSC microenvironment seems to be a factor that inhibits, but does not abolish the commitment of precursor cells. Therefore, permanent expansion under hypoxic conditions does not exert damaging effects on MSCs and is accompanied by a significant change in their functional properties: an increase in proliferative activity and the ability to form CFU-F, the attenuation of osteo- and adipodifferentiation and the enhancement of chondrogenic potential. These changes are based on a fast metabolic switch to anaerobic glycolysis. All these events are accompanied by the up-regulation of the so-called “stemness” genes, possibly determining the undifferentiated MSC state (Fig. 1). What could be the keystone of the described phenomena? Nowadays, the concept of hypoxia, as a “physiological normoxia” for MSCs is becoming increasingly popular (Ivanovic, 2009). Respectively, MSC capacities, revealed in vitro, are interpreted as their inherent in situ capacities. However, reasonable care should be taken when performing a direct extrapolation of in vitro findings to the situation in vivo. In vivo, MSCs are integrated into specific tissue niches where their homeostasis is regulated by a balanced set of physical factors, cell–cell interactions, paracrine mediators, etc. In cell culture, MSCs are present in strictly determined conditions, the components of which may stimulate their proliferation (by growth factors) or differentiation into a particular lineage (in the presence of specific inducers). Therefore, one can assume that, in vitro, MSCs do not display the properties that they possess in situ, but rather the potencies to be realized under certain circumstances. The reversibility of hypoxic effects on MSCs when they are returned to normoxia and vice versa is a good experimental confirmation of the abovementioned assumption. On the other hand, if hypoxia is considered a damaging factor, it is unclear as to why there are no hampered effects of hypoxia on MSCs when permanently cultured at low oxygen. Perhaps the answer to this question is in the methodological approach itself. The short-term exposure of MSCs with severe hypoxia provokes cellular stress. The high degree of MSC plasticity ensures prompt feedback through HIF-mediated and other pathways and further development of an adaptive response. The result of this process can be revealed after long-term MSC expansion in hypoxia.

4. Energy producing pathways in MSC The hypoxic microenvironment of MSCs implies low mitochondrial activity in these cells. However, the balance between glycolytic and mitochondrial energy production in MSCs has been shown to be quite dynamic. It has been found, for instance, that embryonic stem cells have a low rate of aerobic metabolism when they are maintained undifferentiated, but stimulation of mitochondrial activity is crucial for their successful differentiation (Cho et al., 2006; Chung et al., 2007). Undifferentiated hMSCs show higher levels of glycolytic enzymes and increased lactate production, suggesting that hMSCs rely more on glycolysis for energy as compared to MSC-differentiated osteoblasts. Osteogenic induction is accompanied by the transition of ATP production from glycolysis to oxidative phosphorylation. Differentiated MSCs demonstrate high aerobic mitochondrial metabolism, assessed by an increase in the copy number of mitochondrial DNA, protein subunits of respiratory enzymes, oxygen consumption rate, and intracellular ATP content. Interestingly, upon differentiation, a dramatic decrease in intracellular ROS has been observed. This suppression of ROS was due to the upregulation of two antioxidant enzymes, manganese-dependent superoxide dismutase and catalase. Moreover, exogenous H2O2 and mitochondrial inhibitors can retard osteogenic differentiation (C.T. Chen et al., 2008; Chen et al., 2012). Thus, it is obvious that osteogenic differentiation of

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hMSCs needs coordinated regulation of mitochondrial biogenesis and antioxidant enzymes. Hypoxia, through suppression of mitochondrial activity can keep MSCs in an undifferentiated state. Indeed, hypoxic conditions have been shown to impair osteogenic differentiation, as assessed by the activity of alkaline phosphatase and the expression of osteogenic markers core binding factor a-1 and osteopontin (Hsu et al., 2013). In addition, hypoxia was found to compromise differentiation-induced mitochondrial activation, assessed by a decrease in the expression of respiratory enzymes and the oxygen consumption rate. At the same time, glycolytic enzymes were upregulated and lactate production was increased, forcing the cells to rely more on anaerobic glycolysis for energy. Under these circumstances the intracellular level of ATP was sensitive to a glycolytic inhibitor, 2-deoxyglucose, than to antimycin, an inhibitor of the mitochondrial respiratory chain. The glycolytic response of hypoxic cells is primarily mediated by HIF-1 (Hsu et al., 2013).

