Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton

Authors

  • Grit Kasper,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Berlin-Brandenburg Center for Regenerative Therapies, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Lei Mao,

    1. Institute for Human Genetics, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Sven Geissler,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Berlin-Brandenburg Center for Regenerative Therapies, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Albena Draycheva,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Institute for Human Genetics, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Jessica Trippens,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Jirko Kühnisch,

    1. Institute of Medical Genetics, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Miriam Tschirschmann,

    1. Department of Medical Biotechnology, Berlin University of Technology, Berlin, Germany
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  • Katharina Kaspar,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Carsten Perka,

    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Berlin-Brandenburg Center for Regenerative Therapies, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Georg N. Duda,

    Corresponding author
    1. Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Berlin-Brandenburg Center for Regenerative Therapies, Charité—Universitätsmedizin Berlin, Berlin, Germany
    • Julius Wolff Institute and Center for Musculoskeletal Surgery Berlin, Charité— Universitätsmedizin Berlin, Berlin, Germany
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    • Telephone: ++49-30-450659079; Fax: ++49-30-450559969

  • Joachim Klose

    1. Berlin-Brandenburg Center for Regenerative Therapies, Charité—Universitätsmedizin Berlin, Berlin, Germany
    2. Institute for Human Genetics, Charité—Universitätsmedizin Berlin, Berlin, Germany
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  • Author contributions: G.K., C.P., G.N.D., and J.K.: designed research; L.M., S.G., J.T., M.T., A.D., and K.K.: performed research; L.M., S.G., J.T., A.D., and J.K.: analyzed data; G.K.: wrote paper.

  • First published online in STEM CELLSExpress February 26, 2009

Abstract

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague–Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation. STEM CELLS 2009;27:1288–1297

INTRODUCTION

Mesenchymal stem cells (MSCs) have gained increasing interest for application in cell-based therapies and tissue engineering approaches [1]. This is based on their potential for self-renewal, as well as multilineage differentiation, combined with an easy accessibility [2]. MSCs reside in several tissues, e.g., bone marrow, fat, and muscle tissue, and contribute to physiological regeneration in organs such as bone, skin, liver, and muscle. However, clinical experience as well as animal studies prove that the regeneration potential of bone and other tissues declines with age [3]. Consequently, treatment with MSCs from older donors appears to be less effective than application of their younger counterparts [4]. If the age-dependent decrease in regenerative potential is caused by intrinsic changes in MSCs themselves, autologous approaches are prone to be suboptimal in elderly patients, who have the most need for such therapies. Understanding the underlying mechanisms of the age-associated decrease in regeneration potential might pave the way for the development of innovative treatment strategies, e.g., for bone defect healing.

Age-related changes in MSCs may not only account for delayed regeneration in the case of an acute trauma but also for a limited quality of the regenerated tissue. Indeed, aging of stem and progenitor cells was suggested to account for aging of tissue and whole organisms [5]. In principle, MSCs from aged individuals may be altered in quality or quantity (for details see review [6]). However, studies investigating the influence of age on MSCs are contradictory, probably due to differences in experimental parameters such as donor species, sex, age, cell isolation, and cell culture protocols. For example, most studies point to a decrease in MSC number with age [7–9], whereas others find no changes [10, 11]. Concerning the differentiation potential of MSCs, it has been demonstrated that MSCs lose osteogenic in favor of adipogenic potential (the so-called “adipogenic switch”) [12], while others show the opposite [13]. Regarding growth rates, a significant decrease in MSCs from older donors was reported [14, 15], while another study shows an increase of more than three times in the proliferation rate of MSCs from older animals [16]. Further characteristics, such as number of colony-forming units (CFUs), telomere length, and telomerase activity, have been examined with a similar heterogeneity [6]. In conclusion, the impact of age on MSC behavior and availability is still controversial. Furthermore, the molecular basis for potential functional changes remains even more elusive.

However, there are some insights into adult stem cell aging from the hematopoietic stem cell (HSC) system. For these cells, the numbers do not necessarily decline with age, but for that cellular function is clearly compromised, for example with regard to mobilization, homing, and lineage choice [5, 17, 18]. Cellular aging of HSCs has been attributed to various mechanisms that exhibit a partial cause and effect relationship to each other. For example, telomere shortening, as a cell-intrinsic trigger for replicative senescence, was shown to be associated with impaired HSC function due to reduced long-term repopulation capacities and increased genetic instability [19].

