Senescence suppressors: their practical importance in replicative lifespan extension in stem cells

Abstract

Recent animal and clinical studies report promising results for the therapeutic utilization of stem cells in regenerative medicine. Mesenchymal stem cells (MSCs), with their pluripotent nature, have advantages over embryonic stem cells in terms of their availability and feasibility. However, their proliferative activity is destined to slow by replicative senescence, and the limited proliferative potential of MSCs not only hinders the preparation of sufficient cells for in vivo application, but also draws a limitation on their potential for differentiation. This calls for the development of safe and efficient means to increase the proliferative as well as differentiation potential of MSCs. Recent advances have led to a better understanding of the underlying mechanisms and significance of cellular senescence, facilitating ways to manipulate the replicative lifespan of a variety of primary cells, including MSCs. This paper introduces a class of proteins that function as senescence suppressors. Like tumor suppressors, these proteins are lost in senescence, while their forced expression delays the onset of senescence. Moreover, treatments that increase the expression or the activity of senescence suppressors, therefore, cause expansion of the replicative and differentiation potential of MSCs. The nature of the activities and putative underlying mechanisms of the senescence suppressors will be discussed to facilitate their evaluation.

Introduction

Mesenchymal stem cells (or marrow stem cells; MSCs) are able to differentiate to become osteoblasts, chondrocytes, adipocytes, myoblasts, and neurons. This pluripotency makes them excellent resources for cell replacement therapy or stem cell therapy, an approach to regenerate the functions of damaged tissues or organs by supplying stem cells. MSCs have advantage over embryonic stem cells in many aspects, such as availability and feasibility and low risk of cancer development. However, the low cancer risk, by nature, also raises the problem of a limitation to the number of available cells. The in vitro life span of stem cells isolated from human bone marrow (BMSCs) reportedly ranges from approximately 20–40 doublings, with Banfi et al. [1] reporting 22–23 doublings while Bruder et al. [2] reported 38 doublings, which is possibly dependent on many variables, including donor age and the efficiency of MSC preparation. In fact, current MSC preparations used in therapies are mixtures of heterogeneous stromal cells that include a subpopulation of stem cells. In order to be useful for therapy, MSCs should be expanded in vitro to a number sufficient for transplantation. In a consensus, at least 1 × 10⁶/kg or 10⁸ total cells are required to be transplanted for a successful MSC therapy [3]. This number of cells is reached after 27 doublings from a single cell, which consumes a large portion of the proliferative potential. Even if the population does not reach the end of its life span, i.e., replicative senescence, many cells in the late passage population show slow proliferation and express a senescence phenotype. Transplantation of such cells would not produce good results and may even be harmful. Senescence also imposes a limit to the stemness capacity of MSCs in differentiation and homing. As cells approach the end of their life span, they tend to lose osteogenic potential and gain in adipogenesis, the phenomenon termed “osteogenic to adipogenic shift” [1, 4–7]. Late passage MSCs express smaller amounts of homing receptors [8, 9] dropping down the efficacy of therapy.

Replicative senescence becomes more problematic when cell therapy is considered for aged individuals for whom the practice would be in greater demand. BMSCs from older human donors have a lower growth rate and capacity as well as decreased potential for differentiation [10–13]. The decreased proliferation is due to an increase in gate-keeping tumor suppressor activity and likely functions in reducing the incidence of cancer in aging tissues, but it also causes a decrease in regenerative capacity [14, 15]. This would certainly impose a limitation on the applicability of stem cell therapy in aged donors.

Replicative senescence may simply be viewed as an outcome of the chronic activation of DNA damage response triggered from short and deformed telomeres. p53-Rb and p53-Rb2 (probably functions more importantly than p53-Rb in human MSC), gate-keeping tumor suppressor pathways, play central roles in senescence imposition by linking damage sensing and execution of cell cycle arrest [17–19]. DNA damage is exacerbated by reactive oxygen species (ROS), and ROS-mediated single-strand breaks in telomeres have been shown to cause earlier imposition of senescence [16]. ROS are produced at a high level by damaged mitochondria, causing even higher levels of damage [20]. Thus, the cellular ROS level increases exponentially during continued cell cultivation in vitro [21]. Therefore, attenuating the accumulation of ROS or damage to cellular components can be an excellent strategy to slow down the cellular progression to replicative senescence. Indeed, cultivation of human fibroblasts and MSCs under low oxygen tension results in decreased levels of DNA damage and an extended replicative lifespan [22–24].