5. MSCs and cell death Two of the consequences of mitochondrial silencing under hypoxic conditions are suppression of mitochondrial pathways in apoptosis and increased resistance of tumor cells to conventionally used anticancer drugs (Graeber et al., 1996). Thus, incubation of multipotent mesenchymal stromal cells from human adipose tissue with etoposide for 24, 48, and 72 h under standard conditions (20% O2), at a “physiological” oxygen content (5% O2), and under hypoxic conditions (1% O2) did not induce cell apoptosis and only slightly increased the number of necrotic cells. The absence of growth factors in the culture medium did not potentiate the damaging effect of etoposide on multipotent mesenchymal stromal cells under standard and hypoxic conditions (Rylova et al., 2012) (Fig. 2). Apparently, a key role in the response of MSCs to a hostile environment is played by HIF-1α. Modulation of the level of this protein alters the resistance of MSCs to hypoxic and oxidative stresses. Whereas overexpression of HIF-1α in MSCs is protective against cell death and apoptosis triggered by hypoxic and oxidative stress conditions, its downregulation increases apoptosis and the death rate (Kiani et al., 2013). Stimulation of HIF-1α expression by deferoxamine treatment enhances the in vitro migration of MSCs as well as in vivo homing of DFO-treated MSCs (Najafi and Sharifi, 2013). However, in a number of publications, hypoxic conditions were shown to cause death in MSCs, which was presumably apoptotic (Potier et al., 2007; Zhang et al., 2009; Zhu et al., 2006), confirmed by cytochrome c release and caspase-3 activation (Chang et al., 2009; Zhang et al., 2009). A plant-derived antioxidant, berberine, was able to protect MSCs against hypoxia-induced apoptosis (Zhang et al., 2009), which implies the involvement of ROS. It should be mentioned that in many of publications, hypoxic conditions are co-applied with serum withdrawal, thus

Hypoxia MSCs

+serum deprivation Apoptosis

Autophagy

+serum deprivation

Necrosis

Fig. 2. Hypoxia-mediated modulation of various modes of cell death. Hypoxia alone enhances viability of MSCs. Hypoxia combined with serum deprivation can initiate cell death. Autophagy can be a protective factor in hypoxia/serum deprivation-induced apoptosis in MSCs.