Furthermore, abundant evidence in different model systems supports a connection between oxidative metabolism, stress resistance, and aging. It has been shown that lifelong dietary restriction increases HSC frequencies and improved HSC function. The self-renewal capacity of HSCs depends on the control of oxidative stress [20], and additionally, progressive bone marrow failure is associated with elevated reactive oxygen species (ROS). Concordantly, treatment with antioxidative agents has prevented bone marrow failure and restored the reconstitutive capacity of HSCs deficient of Atm, whose gene product inhibits oxidative stress.

Recently, proteomic approaches analyzing the MSC membrane fraction by mass spectrometry revealed novel MSC-specific markers [21] In a another study, PI3K was discovered as a control point for osteogenic differentiation of MSCs [22]. Thus, proteomics was considered in this study as valuable tool to investigate stem cell aging and molecular changes altering cellular behavior. By employing MSCs from young (yMSCs) and old (oMSCs) murine donors, it was demonstrated that a decrease in cell number with donor age is paralleled by increased senescence and decreased migration capacity. A high-resolution large-gel two-dimensional electrophoresis (2DE) screen, performed concurrently, revealed several candidate proteins for mediating these changes. Functional annotation clustering of proteins differently expressed with age and subsequent in vitro assays indicated that processes related to cytoskeleton organization and antioxidant defense are important cellular mechanisms associated with MSC aging.

MATERIALS AND METHODS

MSC Culture

MSCs were isolated from the tibial and femoral bone marrow of 83-week- and 12-month-old Sprague–Dawley rats (Fa. Harlan Winkelmann, Eystrup, Germany, http://www.harlan-winkelmann.de). Male animals were chosen to avoid potential influences of estrogen levels. Isolated cells were cultured in standard expansion medium (EM) [23]. For flow cytometry the following antibodies were employed: mouse (α-rat CD44) (Serotec, Oxford, U.K., http://www.serotec.com), mouse (α-rat CD45) (Acris Antibodies, Herford, Germany, http://www.acris-antibodies.com), mouse (α-rat CD73) (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com), mouse (α-rat CD90) (Acris Antibodies), rat(α-mouse IgG):PE (BD Biosciences).

Functional Assays

All assays were carried out with cells in passage two (P2). Senescence was additionally investigated in P6. Senescence: At a confluence of 80%–90%, cells were stained by the Senescent Cells Staining Kit (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) according to manufacturer's instructions. Migration: A modified Boyden chamber assay was used. MSCs (1 × 104) were seeded and incubated for 5 hours at 37°C. Migrated cells were stained with 10 μg/ml Hoechst. Differentiation: Confluent MSCs were exposed to osteogenic and adipogenic differentiation media [2]. Alkaline phosphatase (AP) activity was detected colorimetrically by para-nitrophenylphosphate and mineralization by Alizarin red staining. Adipogenic differentiation was visualized by Sudan IV staining. Proliferation: Short-term proliferation assays were carried out for 3 days before measuring cell numbers by CellTiter 96 AQueous test (Promega, Madison, WI, http://www.promega.com). Population doubling times were determined by seeding 3,000 cells/cm2 and determination of cell numbers after reaching 80% confluence.

2DE and Mass Spectrometry

For 2D gel electrophoresis, MSCs from young and old animals (passage 2) were cultivated to 80% confluence. For osteogenic stimulation, cells were additionally incubated with osteogenic medium (OM) for 5 days. Subsequent to washing cells with phosphate-buffered saline (PBS), they were harvested with PBS/5 mM EDTA by cautious scraping on ice. Cells were washed again with PBS and the remaining buffer was quantitatively removed before shock freezing of cells in liquid nitrogen. Seventy micrograms of protein lysates were separated by 2DE as described previously [24]. Gel evaluation was performed using Delta2D (version 3.4, Decodon, Greifswald, Germany, http://www.decodon.com) [25]. Briefly, corresponding gel images were first warped using “exact mode”. A fusion image was subsequently generated. Spot detection was carried out on this fusion image automatically, followed by manual spot editing. Subsequently, spots were transferred from the fusion image to all gels. The signal intensities (volume of each spot) were computed as a weighted sum of all pixel intensities of each protein spot. Percent volume of spot intensities to the total spot volume of the parent gel was used for quantitative analysis of protein expression level. 2DE was repeated seven times for EM and eight times for OM. For protein identification, spots of interest were in-gel trypsin digested and analyzed by LCQ Deca XP nano HPLC/ESI ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com). Monoisotopic mass values of peptides were searched against NCBInr sequence database (taxonomy: Mus musculus), allowing one missed cleavage [26]. To conduct functional categorizing, all differentially expressed proteins were submitted to the Database for Annotation, Visualization and Integrated Discovery [27].