A number of proteins that function in damage response or defense against oxidative stress are involved in modulation of the lifespan of human primary cells. Some of these proteins, when overexpressed in human MSCs or human fibroblasts, induce substantial expansion of the proliferative potential and delay replicative senescence to an extent worthy of practical consideration. Interestingly, they are commonly lost during the progression to senescence. In this review, these proteins are termed as “senescence suppressors” and are introduced as promising targets for manipulation to enhance the proliferation and differentiation capacities of MSCs.

Senescence suppressor proteins

Tumor suppressor proteins such as p53 and Rb are frequently lost in cancer cells. Their forced expression puts a strong brake on the proliferation of cancer cells by causing either senescence or apoptosis. Therefore, tumor suppressors are characterized as proteins whose expression or activity needs to be attenuated for a cell to become cancerous [21]. An analogy can be found for cellular senescence. Certain proteins are highly expressed in early passage primary cells, but their expression declines during continued proliferation and is lost at senescence. Furthermore, their forced expression delays senescence and extends replicative lifespan, while the attenuated expression or activity leads to the development of the senescence phenotype. Here, these are analogously termed senescence suppressors, despite a difference in the etiology. Unlike tumor suppressors, the change in the levels of senescence suppressors is not known to be caused by loss of function mutations, and therefore, cellular senescence does not arise as an outcome of genetic selection against them. Importantly, the forced expression of a list of senescence suppressors has been shown to allow MSCs to maintain the phenotype of actively proliferating cells and the property of stemness.

Nrf2: master regulator of antioxidative defenses

Nf-E2 related factor 2 (Nrf2) is a transcription factor that, in response to oxidative stress, activates various antioxidant responsive element (ARE)-dependent genes encoding cellular redox regulators such as NAD(P)H quinone oxidoreductase 1, glutathione S-transferase, and peroxiredoxins [26]. It also activates the expression of multiple subunits of the 20S proteasome [27, 28]. Nrf2 has been shown to critically control stem cell survival under oxidative damage, which is likely mediated through cellular redox and proteome homeostasis by the aforementioned proteins [29–31].

The activity of Nrf2 declines during replicative senescence and aging [27, 32], and this has been proposed to contribute to the age-associated decline of antioxidative capacity. Furthermore, Nrf2 silencing causes premature senescence in human fibroblasts [27], while its activation induces lifespan extension of human fibroblasts and BMSCs [27, 31]. Considering that BMSCs are highly sensitive to oxidative stress [30], Nrf2 activation (through certain dietary phytochemicals and synthetic chemicals [33–35]) may be an excellent strategy to extend the lifespan of BMSCs in vitro.

20S proteasome: protein homeostasis

The 26S proteasome complex removes extra or damaged proteins. In cells at late passage or those isolated from aged donors, highly oxidized proteins are abundantly present and frequently cross-linked to each other, forming aggregates that are resistant to proteasome-mediated proteolysis [36]. Hydrophobic surface patches appear to be a major recognition motif in the substrates of proteasomes, and their oxidative modification is thought to help the proteins escape proteolysis [37, 38]. Such a change in susceptibility to proteolysis has been suggested to contribute to senescence and various age-related neural disorders such as Parkinson’s disease [39, 40]. Proteasome dysfunction or reduced activity of the 20S proteasome, the active component of 26S proteasome complex, has been observed in cells at late passage or from aged donors [31, 40–42]. Meanwhile, inhibition of proteasome activity using antagonists, such as MG132, induced senescence in early passage MSCs [41, 43].

While proteasome antagonists have attracted attention for their antitumoral effects [44], proteasome agonists may be the subjects of further focus for their anti-senescence effects. A triterpenoid, 18α-glycyrrhetinic acid (18α-GA) increased the level of the 20S proteasome subunit and its activity probably through the activation of Nrf2 [27, 45]. The treatment of human fibroblasts with 18α-GA provided cytoprotection against oxidative stress in a manner dependent on proteasome activity. It also caused substantial extension of the replicative lifespan in human fibroblasts and BMSCs [27, 31], and helped neuronal cells maintain expression of stemness markers [31].

