SEARCH

SEARCH BY CITATION

Keywords:

  • Mesenchymal stem cell;
  • Akt;
  • Mammalian target of rapamycin;
  • Rapamycin;
  • Self-renewal;
  • Aging

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

The decline in mesenchymal stem cell (MSC) self-renewal and function with aging contributes to diseases associated with impaired osteogenesis. MSC donor age in prolonged culture also limits the therapeutic potential of these cells for tissue engineering and regenerative medicine. Here, we demonstrate an intervention to preserve the immature state MSC and consequently maintain self-renewal and differentiation capacity during in vitro aging. We showed that blocking of phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) prevents the development of an age-related phenotype and maintains MSC morphology of early passage cells with high clonogenic frequency and enhanced proliferative capacity. MSC cultured in the presence of inhibitors of Akt or mTOR also robustly maintain their osteogenic potential, that is otherwise lost during in vitro aging. We further report that these effects may be mediated by induction of expression of pluripotency genes Nanog and Oct-4 and by the reduction in the production of cytoplasmic reactive oxygen species (ROS). Additionally, loss of Akt/mTOR and ROS was accompanied with lower levels of DNA damage. These results provide an insight into mechanisms involved in MSC aging and suggest possible interventions to maintain quiescence and function of MSC prior to in vivo transplantation or as pharmacological agents in diseases associated with loss of MSC function. Stem Cells 2014;32:2256–2266


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Aging is associated with the reduction in self-renewal capacity and differentiation potential of mesenchymal stem cells (MSCs). MSCs isolated from old individuals or from aged animals have reduced osteogenic differentiation capacity, bone marrow frequency, proliferation rate and show greater levels of senescence and senescence-associated changes [1, 2]. Accordingly, donor age may adversely affect the efficacy of MSC transplantation for tissue regeneration [3]. The decline in proliferation and stem cell properties of MSC with age has also been proposed as a cause of the progressive reduction in bone mass, incidence of osteoporosis, and risk of fracture seen in elderly [4, 5]. These age-related changes appear to be similar or even more pronounced during cultivation of MSC in vitro. Our previous studies, as well as those from others, indicate that MSCs show a progressive and rapid loss of differentiation potential and reduction in proliferation rate with prolonged culture [6, 7]. Interestingly, the observed decrease in stem cell properties may be even more pronounced in MSC expanded in the presence of factors such as fibroblast growth factor-2 (FGF)−2 and platelet-derived growth factor (PDGF)-BB [6].

The decline in stem cell number and function with age may be associated with accumulation of cellular damage [8, 9]. In particular, the level of oxygen tension and generation of reactive oxygen species (ROS) may be particularly implicated in MSC aging. MSCs exposed to atmospheric oxygen show a reduced life span and function, while hypoxia has been shown to inhibit senescence and maintain their self-renewal capacity. This has been attributed mainly to the resultant reduced oxygen tension and lower level of ROS production [10-12]. In fact, a hypoxic microenvironment is considered to be an important component of a stem cell niche such as in bone marrow. However, even at these sites ROS production increases with age and causes oxidative DNA damage resulting in reduced stem cell function [1]. Recent studies also suggest that persistent long-term activation of intracellular signal pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) is associated with similar loss of quiescence, premature aging, and a diminishing pool of the self-renewing stem cell population [13-17]. mTOR and its upstream activator Akt play key roles in many vital processes in stem cells, including cell growth, proliferation, differentiation, and survival [18-22]. The Akt/mTOR pathway can be induced by a variety of mechanisms including cytokine receptors such as PDGF receptors and by environmental cues. Once initiated, the signal results in activation of the catalytic subunit of PI3K and conversion of phosphatidylinositol-4,5-biphosphate into phosphatidylinositol-3,4,5-triphosphate, which subsequently binds 3′-phosphoinositide-dependent kinase 1 and Akt, causing Akt phosphorylation and activation. Akt is the key mediator of the PI3K signal that regulates vital cellular processes by activating the TOR complex 1 (TORC1) and mTOR signaling. Activation of mTOR propagates the signals by phosphorylation of its downstream effector targets, notably eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1) [23]. Interestingly, links are emerging between mTOR and ROS pathways with evidence suggesting reciprocal regulation of these pathways by each other [24]. For instance, high levels of mTOR have been shown to correlate with increase in ROS generation and reduction in hematopoietic stem cell self-renewal [25]. In the study reported here, we demonstrate that inhibition of Akt/mTOR pathways could provide an environment to maintain MSC in their immature undifferentiated state during long-term culture expansion. MSC cultured in the presence of inhibitors of Akt and mTOR showed morphology of cells in their early passages, retained their clonogenic ability, showed a high proliferative rate and osteogenic potential, which were otherwise lost during in vitro aging either in the presence or absence of FGF-2. We further demonstrate that loss of Akt/mTOR may mediate these effects by regulating the production of cytoplasmic ROS, expression of pluripotency genes Nanog and Oct-4, and by reducing accumulation of DNA damage during aging of MSC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Cell Culture