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it is not clear whether hypoxia per se or the absence of serum in addition to hypoxic conditions induces apoptosis in MSCs. Separate administration of these deleterious factors revealed that cell death is not stimulated during 48 h of hypoxia, but increased markedly after serum withdrawal, suggesting that between the two components of ischemia, nutrient deprivation is the stronger factor (Potier et al., 2007). An analysis of the mechanisms involved in apoptosis caused by hypoxia and serum deprivation revealed that it can be suppressed by HSP 90 via the PI3K/Akt and ERK1/2 pathways (Gao et al., 2010). Hypoxiaand serum deprivation-induced apoptosis has been successfully prevented by lysophosphatidic acid (J. Chen et al., 2008) or lovastatin, a member of the drug class of statins (Xu et al., 2008). Lovastatin (0.01– 1 μM) prevented MSCs from hypoxia/serum deprivation-induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to the attenuation of caspase-3 activity. The loss of mitochondrial membrane potential and cytochrome c release from mitochondria were significantly inhibited by lovastatin. The mitochondrial apoptotic pathway was effectively abrogated by both a PI3K inhibitor, LY294002, and an ERK1/2 inhibitor, U0126 (Xu et al., 2008). The information on the sensitivity of MSCs to conventional anticancer drugs is rather controversial. Thus, human MSCs are resistant to chemotherapeutic agents commonly used in bone marrow transplantation (i.e. busulfan, cyclophosphamide and methotrexate); in contrast, cytotoxic agents, such as paclitaxel, vincristine, etoposide and cytarabine are quite toxic (Li et al., 2004). In vitro, human MSCs show significant resistance to cisplatin, vincristine, and etoposide compared with sensitive tumor cell lines, particularly at apoptosis-inducing doses (Mueller et al., 2006). The authors explained the elevated threshold for cisplatininduced apoptosis in MSCs by a lack of caspase-9 activity in apoptotic cells, although the mechanism behind the absence of this activity is unclear. In vitro exposure of MSCs to cisplatin, vincristine, and etoposide results in increased p53 expression, independent of apoptosis induction. Apoptosis induced by cisplatin or etoposide in MSCs is accompanied by expression of TAp63α, a subtype of p63, highly similar to p53. Downregulation of TAp63α does not influence the phenotype, proliferation capacity, and differentiation potential of MSCs, but markedly decreases apoptosis (Lu et al., 2011). Overexpression of p73, which is also a member of the p53 family, in human bone marrow MSCs significantly enhances sensitivity to cisplatin. Treatment of p73 overexpressing cells with cisplatin results in co-activation of the pro-apoptotic factors Bax and p21, suggesting that p73α is an important determinant of human bone marrow MSCs to treatment (Liang et al., 2010). Moderate hypoxia (0.1–3% oxygen) was shown to stimulate autophagy involved in a cell survival response in various types of cells (Mazure and Pouyssegur, 2010) (Fig. 2). Recently hypoxia-mediated autophagy was demonstrated in human placental chorionic plate-derived MSCs (Lee et al., 2013), as well as bone marrow MSCs (Wu et al., 2014). Autophagy was mediated by the ERK pathway, since ERK1/2 inhibitor U0126 (5 μM) significantly repressed hypoxia-induced expression of LC-3 and Atg5, as well as conversion of LC3B-I to LC3B-II (Wu et al., 2014). Stimulation of autophagy by atorvastatin (Li et al., 2014) or rapamycin as well as hypoxic preconditioning (Wang et al., 2014) can effectively enhance MSC survival and suppress apoptosis during hypoxia and serum deprivation, although the mechanisms involved are different. Atorvastatin stimulated ERK1/2 phosphorylation, autophagosome formation, and enhanced LC3-II/LC3-I ratio. In the case of hypoxic preconditioning or rapamycin treatment, protection can be explained by modulating both AMPK and mTOR pathways (Wang et al., 2014). Thus, autophagy can be a protective factor in hypoxia/serum deprivation-induced apoptosis in MSCs. Recent observations revealed that MSCs can modulate various forms of programmed cell death. Resistant to treatment bone marrow SCs can protect other cells from both apoptosis and necrosis (Fig. 2). Thus, in cardiomyocytes, exposed to hypoxia for 30 h, LDH release was observed and MTT uptake was decreased, which were prevented by co-culturing