SIRT1: promotion of cell survival and differentiation in response to genomic stress

SIRT1, an NAD⁺-dependent protein deacetylase, is a major mediator in the effect of calorie restriction and plays a preventive role in many aging-associated disorders [46, 47]. It also exerts positive effects on lifespan and differentiation of various stem cells. SIRT1 expression is reduced in senescent stem cells [48] and fibroblasts [49–52]. In addition, its knockdown suppresses cell growth and accelerates senescence of MSCs, while its overexpression delays the onset of senescence and the loss of differentiation capacity [51].

The effects of SIRT1 on the cellular lifespan appear to manifest through multiple different ways. First, it modulates p53 activity. Deacetylation by SIRT1 antagonizes p53 transcriptional activity [53]. It also induces p53 translocation to mitochondria [54], thereby attenuating the biological response to DNA damage [55, 56]. An outcome of this may be the attenuation of stress-induced premature senescence. SIRT1 activation prevented premature senescence induced by oxidative stress [57], oncogene expression [53, 58], or PML upregulation [53]. SIRT1 may also extend cellular lifespan by suppressing triggering of the growth-arrest signal derived from telomere shortening or dysfunction. SIRT1 overexpression attenuated telomere shortening, while its deficiency caused increased chromosome fragility at telomeres and telomere dysfunction in MEFs [59]. Moreover, it upregulates the hTERT gene, the catalytic subunit of telomerase (possibly through c-Myc activation [60]) [61]. SIRT1 is also involved in the management of oxidative stress. It activates FOXO3a, which upregulates the expression of antioxidant proteins such as MnSOD, catalase, thioredoxin 2, thioredoxin reductase 2, and peroxiredoxins [62], and has been shown to protect against cigarette smoke-induced oxidative stress in a FOXO3-dependent manner [63]. In addition, SIRT1 is involved in basal level autophagy. Through deacetylation, it activates key molecules in autophagosome formation such as Atg5, Atg7, and Atg8 [64]. It was shown that calorie restriction-mediated SIRT1 activation in the kidney of aged mice attenuates renal dysfunction possibly through the activation of autophagic disposal of damaged mitochondria [65]. These suggest that SIRT1 activation can promote stem cell longevity by facilitating better maintenance of mitochondria quality through augmentation of basal level autophagy.

Intriguingly, SIRT1 also imposes a limit to cellular lifespan. This appears to be a response to chronic genotoxic stress. SIRT1-deficient MEFs showed increased replicative lifespan and proliferative capacity under chronic sub-lethal oxidative stress [66]. In these cells, the level of p19ARF, which stabilizes p53, was low, and its increase during serial passage was attenuated, resulting in a reduction in p53 protein levels. SIRT1 antagonizes REGγ, a proteasome activator, which promotes degradation of p19ARF along with other cyclin-dependent kinase inhibitors (CDKIs), p21WAF1 and p16INK4a [67]. Therefore, the absence of SIRT1 might cause reduction in the levels of CDKI and p53. Together, these findings suggest that, in chronic sub-lethal genotoxic stress such as that generated during serial cultivation under high oxygen conditions, SIRT1 potentiates p53-growth-inhibitory pathway and causes early onset of cell cycle arrest and senescence. These contradictory findings on the effect of SIRT1 on cellular lifespan suggest that cells have evolved diverse responses to cope with different levels of genotoxic insult.

SIRT1 appears to modulate stem cell differentiation primarily by antagonizing p53 suppression of the Nanog gene, which functions in self-renewal and maintenance of a differentiated state [54, 68]. In addition, the direction of differentiation may also be affected by SIRT1. Under oxidative stress, SIRT1 deacetylates histones at the promoter region of Mash1, which is responsible for the activation of a neuron-specific transcriptome [69], thereby switching neural differentiation to gliogenesis [70, 71]. Whether SIRT1 exerts this kind of regulatory effect on other types of differentiation is not known.