Primary human MSCs isolated from bone marrow of healthy young males (n = 2) and female (n = 1) adults (age between 21 and 25 years) were obtained from Lonza (Slough, U.K., http://www.lonza.com/) and all experiments were conducted with MSCs of both sexes. Cells already at passage 2 when purchased were aged in normal growth medium consisting of α-minimal essential medium, penicillin (50 U/ml), streptomycin (50 µg/ml) (all from Sigma-Aldrich, Poole, Dorset, U.K., http://www.sigmaaldrich.com/united-kingdom.html), Glutamax (2 mM) (Invitrogen, Paisley, U.K.), and 10% fetal bovine serum (FBS) (Sigma-Aldrich) and maintained at 37°C in a humidified 5% CO2:95% air atmosphere for 5 weeks to P6. Cells were subsequently seeded at a density of 500,000 in T-75 flasks and grown in normal medium alone or medium supplemented with 10 ng/ml FGF-2 (F, fibroblast growth factor-2) (Peprotech, London, U.K., http://www.peprotech.com) in the presence or absence of the mTOR inhibitor, rapamycin (F+R) at 10 nM (Cayman Chemical, Cambridge BioscienceCambridge, UK http://www.bioscience.co.uk) or the PI3K/Akt inhibitor, LY294002 (F+L) at 10 µM (Sigma-Aldrich). DMSO was used as vehicle control. Cultures were maintained in continuous culture for a further four passages (for 5 weeks).

Differentiation Potential

For osteogenic differentiation, cells were seeded at a density of 15,000 cell per square centimeter and induced with growth medium supplemented with 0.1 µM dexamethasone (Dex), 0.05 mM ascorbic acid 2-phosphate, and 10 mM glycerophosphate (Sigma-Aldrich). To assess osteogenic differentiation, RNA was isolated after 14 days of incubation and mRNA expression of markers of differentiation (i.e., Runx-2, alkaline phosphatase [ALP], and osteopontin) was determined by quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and accumulation of calcium deposits was visualized and quantified by staining with Alizarin Red dye as described previously [26]. Adipogenic differentiation was induced at a density of 40,000 cell per square centimeter with growth medium supplemented with 1 µM Dex, 0.25 mM isobutylmethylxanthine, 50 µM indomethacin, and 10 µg/ml insulin (Sigma-Aldrich). Adipogenesis was assessed by qRT-PCR analysis of markers of differentiation (i.e., peroxisome proliferator-activated receptor γ [PPAR-γ], fatty acid binding4 [FAB-4], and lipoprotein lipase) and lipid accumulation was visualized and quantified following staining with oil red O dye as described previously [26].

qRT-PCR Analysis

Total RNA was extracted using TRI reagent (Ambion, Warrington, U.K., http://www.lifetechnologies.com) and Phase Lock Gel Heavy tubes (5 Prime, VWR, Leicestershire, U.K.) according to the manufacturer's instructions. RNA purity and quantity were assessed by nanodrop (Fisher Scientific) (A260/A280 1.8–2 was considered suitable for further analysis), possible contaminating DNA was removed and cDNA prepared from 1 µg RNA using QuantiTect Reverse Transcription Kit (Qiagen, West Sussex, U.K., http://www.qiagen.com/) according to the manufacturer's instructions. qRT-PCR was performed on a Mx3000P real-time PCR system using Brilliant III Ultra-Fast SYBR Green qPCR Master mix (Stratagene, Agilent Technologies, Cheshire, U.K., http://www.genomics.agilent.com) and primer pairs as listed in Supporting Information Data (Supporting Information Fig. S1A). PCR conditions consisted of 1 cycle of 95°C for 3 minutes and 40 cycles of 95°C for 10 seconds and 60°C for 20 seconds. RPL13a was used as an invariant housekeeping gene.

5-Ethynyl-2′-Deoxyuridine Incorporation Assay

MSCs were seeded at a density of 150,000 in six-well plates and incubated for 24 hours in normal growth medium. Medium was changed with reduced (1%) FBS culture medium and cells were incubated for an additional 12 hours prior to refeeding the cells with medium with 10% serum. Cell proliferation (DNA synthesis) was assessed by measuring 5-ethynyl-2′-deoxyuridine (EdU) DNA incorporation using the Click-iT EdU Alexa Fluor 647 cell proliferation assay kit (Invitrogen). Briefly, cells were treated with EdU at 10 µg/ml for 48 hours, harvested by trypsinization, washed in PBS/1% BSA, and fixed with Click-iT fixative. The cells were then permeabilized using saponin-based permeabilization reagent, treated with the Click-iT EdU reaction cocktail in the dark, and washed with saponin-based permeabilization reagent. The number of EdU-positive cells was determined using a FACS-Canto II flow cytometer, and data analysis was performed using DIVA software (Becton Dickinson Biosciences, San Jose, CA, http://www.bdbiosciences.com/).