Western Blot

The Novex system was employed according to the Invitrogen NuPAGE protocol. Primary antibodies were mouse (α-human calponin) (1:1,000; Sigma-Aldrich), mouse (α-human galectin-3) (1:1,000, Biozol, Eching, Germany, http://www.biozol.de), goat (α-human transgelin) (1:200, Santa Cruz Biotechnology, Santa Curz, CA, http://www.scbt.com), mouse (α-human peroxiredoxin-5) (1:5,000, Biozol) and mouse (α-rabbit glyceraldehyde-3-phosphate dehydrogenase) (1:10,000, Biozol). Secondary antibodies were donkey (α-goat IgG) peroxidase and goat (α-mouse IgG) peroxidase. Band intensities were quantified by NIH ImageJ software package (http://rsb.info.nih.gov/ij).

Antioxidant Activity and Cytoskeleton Dynamics

Cell lysates were investigated by the Antioxidant Assay Kit (Sigma-Aldrich) following manufacturer's instructions. Staining of actin fibers of (para)formaldehyde-fixed and permeabilized cells was achieved by an incubation with Alexa 488-conjugated phalloidin (6.6 nM; Invitrogen, Carlsbad, CA, http://www.invitrogen. com). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Jasplakinolide (Merck, Darmstadt, Germany, http://www.merck.de) was delivered at a final concentration of 250 nM. Confocal fluorescence imaging used an LSM 510 META microscope system (Carl Zeiss, Jena, Germany, http://www.zeiss.de) under identical excitation and exposure conditions.

Statistics

Functional data were analyzed by Mann–Whitney U test. Results from expression analysis were tested by the Student's t test. All tests were analyzed two-sided and at a significance level of p < .05.

RESULTS

The Number of MSCs Is Reduced in Aged Animals

To investigate the age-dependent variation of osteoprogenitor cells in bone marrow, CFUs were determined (Fig. 1). As bone, and hence bone marrow, volume is smaller in young animals (bone marrow volume femur: medianoMSCs = 180 μl, medianyMSCs = 40 μl; bone marrow volume tibia: medianoMSCs = 78 μl medianyMSCs = 30 μl), results were normalized to the corresponding bone marrow volume. The total number of CFUs was significantly lower in bone marrow of old animals compared with younger ones (femora: noMSCs = 1.1/μl, nyMSCs = 16.7/μl, p = .004; tibiae: noMSCs = 0.3/μl, nyMSCs = 18.0/μl, p = .003). Similarly, the number of AP-positive CFUs was reduced in aged animals (femora: noMSCs = 0.2/μl, nyMSCs = 7.6/μl, p = .004; tibiae: noMSCs = 0.0/μl, nyMSCs = 8.6/μl, p = .003). In addition to the absolute number, the ratio of AP-positive CFUs in relation to total CFU numbers was significantly decreased in bone marrow from tibiae of aged animals (ratiooMSCs = 0.0%, ratioyMSCs = 51.2%, p = .006). A similar tendency could be observed in femora (ratiooMSCs = 15.3%, ratioyMSCs = 44.1%, p = .109). Subsequent to the finding that the quantity of MSCs is diminished with age, we pursued the question of functional alterations in the following experiments.

Figure 1.

Quantity of total and AP-positive CFUs is decreased in oMSCs. (A): Typical photographs of CFUs derived from bone marrow of old and young animals. (B): Absolute numbers of CFUs and of AP-positive CFUs per volume of bone marrow. (C): Percentage of AP-positive CFUs relative to total CFUs. CFUs were considered as AP-positive when more than 50% of cells stained AP. *, indicates statistical significance. Abbreviations: AP, alkaline phosphatase; CFU, colony-forming unit; oMSC, old mesenchymal stem cell; yMSC, young mesenchymal stem cell.