NAMPT and nicotinamide: regulators of NAD redox and SIRT1 activity

Nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin) converts nicotinamide (NAM) to nicotinamide adenine mononucleotide (NMN), in the rate-limiting step of the salvage pathway for NAD⁺ biosynthesis [72]. It thereby provides cells the key coenzyme for metabolism. The concomitant increase in the NAD⁺/NADH ratio also affects the activity of several key enzymes including SIRT1 [73, 74]. The level of Nampt protein in smooth muscle cells decreases after continued proliferation, and antagonist-mediated downregulation of its activity causes premature senescence [75]. Meanwhile, transduction of the Nampt gene lengthened the lifespan of smooth muscle cells (extension of 34–71 % depending on the cell strains) and fibroblasts. This effect is likely to be mediated by SIRT1 activation, since the expression of the dominant-negative form of SIRT1 abrogated the effect of Nampt overexpression [75]. Furthermore, the lifespan-extending effect of SIRT1 overexpression was significantly potentiated by Nampt overexpression [76].

Meanwhile, prolonged administration of 5-mM NAM effectively extends the replicative lifespan of human fibroblasts and keratinocytes [77]. The lifespan was extended by 60 %, while the increase in ROS was substantially delayed. In the NAM-treated cells, mitochondrial autophagy was augmented while the level of mitochondrial ROS decreased. These changes were reproduced by the treatment of SIRT1 activators, and abrogated by co-treatment with FK866 [77, 78], an antagonist of Nampt, indicating that at least a part of the effect of NAM is mediated by an enhancement of cellular levels of NAD⁺ or the ratio of NAD⁺/NADH, and, thereby, activation of SIRT1. Meanwhile, in a recent study, NAM and its methyl derivative, methylnicotinamide extended the lifespan of Caenorhabditis elegans even in the absence of SIR-2.1 [79] suggesting a SIRT1-independent effect of NAM. NAM has been reported to have strong antioxidant activity [80], and whether this contributes to the prolongevity effect of NAM that needs to be determined.

Klotho: attenuation of senescence-associated inflammation

A defect in the Klotho gene causes premature aging [81], and the anti-aging effect of the Klotho gene has been demonstrated in mice [82]. α-Klotho protein, an obligatory co-receptor for FGF23, a hormone involved in Ca²⁺ and phosphate homeostasis [82, 83], was shown to interact with retinoic acid-inducible gene-I (RIG-1) protein [84] and inhibit RIG-1-mediated expression of IL-6 and IL-8, which are key mediators of inflammation and factors involved in the senescence-associated secretory phenotype (SASP) [85]. Expression of Klotho declines in senescent cells while that of RIG-1 increases inversely, possibly contributing to the expression of SASP and senescence-associated inflammation in senescent cells [84]. Extension of this line of investigation on Klotho may help clarify the possible link between replicative lifespan and SASP. Meanwhile, knocking down Klotho expression caused premature senescence in a p53-dependent manner, suggesting that Klotho normally suppresses senescence by interfering with the damage accumulation or the p53-damage response pathway [86].

HO-1, BVR-A, and Nrf2: primary antioxidative defense network

Heme oxygenase-1 degrades heme to produce biliverdin and carbon monoxide [87]. It upregulates the expression of the key anti-inflammatory modulators IL-10 and IL-1Ra (IL-1R antagonist) [88], and thereby, plays an anti-inflammatory role. For instance, HO-1 protected osteoarthritis chondrocytes, synoviocytes, and osteoblasts against damage caused by cytokine-induced inflammation and apoptosis [89, 90]. HO-1 also exerts strong positive effects on the osteogenesis. Its overexpression potentiated BMSCs and periodontal ligament cells for osteogenic differentiation while suppressing adipogenesis [91]. It also prevented the expression of the senescence phenotype as well as the reduction of bone formation in osteoblasts treated by IL-1β, a potent pro-inflammatory molecule [92]. Therefore, HO-1 manipulation would certainly provide a means to block the osteogenic to adipogenic shift that takes place during MSC expansion in vitro.

Bilirubin is an important intrinsic antioxidant that quenches inflammation-induced free radicals by being oxidized to biliverdin. Biliverdin reductase A (BVR-A) converts biliverdin to bilirubin [93]. In addition, it facilitates the expression of HO-1 by forming a transcription factor along with hematin [94]. Therefore, BVR-A constitutes a strong cellular antioxidative defense along with HO-1. The upregulation of the HO-1/BVR-A antioxidant system has been identified as one of the early responses to counteract oxidative stress in the brain and proposed as a useful mechanism to counteract Alzheimer disease-induced oxidative damage [95]. The activity of BVR-A declines in aging and its upregulation upon oxidative stress is impaired in senescence [96, 97], possibly due to the decrease in Nrf2 activity, which upregulates the expression HO-1 [98].