Colony-Forming Unit assay

MSCs were seeded at a density of 40 cell per square centimeter (400 cells/well) in six-well plates (Nunc, Fisher Scientific, Loughborough, U.K., http://www.fisher.co.uk/) and cultured for up to 14 days with media which was replenished every 3–4 days. Cells were washed with PBS, fixed for 15 minutes with 4% formaldehyde in PBS, stained for 30 minutes with 0.5% Crystal Violet, and washed with PBS.

Western Blotting Analysis

Cells were seeded at a density of 20,000 cells per square centimeter, incubated for 24 hours in normal growth medium, serum starved for 6 hours, and stimulated as indicated. Following incubation for the indicated period, the reaction was terminated by two quick washes in ice-cold PBS containing 1 mM sodium orthovanadate and cells were lysed in ice-cold radioimmunoprecipitation assay buffer [50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloric acid (HCl), pH 7.5, 150 mM sodium chloride (NaCl), 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate] containing a protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium orthovanadate and 0.1 mg/ml phenylmethylsulfonyl fluoride. Cell lysates (10–15 µg of protein) and biotinylated protein ladder (Cell Signaling, New England Biolabs, Hitchin, U.K., http://www.neb.uk.com/) were mixed with Laemmli buffer (Bio-Rad, Hempstead, U.K., http://www.bio-rad.com/) and subjected to SDS-PAGE. Proteins were transferred onto PVDF membranes and incubated overnight at 4°C with primary antibodies against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-Akt (Ser473), phospho-p70 S6 Kinase (Thr389), and phospho-4E-BP1 (Thr37/46) (Cell Signaling). Secondary antibodies (1:2,000) conjugated to horseradish peroxidase were then applied for 1 hour at room temperature, and proteins visualized and photographed using ECL Prime detection reagent (GE Healthcare, Bucks, U.K.) and Molecular Imager Gel Doc XR+ documentation and analysis system with Image Lab Software (Bio-Rad).

Flow Cytometric Analysis of ROS

Accumulation of ROS was assessed after 10 days and 35 days of culture in the presence or absence of FGF-2 or inhibitors in aged MSC. To determine the level of total ROS, cell were stained with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (Invitrogen), incubated for 30 minutes at 37°C, trypsinized, washed, and resuspended in 300 µl of fluorescence-activated cell sorting (FACS) buffer. ROS formation was assessed using a FACS-Canto II flow cytometer (Becton Dickinson Biosciences) at the FITC channel. A total of 10,000 events were acquired for each sample and data analysis was performed using DIVA software (Becton Dickinson Biosciences). Similarly, mitochondrial ROS generation was determined following staining with Mito-SOX-Red (Invitrogen) at 5 µM.

Acridine Orange

Morphological analysis to assess lysosomal membrane integrity and distribution of nucleic acids within the cell compartments was achieved using Acridine Orange staining. Cells were stained with 10 µg/ml of Acridine Orange (Sigma) for 30 minutes at 37°C, washed with media, and assessed using a fluorescent microscope. Acridine Orange emits a red fluorescence when highly concentrated in acidic lysosomes with an intact membrane and emits a green fluorescence at lower concentrations when bound to double stranded DNA in the cytoplasm and nucleus.

Senescence β-Galactosidase Staining

Cellular senescence was assessed using the β-galactosidase staining kit following the manufacturer's instruction (Cell Signaling). Briefly, cells were washed with PBS, fixed with 4% formaldehyde in PBS for 15 minutes, and stained with the β-galactosidase staining solution at pH 6.0. After an over-night incubation, cells were analyzed for blue staining under a phase-contrast microscope (×200 magnification).

Comet Assay

DNA damage was assessed by single-cell gel electrophoresis alkaline comet assay using the Trevigen assay kit (Abingdon, U.K.) according to the manufacturer's instructions. In brief, cells were washed in cold PBS, suspended at a ratio of 1:10 in 0.5% low melting point agarose (37°C), spread at density of 500 cells on comet slides, lysed, and subjected to electrophoresis under alkaline condition on a horizontal electrophoresis apparatus (1 V/cm) for 30 minutes. Slides were stained with SYBR Green I, visualized, and imaged with a fluorescent microscope. DNA damage was scored by visual classification of tail content in five comet categories based on increase in tail moment (CC1–CC5) as illustrated in Supporting Information Data (Supporting Information Fig. S1B). An overall score was calculated as percentage of cells in each category for each treatment.

Oxidative DNA Damage Analysis

8-Oxodeoxyguanosine (8-OxodG) is commonly used as a marker of ROS-induced DNA damage. Avidin binds with high specificity to 8-OxodG in DNA and is used here to detect 8-oxoG residues in the cells. Briefly, MSCs were fixed in 4% paraformaldehyde in PBS and permeabilized with 1% Triton for 15 minutes. After blocking with PBS containing 1% (wt/vol) BSA, cells were stained with FITC-conjugated avidin for 30 minutes and visualized under fluorescence microscopy.