Aged MSCs Are More Susceptible Toward Senescence and Display a Lower Migratory Capacity

Senescence is a phenomenon of cellular aging, which can be monitored by β-galactosidase activity. MSC populations showed no difference in the proportion of senescent cells in second passage (ratiooMSCs = 6.5%, ratioyMSCs = 4.5%, p = .827, Fig. 2A). However, after six passages in vitro, a significantly higher percentage of oMSCs were β-galactosidase positive compared with cultures of yMSCs (ratiooMSCs = 8.8%, ratioyMSCs = 3.2%, p = .0495).

Figure 2.

Number of senescent cells and migratory capacity of mesenchymal stem cells (MSCs) depends on donor age. (A): Photographs of ß-galactosidase staining (blue) of MSCs in passage 6 and the percentage of senescent cells in MSC populations from passage 2 and 6 in relation to total cell numbers. (B): Two representative Transwell filters with migrated cells and the ratio of migrated cells (after 5 hours) normalized to the internal standard (number migrated after 32 hours). Filters were either untreated or coated with Matrigel. (C): Osteogenic differentiation determined by matrix mineralization (Alizarin Red) and AP activity. Values were normalized to optical density values from CellTiter 96 AQueous assay. At least three independent experiments were carried out for all assays. *, indicates statistical significance. Abbreviations: AP, alkaline phosphatase; oMSC, old mesenchymal stem cell; P, passage; yMSC, young mesenchymal stem cell.

To investigate age-dependent changes in functional behavior of MSCs, their migration speed, differentiation, and proliferation was analyzed. Motility of oMSCs was lower relative to yMSCs on uncoated filters (migrated cellsoMSCs = 32.7%, migrated cellsyMSCs = 54.4%, p = .031, Fig. 2B). However, in the presence of Matrigel, oMSCs and yMSCs exhibited slightly elevated, but comparable, migration rates (migrated cellsoMSCs = 59.7%, migrated cellsyMSCs = 84.5%, p = .289). Both oMSCs and yMSCs responded similarly to osteogenic stimulation, as determined by the amount of matrix mineralization as well as cellular AP activity (Fig. 2C). There was a trend toward lower AP activities in oMSCs (oMSCs: ODAP/ODMTS = 1.2, yMSCs: ODAP/ODMTS = 2.3, p = .157). However, this tendency did not reach statistical significance, possibly due to the high interindividual variations that were generally observed in differentiation assays. In addition, there was a similar capability of oMSCs and yMSCs to differentiate into adipocytes (percentageoMSCs = 13.2%, percentageyMSCs = 14.3%, p = .275, data not shown). Furthermore, their proliferative capacity was unaffected by donor age. This was deduced from results of short-term proliferation assays (ODoMSCs = 2.4, ODyMSCs = 2.0, p = .827, data not shown) as well as by determining the population doubling time over a culture period of about 10 weeks.

To clarify whether alterations in the functional behavior of aged MSCs are due to changes in the composition of the MSC populations, their cell surface marker protein expression [2] was determined and found to be similar on cells from young and old rats (CD44+, CD73+, CD90+, CD45-, data not shown). Further, the pattern remained stable over serial passaging (analyzed until passage 17). In addition, oMSCs and yMSCs displayed a similar and typical fibroblastic morphology. Having established that old MSCs show increased susceptibility to senescence and decreased motility, we aimed to determine candidate proteins responsible for these cell physiological alterations.

Aging Alters the Cellular Proteome of MSCs

Lysates from MSCs with and without osteogenic stimulation were resolved by high-resolution 2DE. Around 5,000 protein spots were detected. Among the detected proteins, 10 were significantly and reproducibly downregulated and 26 upregulated under EM (Table 1, supporting information). In OM, 29 proteins were decreased and 40 proteins increased in their expression. Taken together, 14 proteins were age-dependently expressed under both conditions (Table 1). Differences between oMSCs and yMSCs were more prominent after osteogenic stimulation, reflected by a higher number of affected proteins (64 vs. 34), higher ratios of differential expression (maxEM = 6.94, maxOM = 10.00, minEM = 0.55, minOM = 0.29) and lower p values (median p valueEM = .013, median p valueOM = .004).