PPARγ and δ: transcriptional activation of antioxidative and anti-inflammatory functions

Peroxisome proliferator-activated receptors (PPAR)γ and δ, members of the PPAR transcription factor family, have antioxidative and anti-inflammatory functions through the expression of a battery of downstream antioxidative proteins [99, 100]. PPARγ induces the expression of Cu/Zn-SOD [101] and Klotho [102]. It also inhibits inflammatory gene expression by directly or indirectly interfering with the pro-inflammatory transcription factor NF-kB [103]. Expression of PPARγ decreases during aging of rats [104], while its activation rescues the functions of tissues damaged by oxidation or inflammation [104] and reduces the senescence phenotype in fibroblasts in inflammatory skin [105]. Meanwhile, PPARδ induces the expression of glutathione peroxidase 1 (GPx1), thioredoxin 1 (Trx1), MnSOD, and HO-1 [106]. It also suppresses the inflammation of vascular smooth muscle cells (VSMCs) through upregulation of TGF-β1, an anti-inflammatory mediator [107, 108]. PPAR δ activation attenuates ROS generation and prevents premature senescence of VSMCs [109–111]. Overall, activation of the PPAR transcription factors by agonists such as thiazolidinedione compounds (for PPARγ) [112, 113] and GW501516 (for PPARδ) [109] may be a strategy worthy of trial for functional expansion of MSCs in vitro.

Most of the antioxidative genes induced by PPARγ are also activated by Nrf2 [114]. Further, Nrf2 modulates PPARγ expression by binding to a putative ARE in its promoter [115]. This suggests a possible link between the pathways of these transcription factors for cellular antioxidant function.

Prion protein: enhancing cellular antioxidation capacity

Cellular prion protein, PrP^C, is expressed in multipotent neural precursors and plays roles in neurogenesis [116], while certain variants, collectively designated PrP^SC, are culprits of Creutzfeldt-Jakob disease (CJD) and other transmissible spongiform encephalopathies [117]. PrP^C expression decreases in MSCs during expansion in vitro, and its knockdown causes a significant reduction in proliferation as well as differentiation [118]. Meanwhile, maintenance of its expression resulted in an extended lifespan [108]. The antioxidant activity of PrP^C has been recognized [119–121]. PrP^C increases cellular SOD activity through increased incorporation of copper into CuZn-SOD, or the formation of a complex with copper that mimics cellular SOD activity at the cell surface [122, 123]. It also causes a substantial increase in the activity of glutathione reductase and, thereby, the level of glutathione [123], although the underlying mechanism is not known. A chemical named 3/689 has been shown to cause sustained expression of PrP^C protein [118], while inhibiting PrP^SC accumulation during continued cell passage in human MSC [124]. In addition, this treatment extended the replicative lifespan of hMSCs (near 25 % increase in PD number) and increased their engraftment efficiency after bone marrow transplant in a manner dependent on PrP^C expression [118]. An increase in SOD2 activity was observed in the late passage hMSC treated with the chemical [118].

Statins: senescence suppressor-like effectors modulating cell cycle and cellular oxidative level

Statins (HMG-CoA reductase inhibitors) such as atorvastatin and mevastatin were shown to inhibit the onset of senescence as well as apoptosis in endothelial progenitor cells (EPCs) [125]. Atorvastatin treatment of human EPCs was found to activate the PI3-kinase/Akt pathway, which is known to regulate the senescence of endothelial cells [126] and also to mediate the angiogenic effect of VEGF by conferring endothelial cell survival and neovascularization [127, 128]. PI3K/Akt activation-mediated suppression of FOXO4 and its downstream gene, p27Kip1, a CDK inhibitor, has been suggested to be involved in this effect [129–132]. Statins may also delay senescence through antioxidative effects. Atorvastatin downregulates the expression of NOX1 and p22phox, which are key subunits of NAD(P)H oxidase, a predominant producer of superoxides in the endothelium [133, 134], and upregulates the expression of catalase and SIRT1 in rat aortic vascular smooth muscle cells [135] and PPAR-α/β/δ/γ in the rat heart [136]. Furthermore, atorvastatin has been shown to delay the onset of replicative senescence of endothelial cells caused by homocysteine-induced inactivation of telomerase [137]. Meanwhile, simvastatin has been reported to induce melanoma cell senescence, possibly by increasing oxidative stress [138]. This finding suggest that certain statins may have pro-oxidative effects.