Data Analysis

Statistical comparisons between means were made by one-way ANOVA (SPSS 17, SPSS) and post hoc analyses using the Tukey test to evaluate the differences among the mean values between groups. If comparisons were made only between two groups Student's t test (SPSS 16, SPSS) was used. A p-value of less than .05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Inhibition of Akt/mTOR Maintains the Self-Renewal, Proliferation, and Osteogenic Differentiation Potential of MSC in Long-Term Expansion In Vitro

During in vitro aging, MSCs retain their stem-cell properties up to 6 passages, while cultivation for up to 10 passages results in loss of self-renewal and differentiation capacity even when expanded in the presence of a growth factor such as FGF-2 [6]. Activation of intracellular signal pathways including PI3K/Akt and mTOR has been shown to be associated with loss of stem cell quiescence and depletion of stem cell pool in vivo [13-15]. To determine whether persistent activation of these signal cascades is associated with loss of self-renewal and differentiation of MSC with long-term culture, we blocked the Akt and mTOR pathways during FGF-2-induced expansion of MSC using LY294002 and rapamycin, respectively, from P6 to P10 and subsequently analyzed self-renewal and differentiation capacity of MSC. As previously observed [6], FGF-2 markedly increased the number of cell doublings up to P10 when compared with control cultures, but both sets of cultures reached functional senescence at this time with loss of clonogenic potential, reduced proliferation rate, and differentiation capacity as well as phenotypic changes in size and shape. However, when cells were continuously treated with FGF-2 and LY294002 or rapamycin, the number of cell doublings was reduced but remained higher than control cultures (Fig. 1A).

image

Figure 1. Inhibition of Akt/mammalian target of rapamycin maintains the clonogenic capacity and enhances cell cycle frequency of mesenchymal stem cell (MSC) aged in vitro. MSCs already aged for six passages were treated with NM, FGF-2 or FGF-2 (10 ng/ml) (F) in presence of LY294002 (10 µM) (F+L), rapamycin (10 nM) (F+R), or vehicle (DMSO in NM and F cultures) for further four passages (35 days). (A): Cumulative cell doubling per passage was calculated by cell counting. Subsequently cells were cultured in NM and (B) differences in rate of cell cycle were determined by analyzing the proportion of cells that incorporated EdU following 48 hours of incubation using flow cytometry (n = 4 independent experiments). (C): The frequency of colony formation by MSC was determined by colony forming unit assay and crystal violet staining (representative images from four independent experiments). (D, E): Cell size and granularity were analyzed by flow cytometry using forward and side scatter, (D) representative dot plot, and (E) mean value (n = 4 independent experiments). Each bar indicates mean ± SEM (**, p < .01; and ***, p < .001). Abbreviations: FGF, fibroblast growth factor; NM, normal medium; R, rapamycin.

Download figure to PowerPoint

To assess the growth fraction of cells at P10, serum-stimulated cultures were continuously labeled with EdU for 48 hours. The proportion of cells still cycling was 6.6- and 4.1-fold greater in LY294002 and rapamycin expanded cells, respectively, in comparison to untreated cultures (Fig. 1B). MSC cultured with FGF-2 or in normal media also showed complete loss of colony formation capability, whereas in cultures treated with rapamycin or LY294002 many large colonies were seen (Fig. 1C). Cells cultivated in the presence of inhibitors were also distinguishable by their lower size and granularity based on analysis of side and forward scatter using flow cytometry, compared to other cultures where cells became large and granular as expected with extensive in vitro passaging (Fig. 1D, 1E).

Inhibiting Akt and mTOR also significantly reversed the age-dependent loss of osteogenic differentiation observed with long culture seen both in control and FGF-2 expanded MSC (Fig. 2A, 2B). Following induction of differentiation, expression of the osteogenic differentiation markers of osteogenesis Runx-2, ALP, and osteopontin was significantly higher and matrix mineralization was retained in rapamycin and LY294002-treated cells. These effects were particularly marked in rapamycin expanded cells (Fig. 2A). In contrast, there was only a negligible effect on differentiation toward the adipocyte lineage (Fig. 2C, 2D). This is interesting as evidence suggests an inverse relationship between adipocytes and osteoblasts in aging with negative effects on bone mass and osteoblast production and increase in marrow adiposity [27, 28]. Taken together, these data demonstrate that inhibition of Akt and mTOR pathways during in vitro aging maintains stem cell properties including self-renewal, colony formation, and osteogenic differentiation potential. In support of this, we also observed that the inhibition of these pathways during induction of cell proliferation leads to reduction in the number and size of colony-forming units and cell doubling and during differentiation results in reduction in differentiation capacity of these cells (Supporting Information Fig. S2).