Table 1. Proteins age-dependently expressed under expansion as well as osteogenic conditions
inline image

From all detected protein spots, roughly 3% (140 of 5,000) were differentially expressed under conditions of osteogenic stimulation. In contrast, from the detected age-affected protein spots, 57% (8 of 14) were altered after osteogenic stimulation (Table 2), indicating that a high number of proteins affected by aging of MSCs are also regulated during osteogenic differentiation of the cells. Among these proteins regulated in response to an osteogenic stimulus, pyruvate dehydrogenase (lipoamide) beta has already been described as related to osteogenic differentiation of MSCs [28]. The high-resolution 2DE could additionally identify fatty acid-binding protein 5, galectin-3, γ-synuclein, heterogeneous nuclear ribonucleoprotein A1, myosin light chain, regulatory B, peroxiredoxin 5 precursor, and transgelin as responding to osteogenesis. These proteins have not been detected previously in other proteomic studies investigating osteogenic differentiation of MSCs [21, 28, 29].

Table 2. Age-dependently expressed proteins associated with cytoskeleton and antioxidant defense
inline image

Functional annotation clustering of the differentially expressed proteins in oMSCs versus yMSCs revealed that processes altered, with and without osteogenic stimulation, seem to be related to antioxidant defense and cytoskeleton organization (Fig. 3B, Table 2): antioxidant activity (pEM < .001, pOM = .005), cytoskeletal protein binding (pEM < .001, pOM = .004), and actin binding (pEM < .001, pOM = .008). Furthermore, age-affected proteins seem to be involved in lipid binding, fatty acid binding, and RNA binding. Interestingly, age-associated proteins were not clustered to biological processes of cell cycle regulation or cell proliferation. This is in line with our functional analysis, demonstrating no age-dependent changes in the proliferative capacity of MSCs.

Figure 3.

Age-related proteomic changes in mesenchymal stem cells (MSCs). (A): Representative two-dimensional protein pattern of MSCs. Enlarged insets show examples of upregulated and downregulated protein spots. (B): Pie charts depicting functional annotation clustering of age-affected proteins in EM and OM. Common proteins between OM and EM are indicated by bold font. Two-dimensional polyacrylamide gel electrophoresis was repeated seven times for EM and eight times for OM. (C): Representative Western blots for galectin-3, calponin, transgelin, and the corresponding GAPDH control blots and quantified signal intensities normalized to GAPDH. Data were determined from four animals. *, indicates statistical significance. Abbreviations: EM, expansion medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; O, oMSC; OM, osteogenic medium; oMSC, old mesenchymal stem cell; Y, yMSC; yMSC, young mesenchymal stem cell.

Differentially expressed proteins, involved in bone physiology or aging, were validated by Western blotting of whole cell lysates (Fig. 3C). Age-dependent expression of galectin-3 and calponin-1 was confirmed in EM and OM and of transgelin in OM (EM: ratiogalectin-3 = 3.33, pgalectin-3< .001, ratiocalponin-1 = 0.17, pcalponin-1 < .001, ratiotransgelin = 2.12, ptransgelin = .122; OM: ratiogalectin-3 = 1.60, pgalectin-3 = .003, ratiocalponin-1 = 0.14, pcalponin-1 < .001, ratiotransgelin = 4.94, ptransgelin = .014). However, peroxiredoxin-5 was not found to be differentially expressed (EM: ratioperoxiredoxin-5 = 1.01, pperoxiredoxin-5 = .897; OM: ratioperoxiredoxin-5 = 0.94, pperoxiredoxin-5 = .380), probably due to the detection of different isoforms in Western blotting versus 2DE.

The mRNA amounts of the age-dependently expressed genes galectin-3, calponin-1, and transgelin were similar in young and old MSCs (Fig. 1, supporting information), indicating an age-associated regulation of protein levels by post-transcriptional mechanisms.

MSC Aging Affects Antioxidant Defense and Cytoskeleton Turnover

To further investigate the ontology-based association of aging and antioxidant defense in MSCs, the antioxidant power of old and young MSCs was determined under different culture conditions (Fig. 4A). Lysates from oMSCs cultured in EM exhibited a significantly reduced antioxidant activity compared with those from young cells (antioxidantoMSC = 56 μM, antioxidantyMSC = 94 μM, p = .043). A similar trend was observed after the application of an osteogenic stimulus, but did not reach statistical significance (antioxidantoMSC = 62 μM, antioxidantyMSC = 108 μM, p = .083).