cardiomyocytes with BMSCs. Similarly, apoptosis in cardiomyocytes subjected to hydrogen peroxide stress was markedly reduced by coculturing with bone marrow SCs. This anti-apoptotic activity was explained by the activation of the HIF-1α signaling pathway in SCs. As mentioned above, HIF-1 regulates the expression of numerous target genes, including VEGF and other cytoprotective proteins. In parallel with a high level of HIF-1α in BMSCs following anoxia or hypoxia, the release of VEGF from BMSCs is significantly increased in a time-dependent manner (Dai et al., 2007). Co-incubation of bone marrow SCs with neuron-like PC12 cells protects PC12 against apoptosis induced by CoCl2, via the production of erythropoietin. Knocking down erythropoietin abrogated the increase in erythropoietin expression induced by CoCl2 and decreased the cytoprotective effect of MSCs. Protection by MSCs might be dependent on erythropoietin expression, at least in part, via the regulation of Bcl-2 family members and caspases (Mo et al., 2012). Reverse transcriptase polymerase chain reaction results showed that EPO siRNA reversed the upregulation of Bcl-2 and Bcl-X(L) expression and the downregulation of Bax, Bak, caspase-9, and caspase-3 expression. It is known that autophagy plays an important role in various neurological disorders, including Alzheimer's disease (AD); dysfunction in the autophagic system may lead to the accumulation of amyloid-β peptides (Aβ). MSC administration has been shown to stimulate autophagosome induction and fusion with lysosomes. Moreover, MSC administration significantly reduces the level of Aβ in the hippocampus, which is elevated in Aβ-treated mice, concomitant with increased survival of hippocampal neurons. Finally, MSC co-culture has been shown to upregulate BECN1/Beclin 1 expression in AD models (Shin et al., 2014). Disturbances in the autophagy system may also lead to α-synuclein accumulation in the pathogenesis of Parkinson's disease. Co-culture with MSCs increases cellular viability in MPP+-treated neuronal cells, attenuates the expression of α-synuclein, and enhances the number of LC3-IIpositive autophagosomes compared with cells treated with MPP+ only (Park et al., 2014). Thus, MSCs not only are resistant to various damaging factors, but also can enhance the viability of neighboring cells. One of the recently demonstrated pathways employed in MSC-mediated cell rescue is mitochondrial transfer between MSCs and alveolar epithelia (Islam et al., 2012). Live optical studies revealed that MSCs form connexin 43-containing gap junction channels with alveolar epithelial cells in mice, releasing mitochondria-containing microvesicles that are engulfed by epithelial cells. This mitochondrial movement is regulated by a mitochondrial Rho-GTPase, Miro1, which regulates intercellular mitochondrial movement. Overexpression of Miro1 in MSC causes enhanced mitochondrial transfer and rescue of epithelial injury, while Miro1 knockdown suppresses this process (Ahmad et al., 2014). 6. Concluding remarks To answer the question raised in the title of this review, we can conclude that the hypoxic effects on MSC behavior are biphasic. Acute short-term oxygen depletion can provoke cell damage involving apoptosis. With an increased duration of hypoxic exposure, MSCs rapidly adapt to the microenvironment by switching their metabolism to anaerobic glycolysis accompanied by the maintenance of an undifferentiated multipotent state. The basis of these phenomena is the activation of signaling pathways providing cell adaptation to low oxygen. The interplay of these pathways allows for fine-tuning of the cellular response to hypoxia, supporting the assumption of a high degree of oxygen-mediated MSC plasticity. This plasticity in vivo can be employed as a mild intrinsic governor of MSC maintenance in their native tissue niches and as a moderator of homing and survival of these cells in injury. The need to further study the effects of hypoxia on stromal precursors is closely related to the functions they perform in tissue repair, particularly given the data on the stimulating effect of low oxygen on cultured MSCs, as well as the possibility of modifying their functional properties with

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hypoxia. These findings are of interest not only for scientific reasons, but also in practical terms. Thus far, the oxygen concentration has been found to be critical as it largely contributes to MSC physiology and should be taken into account in the setting of a protocol for cellular therapy. Silencing mitochondrial activity under hypoxic conditions makes MSCs resistant to cell death inducers. Nonetheless differentiated MSCs can demonstrate sensitivity to treatment due to the stimulation of aerobic mitochondrial metabolism. Understanding the metabolic regulation of stem cells will benefit the characterization and isolation of these cells with improved differentiation potential. The self-renewal ability of stem cells and the great potential of differentiation into various cell lineages provide a promising strategy for cell therapy and tissue engineering.

Acknowledgments The work of the authors was supported by grants from the Russian Science Foundation (14-25-00056, 14-15-00463 and 14-15-00693), Swedish (130463) and the Stockholm (121252) Cancer Societies, Swedish Childhood Cancer Foundation (12/043), and the Swedish Research Council (B0436801).

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