image

Figure 2. Inhibition of Akt/mammalian target of rapamycin maintains the differentiation potential of mesenchymal stem cell (MSC) in long-term expansion in vitro. MSCs already aged for six passages were treated with NM, FGF-2 (10 ng/ml) (F) or FGF-2 in the presence of LY294002 (10 µM) (F+L), rapamycin (10 nM) (F+R), or vehicle (DMSO in NM and F cultures) for a further four passages (35 days). Cells were subsequently induced to differentiate to osteoblasts or adipocytes in the presence of appropriate differentiation media. (A): Upper and middle panels: matrix mineralization was visualized (representative images from four independent experiments) and quantified (lower panel) following Alizarin Red staining and (B) mRNA expression of the osteogenic markers Runx-2, ALP, and osteopontin was analyzed by quantitative-reverse transcription polymerase chain reaction (qRT-PCR) (n = 4 independent experiments). (C): Upper panels: lipid accumulation was visualized (representative of three independent experiments) and quantified following oil red O staining (lower panel) (×200) and (D) expression of adipogenic markers PPARγ, FAB-4, and LPL mRNA was analyzed by qRT-PCR (n = 4 independent experiments). Each bar indicates mean ± SEM (*, p < .05; **, p < .01; and ***, p < .001). Abbreviations: ALP, alkaline phosphatase; FGF, fibroblast growth factor; NM, normal medium; R, rapamycin.

Download figure to PowerPoint

Inhibition of Akt/mTOR Induces Expression of Nanog and Oct-4 and Inhibits ROS Production in MSC Aged In Vitro

To determine cell intrinsic mechanisms involved in maintaining MSC self-renewal and differentiation capacities in LY294002 and rapamycin-treated cells, we examined responses of intracellular signal cascades to stimulation as these has been shown reduced in aged cells [29]. MSC exposed to FGF-2 showed a reduction in extracellular signal-regulated kinases (Erk) and Akt phosphorylation. This effect was reversed and even enhanced in cells expanded in the presence of LY294002 but not rapamycin (Fig. 3A). Although both LY294002 and rapamycin are potent inhibitors of mTOR substrates, the effect of LY294002 on phosphorylation of S6K appears to be reversed within 24 hours of withdrawal of the inhibitor, while it remained completely suppressed with rapamycin treatment even after withdrawal of the inhibitor (Fig. 3A).

image

Figure 3. Akt/mammalian target of rapamycin inhibition induces expression of Nanog and Oct-4 and inhibits reactive oxygen species (ROS) production of mesenchymal stem cell (MSC) aged in vitro. MSCs already aged for six passages were treated with NM, FGF-2 (F), or FGF-2 (10 ng/ml) in the presence of LY294002 (10 µM) (F+L), rapamycin (10 nM) (F+R), or vehicle (DMSO in NM and F cultures) for further four passages (35 days). (A): MSCs harvested from each treatment were cultured for 24 hours in normal growth medium, serum starved for 6 hours, and intracellular signal cascade responses for p-44/42 MAPK (Erk1/2), p-Akt, p-S6K, and p-4E-BP1 to stimulation by normal culture media containing 10% FCS for 20 minutes, 6 hours, and 24 hours were analyzed by Western blotting. NS—no stimulation. Representative blots from three separate experiments are shown. (B): Expression of pluripotency genes Nanog and Oct-4 and osteogenic and adipogenic lineage commitment master switches Runx-2 and PPARγ was analyzed by quantitative-reverse transcription polymerase chain reaction (n = 4 independent experiments). (C): Total ROS (upper panel) and mitochondrial ROS generation (lower panel) were determined by staining with H2DCFDA (DCF-DA) and MitoSOX-Red, respectively, following flow cytometry analysis (n = 3 independent experiments). Each bar indicates mean ± SEM (*, p < .05; **, p < .01; and ***, p < .001). Abbreviations: FGF, fibroblast growth factor; L, LY294002; NM, normal medium; R, rapamycin.

Download figure to PowerPoint

We next analyzed whether the treated cells showed gene expression associated with stem cell properties. Both rapamycin and LY294002-treated cells expressed higher level of “stemness” genes Oct-4 and Nanog (Fig. 3B). They also displayed higher level of the osteogenic transcription factor Runx-2 required for commitment to the osteoblast lineage, but lower level of adipogenic master switch PPAR-γ and genes associated with a differentiated phenotype ALP and FAB-4 (Fig. 3B and Supporting Information Fig. S3). It has previously been shown that the expression of Oct-4 and Nanog is reduced in MSC under normoxic cultures but is elevated in hypoxic conditions to inhibit senescence and maintain self-renewal capacity of MSC in long-term culture [30, 31]. Therefore, we investigated the generation of ROS in these cultures. Prolonged cultivation of human MSC showed an increase in ROS generation, particularly in the presence of FGF-2. Inhibition of mTOR by rapamycin treatment significantly reduced ROS production. MSC cultured with LY294002 also showed some reduced ROS production but only with longer time in culture (Fig. 3C). We further confirmed that this inhibitory effect was mainly by reducing ROS from cytoplasm rather than mitochondria by staining with Mito-SOX-Red, with MSC treated with rapamycin showing similar and LY294002 significantly elevated mitochondrial ROS levels (Fig. 3C).