Figure 4.

Antioxidant power and cytoskeleton kinetics are decreased with mesenchymal stem cell (MSC) aging. (A): Concentration of antioxidant activity in lysates of MSCs, according to a Trolox standard, investigated in four independent samples. (B): Representative images of phalloidin labeling in oMSCs and yMSCs after jasplakinolide application over a time series. (C): Cells containing a completely intact or a fully destroyed actin cytoskeleton were counted at different time points after Jasplakinolide application. Original magnification of fluorescence microscopy is ×630. Samples from three young and old animals were investigated. *, indicates statistical significance. Abbreviations: EM, expansion medium; OM, osteogenic medium; oMSC, old mesenchymal stem cell; yMSC, young mesenchymal stem cell.

The second major group of proteins over-represented in ontology analysis was related to cytoskeleton organization. To further validate these in silico data, actin fibers were labeled by phalloidin. Micrographs showed that both old and young MSC populations had no gross abnormalities in their actin filament content and organization. As it is known that aging affects the cytoskeletal dynamics, we further investigated the actin cytoskeleton remodeling by applying the actin-stabilizing drug Jasplakinolide in a time-dependent manner (Fig. 4B, 4C). The mean ratio of cells responding with a fully collapsed actin cytoskeleton was lower in oMSCs than in yMSCs at all five time points investigated reaching statistical significance at 96 minutes after Jasplakinolide supplementation (meanoMSCs = 51%, meanyMSCs = 63%, p = .049). Furthermore, the ratio of cells maintaining an intact actin cytoskeleton was higher in oMSCs compared with yMSCs at all time points and reached the significance level at 72 minutes (meanoMSCs = 43%, meanyMSCs = 27%, p = .028). Thus, oMSCs seem to respond to Jasplakinolide in a delayed manner indicating a decelerated actin turnover. Likely, the effect of Jasplakinolide was highest between 72 and 96 minutes after application due to its uptake or degradation kinetics, thereby reaching statistical significance at these time points, while at the other time points, a similar trend was observed.

Finally, it was investigated, whether the detected age-dependent alterations also occur in sexual matured animals by investigating MSCs from 3-month-old animals. CFU numbers, migratory capacity, and antioxidant power as well as cytoskeleton dynamic of MSCs have a similar qualitative tendency of elevation in these cells like MSCs from 3-weeks-old animals. However, the quantitative effects were more prominent in the youngest age group (Fig. 2, supporting information), indicating a gradual loss of number and function of MSCs with the age of the animal.

DISCUSSION

We demonstrated that the age-related decrease in MSC number is paralleled by alterations in the functionality of oMSCs. Moreover, the proteome of MSCs undergoing aging was profiled for the first time, thereby uncovering age-affected proteins that are potentially causative for the described functional changes. Functional annotation clustering pointed to age-dependent alterations in cytoskeleton organization and antioxidant defense in MSCs.

Potential Relevance of Age-Affected Proteins in Bone Physiology

Several proteins belonging to the actin-binding protein family of calponins were age-dependently expressed. Studies of the calponin-1 (CNN-1) knockout mouse revealed early onset of cartilage formation, premature ossification, increased postnatal bone formation, and accelerated fracture healing [30]. These observations were explained by the enhanced response to bone morphogenetic proteins (BMP). Therefore, by blocking BMP signaling, CNN-1 was established as negative regulator of bone formation [30]. As BMP levels decrease with age [30], downregulation of CNN-1 might be compensatory to maintain BMP signaling and thus osteogenesis.

Galectin-3 is a multifunctional protein regulation extracellular matrix adhesion, growth factor activity, intracellular signaling, and nuclear transcription. In murine bone development, loss of galectin-3 has been associated with altered Indian hedgehog expression pattern at the growth plate, increased cell death of hypertrophic chondrocytes, and uncoupling of growth plate vascularization [31–33]. The important role of galectin-3 in osteoblast differentiation and chondrocyte maturation is strengthened as its expression is controlled by Runx2/Cbfa1 [34]. Thus, galectin-3 might be involved in the regulation of MSC differentiation or fate determination. However, as galectin-3 is a multifunctional protein and its biological role seems to be defined by its subcellular localization [35], it will be crucial for further studies to investigate its age-dependent concentrations in different cellular compartments. For example, the identified intracellular upregulation of galectin-3 in aged MSCs indicate either intracellular retention due to an insufficient secretion and accumulation in the nucleus or a general increase in expression affecting all cellular compartments. As one of the extracellular functions of galectin-3 is to enhance migration, increased expression might be required to maintain normal MSC migration.