Extracellular matrix (ECM): cell adhesion signaling

Culture substratum affects the growth and differentiation of MSCs. The proliferative capacity of human BMSCs increased by near eight doublings when cultured on a plate coated with ECM-gel^R (Sigma-Aldrich), a commercially available mixture of basement membrane proteins [139]. ECM-gel^R plate also enhanced differentiation and attenuated the adipogenic switch in late passage MSCs. Individual ECM components, fibronectin, laminin-1, laminin-5, and collagen IV were also effective, albeit to smaller extents. Meanwhile, treatment with antibodies against α6 integrin/CD49f blocked the lifespan-extending effect of ECM-gel^R [139]. This indicates an outside-in signaling generated by the ECM-integrin interaction that positively affects the proliferation of MSCs. No detail of the underlying mechanism has yet been determined, although an increase in telomerase activity has been reported [140]. Meanwhile, a senescence-promoting role for integrin β4 has also been proposed in vascular endothelial cells [141]. Knocking down integrin β4, which is highly expressed in senescent HUVECs, suppressed ROS increase and senescence [141, 142].

Cellular rejuvenation has been made by utilizing the ECM of the cells from young animals. In one study, the cell-free ECM was generated by cultivating BMSCs from 3-month-old mice and removing them by treatment with Triton X-100 and NH₄OH. This young ECM, unlike the one generated from BMSCs of 18-month-old mice (old ECM), supported self-renewal and bone formation of BMSCs isolated from old mice [143]. Importantly, cultivation on the young ECM dramatically attenuated the increase of ROS during continued passage. The young ECM was found to contain higher amounts of collagen and lower amounts of phosphate. Whether these affect the proliferation of BMSC is not known, but their ratio at least appears to affect the osteogenic differentiation [143]. In addition, calcium phosphate particles impair the viability and proliferation of osteoblast progenitor cells [144]. ECM of early passage cells may work similarly. Senescent fibroblasts, when plated on the ECM of early passage fibroblasts, resumed proliferation and gained young cell properties (lower level ROS and longer telomeres) for several doublings [145]. These findings, together, suggest that the way cells attach to ECM not only determines their capacities for proliferation and differentiation, but also modulates the growth-inhibitory machinery that is already in force.

The change in the levels of senescence suppressors

Protein homeostasis is important for cell function and physiology. Cells change their fates through subtle alteration in the level of certain proteins. For example, both the upregulation and downregulation of brahma-related gene 1 (BRG1), a component of chromatin remodeling complex that controls cell cycle control, apoptosis, and differentiation [146], induces senescence in MSCs [147]. This suggests that, for certain proteins, manipulations that affect their expression or activity may cause unexpected or unwanted effects, and therefore, need to be attempted with caution.

A hallmark of senescence suppressors is the decline in their levels during continued passaging of primary cells. Whether this is the outcome of prolonged oxidative damage or driven by an innate program induced by complex signaling processes during prolonged cell proliferation is unknown. A variety of mechanisms are known to involved in the decrease in the levels of senescence suppressor proteins. For example, the decline in the SIRT1 level is caused by changes at multiple different points during pre- and post-translational processes. The increased level of p53 at late passages attenuates SIRT1 expression by inactivating the SIRT1 promoter [148] and/or upregulating the expression of miR-34a [149] that binds to SIRT1 mRNA and facilitates its degradation [150]. SIRT1 mRNA is also destabilized upon dissociation of RBP HuR from its AU-rich elements [150, 151], which is caused by RBP HuR phosphorylation by Chk2 [152], a member of the DNA damage signaling pathway. Meanwhile, the SIRT1 protein level decreases in EPCs because of cleavage by cathepsins B, S, and L [153] which are released from lysosomes upon loss of membrane integrity caused by ROS in senescence [154]. Finally, oxidative stress-induced carbonylation of cysteine residues causes proteasome-mediated degradation of SIRT1 protein [155]. Some conclusions can be derived from these findings: the levels of senescence suppressors may be modulated by multiple mechanisms, and most of these mechanisms are strongly associated with oxidative damage and damage signaling. (In the case with SIRT1, the association of oxidative damage and its signaling has not been demonstrated only in the case with miR-34a). These indications may be highly relevant for safety concerning prolonged modulation of senescence suppressor levels in practical applications. If such decreases are simply a passive outcome of oxidative damage, the reversal of their expression can be safely adopted as a strategy for MSC therapy. However, more caution is required if an innate program drives the change in senescence suppressor levels. Therefore, the mechanisms underlying the decreases in senescence suppressor levels warrant further in-depth investigation.