Inhibition of Akt/mTOR Prevents Senescence-Associated Changes and Reduces DNA Damage Induced by ROS During MSC Aging

ROS production has been shown to cause DNA damage, cell senescence, and aging. We investigated whether reduced ROS production is associated with reduced DNA damage in LY294002 and rapamycin-treated MSC. MSC expanded in the presence of inhibitors showed morphology of early passage cells with a spindle-like shape, small size, and low granularity, while cells cultured without inhibitors became large and flatten with an irregular shape and granular cytoplasm representing the morphology of MSC at their senescence stage (Fig. 4A, 4B). Untreated cells also displayed positive staining for Acridine Orange with an increase particularly in green and to a lower level red fluorescence indicating senescence and DNA damage accumulation (Fig. 4B, 4C). Only negligible differences were observed in β-galactosidase in FGF-2-treated cells in the presence or absence of inhibitors. It should be noted that β-galactosidase itself is not involved in aging or senescence processes and cellular aging is not the only factor that can alter β-galactosidase activity [32]. More importantly, we observed the reduction in expression of genes involved and associated with senescence and aging. MSCs treated with inhibitors showed significant reduction in p16 gene expression. p21 expression was also reduced in MSCs treated with rapamycin (Fig. 4F).

image

Figure 4. Akt/mammalian target of rapamycin inhibition prevents senescence-associated changes and reduces DNA damage during in vitro aging of mesenchymal stem cell (MSC). MSCs already aged for six passages were treated with NM, FGF-2 (F), or FGF-2 (10 ng/ml) in the presence of LY294002 (10 µM) (F+L), rapamycin (10 nM) (F+R), or vehicle for a further four passages (35 days). Morphological changes, accumulation of intracellular vacuoles, cellular debris, DNA damage, and senescence were visualized using (A) light microscopy, following staining with (B, C) acridine orange and (D) for senescence-associated β-galactosidase. Representative images from three independent experiments are shown. Scale bar = 100 µm. (E): Total cellular DNA damage level was determined using alkaline comet single-cell gel electrophoresis assays. Upper panel: representative images of cells following the comet assay. Lower panel: calculated data for the percentages of cells with comet tails of different stages (n = 3 independent experiments). Each bar indicates mean ± SEM. (F): Expression of mRNA for genes involved in MSCs aging and senescence for p16, p21, and p53 was analyzed by quantitative-reverse transcription polymerase chain reaction (n = 4 independent experiments). (G): The level of ROS-induced 8-oxodeoxyguanosine DNA damage was determined by staining with FITC-conjugated avidin (green). Phalloidin-TRITC (red) was used for actin filaments staining. Representative images from three independent experiments are shown. Scale bar = 100 µm. Abbreviations: FGF, fibroblast growth factor; L, LY294002; NM, normal medium; R, rapamycin.

Download figure to PowerPoint

To confirm the protective effect of inhibitors, we next investigated the level of DNA damage by measuring single and double strand breaks in treated and untreated cells using alkaline comet single-cell gel electrophoresis assay. In comparison to LY294002 and rapamycin cultured cells, untreated MSC showed a higher percentage and larger comet-like tails generated by broken DNA, indicating greater and more severe DNA damage (Fig. 4E). MSC expanded in the presence of inhibitors, particularly rapamycin, showed only a very few cells with comet tail moment. To determine whether the observed DNA damage was related to the level of ROS generation, we measured the presence of the most frequent oxidized DNA lesion, 8-oxoguanine, which is directly proportional to the amount of ROS-induced oxidative damage. Consistent with the comet assay, only a small number of LY294002 and rapamycin-treated MSC showed fluorescent signals for 8-oxoguanine, while untreated cells displayed high frequency of 8-oxoguanine DNA lesion localized to cytoplasm of the cells (Fig. 4G).