Osteogenesis by Aged MSCs

Although the number of AP-positive CFUs declined with age, the osteogenic differentiation capacity of the whole population, detected by matrix mineralization as well as AP activity was not affected. This is in concordance with other in vitro studies demonstrating the maintenance of the osteogenic capacity in aged MSCs [10]. However, opposite findings might account for analyzing different species (rat, human), variable output parameter (total AP activity, AP-positive cells, matrix mineralization, ectopic bone formation), and importantly different culture conditions (2D vs. 3D, dexamethasone, or calcitriol supplementation) [6, 7, 36]. Hence, the nonconformity of results could be caused by a potential effect of age on the regulation and timing of the osteogenic process. Apart from the intrinsic alterations identified in this in vitro study, an aged systemic environment providing altered biochemical stimuli has to be considered for interpretations in vivo. Indeed, MSCs under standard in vitro culture conditions have displayed a similar adipogenic and osteogenic potential [37], whereas under similar experimental conditions, MSCs exposed to serum from old donors are actually inhibited in their osteoblastic but not adipocytic differentiation, compared with those cultured with serum from young donors [38].

ROS Defense

We identified the age-dependent upregulation of several proteins involved in antioxidant defense, along with a decreased antioxidant power in aged MSCs. This might represent a response of the cells to higher ROS levels, possibly resulting from age-associated altered metabolic activities, which however seems to be inadequate to defend effectively. These effects seem to occur also in other cells as the expression of peroxiredoxins increases with age in mouse embryonic fibroblasts [39]. Hence, although the differential expression of antioxidative proteins is not likely to be causative for aging phenomena in MSCs, the reduced ROS defense in oMSCs indicates that aging of these cells follows at least partially the oxidative stress theory, stating that oxidizing species cause molecular damage and, over time, cell and tissue dysfunction [40]. This is strengthened by our observation of higher susceptibility toward senescence in oMSCs, for which ROS are a major trigger.

Cytoskeleton Dynamics

On the basis of a proteome and ontology analysis, we establish that aging results in altered expression of actin cytoskeleton-associated proteins in MSCs accompanied by decreased actin turnover. The molecular mechanism of actin stabilization in oMSCs might be due to the increased levels of transgelin, which is an actin cross-linking protein already identified as biomarker of aging.

Also consistent with altered cytoskeletal dynamics is the lower migratory capacity of oMSCs, as cellular movement is tightly coupled to local actin organization and turnover [41]. According to the molecular function of the detected age-dependent proteins, such as calponin-1, transgelin, vinculin, caldesmon-1, myosin light chain regulatory B, and β-actin, virtually each step of the cellular migration cycle could be altered in oMSCs. Interestingly, the cellular migration speed of oMSCs analyzed on Matrigel was elevated up to the level of the young cells indicating (a) a severe influence of the cellular environment, and (b) that oMSCs are still able to respond to external stimuli. This finding supports the feasibility of potential therapeutic approaches focusing on in vivo mobilization of MSCs in elderly patients.

Studies on actin dynamics and life span in budding yeast revealed that a deletion of the transgelin homologue Scp1 leads to increased actin dynamics and cell viability [42]. Considering our results in MSCs that (a) transgelin is upregulated with age and (b) aged MSCs show decreased actin dynamics, along with the finding that transgelin is overexpressed in senescent cells [43], this coupling of actin dynamics and life span might be conserved in mammalian cells, and especially in MSCs, with transgelin as a potential regulator and linker of these processes.

Local remodeling of the actin cytoskeleton is responsible for translating and modifying external biochemical stimuli, via growth factors and extracellular matrix molecules, into internal signals. Thus, a less dynamic actin cytoskeleton would respond inadequately to such signals. In the case of bone, it is intriguing that mechanical signals, which are important regulators of tissue homeostasis as well as regeneration, are also transduced via the cytoskeleton [44]. This is particularly interesting when considering that in vivo age seems to affect mechanical requirements [45]. Hence, we would postulate that aged MSCs are less responsive to environmental cues (both biological and mechanical), due to their lower actin dynamic, and that this has negative effects on their regenerative potential.