Concluding remarks

A class of proteins which can be classified as senescence suppressors by definition, are not included in this review. Bmi-1 and Wip1 (PPM1D) commonly extends cellular lifespan by interfering with the damage-response pathways. Bmi-1 suppresses the expression of p16INK4a and p19Arf, the two major inhibitors of cell cycle progression [156–158]. Its overexpression extends the replicative lifespan of primary cells [159–161], but has also been shown to induce immortalization of epithelial cells and MSCs [162, 163] and bears a risk for tumorigenesis [164, 165]. Wip1 abrogates the ATM/p53-DNA-damage checkpoint function [166] and the p38MAPK–p16INKa pathway [167] by dephosphorylating key members in the pathways. Overexpression of Wip1 in chondrocytes and hMSCs attenuated the growth-inhibitory response to oxidative stress (but not the level of ROS itself) [168, 169] and delayed the onset of replicative senescence in MSCs [168]. Its sustained activation, however, has also been suggested to promote tumorigenesis [166]. The primary function of these damage-response pathways is to prevent accumulation of mutations, and therefore, the modulation of the individual components in the pathway would cause cancer development [170]. However, the senescence suppressors introduced here appear to act differently, i.e., reduce the level of oxidative stress either by enhancing the cellular antioxidative capacity or promoting damage removal as summarized in Fig. 1. Cultivation of cells under low oxygen tension has been shown to extend the replicative lifespan of human MSCs and fibroblasts [22, 23, 171], providing a rationale for approaches to reduce the level of oxidative stress as an effective strategy for stem cell expansion. Meanwhile, the cellular glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples control oxidative stress [172]. These can be manipulated to reduce oxidative stress by adding glutathione or cysteine in culture medium. Variation in the GSH/GSSG and Cys/CySS redox, however, also affects cell-signaling pathways [173], and it is not known if the long-term manipulation of these redox couples indeed causes an extension of cellular replicative lifespan.

Fig. 1.

The growth-arrest pathway towards senescence and four possible ways of intervention. The pathway begins with oxidative stress (inflammation also generates ROS), followed by damage on DNA (including telomere shortening and dysfunction), damage response (damage sensing followed by tumor suppressor pathway that is relayed through p53-p21-Rb or p16/p27-Rb), and the execution of growth arrest, which eventually develops to senescence. In “oxidative stress reduction”, senescence suppressors (azure box) reduce the level of oxidative stress or inflammation. PPARγ and δ, Nrf2, BVR-A directly induce antioxidative proteins and chemicals (dark khaki). SIRT1 does indirectly through activating Foxo3. PrP^C and nicotinamide (NAM) may function as antioxidants. Nampt (and NAM) works by enhancing SIRT1 activity. ECM suppresses ROS generation through integrin-mediated signaling. Statins also suppress ROS generation by inhibiting NAD(P)H oxidase and activating catalase. They also activate SIRT1 and PPAR transcription factors. α-Klotho and HO-1, which are activated by PPARγ and Nrf2, respectively, attenuate inflammation-induced ROS generation. In “damage reduction”, SIRT1 induces hTERT and thereby, activates telomerase, which prevents telomere dysfunction. And, in “damage removal”, Nrf2 enhances proteasome activity by inducing the 20S proteasome subunits, while SIRT1 enhances autophagy by activating autophagosome formation. In “tumor suppressor inhibition”, Wip1 and Bmi-1 attenuate either the p53/p21WAF1- or p16INK4a-tumor suppressor pathways. Statins may activate Akt, which suppresses FOXO3-mediated p27 expression. Finally, young ECM appears to cause reversal of arrest state and induce the progression of the cell cycle