Inhibition of Superoxide Dismutase 1 Increases MSC Aging, But Can Be Reversed by Rapamycin Treatment

To confirm that inhibition of mTOR prevents MSC aging by reducing cytoplasmic ROS generation and DNA damage, we used a small molecule (lung cancer screen 1 [LCS-1]) [33] inhibitor of superoxide dismutase 1 (SOD1), an enzyme responsible for destruction of ROS in cytoplasm. MSC at passage 4 cultured for 15 days in the presence of LCS-1 showed similar functional deficiencies to those seen with long-term culture in the presence of FGF-2. Prior cultivation with LCS-1 lowered the level of colony formation frequency and mineralization (Fig. 5A–5C). However, no significant effect was observed in the level of gene expression for Runx-2, ALP and osteopontin during differentiation (Supporting Information Fig. S4A). Similarly, addition of LCS-1 to cultures increased the percentage of comet-like tails generated by DNA damage in the cells (Fig. 5D) and higher levels of FITC avidin staining indicating the presence of 8-oxoguanine due to oxidative stress (Supporting Information Fig. S4B). Interestingly, rapamycin could partly protect MSC from the effect seen in the presence of LCS-1. In comparison to LCS-1 alone, cells cultured in the presence of a combination of rapamycin and LCS-1 showed higher colony formation and proliferation rate, increased mineralization, and lower levels of DNA damage (Fig. 5A–5D).

image

Figure 5. Inhibition of SOD1 partially reverses the effects of rapamycin on mesenchymal stem cell (MSC). MSCs were treated with NM, in the presence of R (10 nM), LCS-1 (1 µM) (LCS), or combination of both (R+LCS) for two passages (15 days). Subsequently, cells were cultured in NM and (A) efficiency of colony formation by MSC was determined by colony forming unit assay and crystal violet staining (representative images from three independent experiments) and (B) cell cycling was determined by analyzing the level of EdU incorporation using flow cytometry following continuous 48 hours of labeling (n = 3 independent experiments). Cells were also differentiated to osteoblasts or adipocytes in the presence of appropriate differentiation media. (C): Matrix mineralization was visualized (representative images from three independent experiments) and quantified (n = 3 independent experiments) following Alizarin Red staining and (D) total level of DNA damage was determined using alkaline comet assays. Upper panel: representative images of cells following the comet assay. Lower panel: calculated data for the percentages of cells with comet tails of different stages (n = 3 independent experiments). Data are presented as mean ± SEM (*, p < .05; **, p < .01; and ***, p < .001). Abbreviations: NM, normal medium; R, rapamycin.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

MSCs persist throughout life, undergoing continued self-renewal and differentiation to maintain and repair tissues of mesodermal lineage. In the body, they reside in a niche in a quiescent state to preserve their number and stem-ness properties. With age however, changes in niche extrinsic microenvironment and consequently cell-intrinsic mechanisms lead to decline in their number and function [12, 34]. Similarly and more potently when isolated and cultured in vitro they will be affected by various extrinsic factors, most notably high oxygen tension, and rapidly lose their stem-ness potential. In recent years, there has been evidence for involvement of mTOR signaling in premature aging of stem cells with inhibition of mTOR preventing aging in stem cells of hematopoietic and epithelial origins and spermatogonial progenitor cells [14, 16, 35, 36]. Here, we demonstrate that inhibition of mTOR and its upstream signal cascade could also prevent in vitro aging of MSC and provide evidence of the underlying mechanisms that may be acting. We show that inhibition of mTOR signaling during FGF-2 stimulated long-term culture results in maintenance of stem cell properties including self-renewal, clonogenicity, and differentiation potential. Furthermore, we show that inhibition of mTOR signaling is associated with persisting expression of the stem-ness genes Nanog and Oct-4, and reduction of accumulation of ROS, and ROS-associated DNA damage. Finally, we show that inhibition of SOD1, an enzyme which destroys superoxide radicals, results in similar changes to that seen with FGF-2 treatment and that these effects can be reversed by mTOR inhibition by rapamycin. The protective effects of inhibitors were similar between MSCs from male and female; however, all cells were derived from young donors and more extensive studies comparing MSCs from young and old donors from both sexes would be interesting.

In our previous studies, we found that in vitro senescence was determined by time in culture rather than number of cell doublings per se [6]. Similarly, in the experiments here we found that FGF-stimulated and control cultures both showed age changes related to time in culture rather than cell doublings, which differed markedly. Thus, our results here cannot be explained simply by effects on cell division as the number of cell doublings seen with FGF-stimulated cultures treated with mTOR inhibitors remained greater than those seen with our control cultures.

As reported in this study and by others Akt/mTOR is critical for functional activity of MSC, in particular for induction of cell proliferation and cell cycle [20, 37]. Constant activation of mitogenic stimuli could however lead to the loss of quiescence, exhaustion of stem cell pool, and function [6, 13, 17]. It is suggested that stem-ness properties could be retained if cells express higher level of inhibitors of mitogenic factors and retain a more quiescent phenotype [13]. Interestingly, continuous feeding of old mice with rapamycin led to their having an extended life span [38]. In addition, deletion of S6K, a major component of the mTOR signaling pathway and a target of rapamycin and LY294002 in MSC [20], led to resistance to age-related bone loss [39]. Mice lacking S6K showed higher bone volume and trabecular number when aged [39]. However, it is not known how mTOR may protect against bone loss during aging. In this study, we observed that inhibition of mTOR by rapamycin prevents the loss of self-renewal, proliferation, and osteogenic capacities of aged MSC with only a negligible effect on adipogenic potential. Therefore, it is likely that the previously observed protective effect of mTOR/S6K1 on bone loss may be due to the preservation of the pool of quiescent MSC with osteogenic potentials.

A major compartment or niche that is known to regulate quiescence and multipotency of MSC is the level of oxygen [12]. High oxygen tension and generation of ROS are well documented to be major factors in MSC aging and loss of stem cell properties [10-12, 30]. Here, we found that rapamycin and LY294002 potently regulates the cytoplasmic ROS level and reduces the accumulation of oxidative DNA damage. Interestingly, a significantly higher level of mitochondrial ROS was observed in cells treated with LY294002. This was independent of the inhibitory role of LY294002 on mTOR signaling as rapamycin treatment did not elicit such response. Inhibition of mTOR is shown to reduce mitochondrial membrane potential and oxygen consumption, while the role of PI3K/Akt on mitochondrial ROS is controversial with observed inhibition and induction [40-42]. ROS generation and DNA damage increase with age and have been suggested as a cause of bone loss and osteoporosis. In particular, loss of SOD1, which protects against cytosolic oxidative stress, is associated with decreased bone stiffness and strength and increased bone fragility [43-45]. Consistent with this, we observed that inhibition of SOD1 results in loss of self-renewal and osteogenic capacity of MSC, an effect which was partly reversed by rapamycin. We also observed that both inhibitors resulted in reduction in the expression of p16, with rapamycin-treated cell also showing reduction in p21, genes that are associated with aging and loss of function in MSCs [6, 9]. In addition to reduction in DNA damage reduced oxygen tension has recently been shown to induce the expression of pluripotency genes Nanog and Oct-4 in MSC [30, 31]. These genes are essential for the maintenance of MSC self-renewal and differentiation and have been shown to reduce in expression during long-term culture of MSC under normoxic conditions [31]. Loss of Nanog and Oct-4 expression directly coincides with the onset of organismal aging and their knockdown reduces MSC proliferation rate and differentiation, while ectopic overexpression increases self-renewal and osteogenic differentiation and reduces adipogenesis [31, 46]. Our data show that inhibition of Akt/mTOR significantly increases the expression of these pluripotency genes, especially that of Nanog, therefore suggesting that upregulation of these genes may represent at least one of the processes by which LY294002 and rapamycin maintain self-renewal and function of MSC during aging. Intracellular signaling responses to extracellular cues have also been suggested to be impaired in aged cells [29]. We demonstrated here that MSCs exposed to FGF-2 show a reduction in intracellular signal cascade (i.e., Erk and Akt) responsiveness; this effect was reversed and even enhanced in cells expanded in the presence of LY294002. This may explain the higher proliferative capacity seen following stimulation.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

In Conclusion, the ability of MSC to carry out normal tissue regeneration in the body and their potential for use in clinical applications may be impaired by loss of stem cell number and function with age. Various attempts have been made to address challenges associated with aging of MSC including culture in hypoxic conditions and ectopic expression of pluripotency-associated factors [30, 46]. However, these are restricted to application with prior in vitro cultivation and may be associated with undesirable side effects related with genetic engineering. In this study, we show that pharmacological inhibition of mTOR could prevent age-related phenotype and maintain MSC self-renewal and function in long-term culture and provides insight into possible mechanisms (Fig. 6). Strategies based on targeting mTOR by rapamycin or other newly developed compounds could be adapted for maintaining stem cell properties during in vitro cultivation prior to in vivo transplantation and for pharmacological intervention in diseases associated with loss of MSC function.

image

Figure 6. Schematic diagram illustrating suggested mechanisms involved in preventing mesenchymal stem cell (MSC) aging by Akt/mTOR inhibition. Long-term culture of MSC leads to loss of self-renewal and differentiation, accumulation of DNA damage, and senescence. Inhibition of Akt/mTOR pathways maintains MSC in quiescence and regulates cytoplasmic ROS generation and pluripotency gene expression. This results in promoting self-renewal and differentiation potential of MSC that is otherwise lost during aging. Abbreviations: mTOR, mammalian target of rapamycin; ROS, reactive oxygen species.

Download figure to PowerPoint

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

B.G.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; S.F.: collection and/or assembly of data, and final approval of manuscript; M.G.: data analysis and interpretation, manuscript writing, and final approval of manuscript; F.H.: conception and design, financial support, provision of study material or patients, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

This work was supported by research funding from the Guy's & St. Thomas Charity. We acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St. Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Author Contributions
  9. Acknowledgments
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1709-sup-0001-SuppfigS1.tif2932KSupporting Information Figure S1
stem1709-sup-0002-SuppfigS2.tif2932KSupporting Information Figure S2
stem1709-sup-0003-SuppfigS3.tif2932KSupporting Information Figure S3
stem1709-sup-0004-SuppfigS4.tif2932KSupporting Information Figure S4

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.