Finally, the results of a series of studies on telomerase-mediated extension of MSC lifespan have revealed that the differentiation potential of MSCs is tightly associated with their proliferation potential, i.e., once the proliferation potential is secured, multipotency can be maintained [174–176]. This suggests that a manipulation that extends the lifespan of MSCs, once the risk can be ruled out for cell immortalization, might be adopted without much concern for its effect on differentiation. To date, individual senescence suppressors have not been compared in terms of their effect on expanding the replicative capacity of MSCs. Although each of the discussed proteins can be activated to obtain certain levels of effect, some are predicted to be more potent than others. Nrf2 acts by inducing a family of antioxidant enzymes and transcription factors targeting antioxidant genes. It also increases proteasome activity, thereby exerting a strong effect on both prevention and removal of damage. Importantly, there are chemical agonists available for Nrf2 (Table 1). SIRT1 may also function in cellular longevity through multiple pathways. It facilitates induction of antioxidative proteins while suppressing chronic damage signaling. Furthermore, SIRT1 activates autophagy that prevents damage accumulation and helps cells avoid damage-induced senescence [177, 178]. A family of SIRT1 activators have also been examined in vivo [179], although their effects have not been clearly determined because of certain off-target effects [180]. However, they can also be readily utilized for lifespan extension of MSCs in vitro.

Table 1.

Proteins that have senescence suppressor characteristics and positively affect replicative lifespan and differentiation of MSCs

Proteins	Effects and mechanisms	Inducers or agonists
Nrf2	Reduction of oxidative stress by inducing antioxidant enzymes	Dimethyl fumarate [181], α-lipoic acid [182, 183], sulforaphane [184], bardoxolone methyl [185]
	Protection from inflammation-caused damage by inducing anti-inflammatory cytokines
	Damage removal by inducing 20S proteasome
PrPC	Reduction of oxidative stress by potentiating CuZn-SOD and glutathione reductase	3/689 [118]
BVR-A	Reduction of oxidative stress by producing bilirubin and inducing HO-1
HO-1	Protection from inflammation-caused damage by inducing anti-inflammatory cytokines	Atrial natriuretic peptide [186]
	Forms HO-1/BVR-A antioxidant loop
α-Klotho	Protection from inflammation-caused damage by suppressing pro-inflammatory cytokine expression	Rosiglitazone through PPAR γ [112]
PPAR γ & PPARδ	Reduction of oxidative stress by inducing antioxidative and anti- inflammatory enzymes	Rosiglitazone [112], Pioglitazone [113] for PPAR γ; GW501516 [109] for PPARδ
20S proteasome	Damage removal	18α-glycyrrhetinic acid [27, 45]
SIRT1	Reduction of oxidative stress by inducing antioxidant enzymes via FOXO3 activation	SIRT1 activators [179]
	Suppression of damage signaling by inhibiting p53
	Telomere maintenance by hTERT induction
	Damage removal through autophagy activation
NAMPT	SIRT1 activation by increasing NAD+/NADH ratio	Nicotinamide works by potentiating Nampt effect, but, also has antioxidative effect
ECM materials	Increased cell survival signaling and decreased ROS production.
	Cellular rejuvenation (by young cell ECM)
Statins	Reduction of oxidative stress by downregulating NAD(P)H oxidase and upregulating catalase	Mevastatin [129] atorvastatin [135] but, simvastatin has pro-senescence effect [138]
	Suppression of damage signaling by inhibiting CDK inhibitor
	Upregulation of SIRT1 and PPAR α/β/δ/γ
Bmi-1a	Suppression of tumor suppressor function: repression of INK4a-Arf locus
Wip1a	Suppression of DNA damage signaling
hTERTa	Damage reduction by telomere maintenance (not-senescence supprossor)

^a Bm-1, Wip1, and hTERT are not suitable for in vitro expansion of MSC for stem cell therapy since their activation causes cell immortalization which can lead to tumorigenesis in vivo

Abbreviations

MSCs

Mesenchymal stem cells

BMSCs

Bone marrow stem cells

ROS

Reactive oxygen species

ARE

Antioxidant responsive element

18α-GA

18α-Glycyrrhetinic acid

MEF

Mouse embryonic fibroblast

CDKI

Cyclin-dependent kinase inhibitor

Nampt

Nicotinamide phosphoribosyltransferase

NAM

Nicotinamide

HO-1

Heme oxygenase-1

BVR-A

Biliverdin reductase A

PPAR

Peroxisome proliferator-activated receptor

VSMC

Vascular smooth muscle cell

EPC

Endothelial progenitor cell

HUVEC

Human umbilical vein endothelial cell

ECM

Extracellular matrix