The two main findings on MSC aging, namely decreased ROS defense and reduced actin dynamics, might well affect each other and could potentially act additively or even synergistically. In yeast it has been shown that actin dynamics and ROS production seem to be coupled inversely [42]. The underlying mechanisms remain elusive, but a conceivable linkage could be the regulation of ion channels within the mitochondrial membrane by actin remodeling, in turn affecting ROS release. On the other hand, increased oxidative stress could damage actin molecules themselves or their binding proteins, for example by irreversible carboxylation, thereby decreasing the kinetics of remodeling. Future studies will need to determine whether the two processes are linked to each other, and if so, which mechanisms are causative.

Aging Model

Aging can be defined as a progressive loss of function and thus increased risk of death with time. Decreased bone regeneration with age reflects the systemic aging phenomenon [46], which we hypothesize is due to (a) the number of MSCs lost over time and (b) the gradual accumulation of nonfunctional senescent MSCs. Aging of the MSC population does not necessarily mean that there is a qualitative decline of each individual MSC. Rather, our observations are quantitative expressions of the whole MSC population, potentially consisting of a mixture of nonfunctional and functional MSCs. Hence, it will be important to determine whether all cells in the MSC population age in a synchronized manner, to a similar extent, or whether all functional changes are attributable to a small number of cells. If the last point is the case, future studies will have to clarify questions such as: how can we identify the “bad guys?” Are there ideal cell surface markers distinguishing functional versus nonfunctional oMSCs?

Potential Clinical Relevance

The smaller number of MSCs observed in the bone marrow of old rats confirms observations from other studies, describing that aging of individuals is associated with a decrease in MSC quantity [6]. This shortage of progenitor cells could be exacerbated at the site of injury by the lower migratory potential of oMSCs. In addition, because oMSCs have a weaker capacity to defend against the reactive oxidant species especially prevalent in the initial phase of healing and are more susceptible to senescence, the pool of progenitor cells at regeneration sites could be further diminished (Fig. 5). This reduction in MSC number could account for the clinically recognized decrease in regeneration potential with age and might be overcome by cell therapies based on enrichment or ex vivo expansion of MSCs before retransplantation. Interestingly, there was no age-dependent change in adipogenic differentiation potential and only a mild trend in osteogenic differentiation. Based on these and the previously described observations, one could speculate that potential novel strategies for augmentation of regeneration should focus on attracting progenitor cells to the site of injury, e.g., by chemotactic agents, and/or on ROS protection, rather than simply to stimulate cell differentiation.

Figure 5.

MSC-associated changes in aged individuals might cause a decrease in regeneration capacity. MSC numbers are reduced in the bone marrow of old individuals. Also, due to the lower migratory speed of old mesenchymal stem cells (oMSCs), fewer cells might be capable of homing to the site of injury, thereby further depleting their numbers in the hematoma. Reactive oxygen species, present especially in the early phases of healing, might affect oMSCs more dramatically than young mesenchymal stem cells, because of the age-related reduction in antioxidant power. A higher susceptibility toward senescence in oMSCs might additionally contribute to a shortage of functional progenitor cells able to augment a fast and uneventful regeneration. Abbreviation: MSC, mesenchymal stem cell.

CONCLUSIONS

Results of this study are significant for the understanding of MSC aging, appearing to concur with current aging models involving ROS defense and cytoskeleton dynamics. Profound knowledge about these events might enable us to better understand age-related changes in tissue regeneration processes and thereby rationally design innovative approaches for regenerative therapies. Investigating stem cell aging might also give insights into organism aging and has been postulated as the other side of the coin in cancer protection [5]. This hypothesis aligns with our results because ROS protection and senescence control are two crucial physiological strategies of anti-tumor defense. A further understanding of the molecular basis of aging might allow for rational manipulation of cells, in a way that deleterious effects of aging are compensated without increasing the risk of tumorigenesis.

Acknowledgements

This study was supported by the Federal Ministry of Education and Research (BMBF) excellence cluster Berlin-Brandenburg Center for Regenerative Therapies and the German National Genome Research Network (NGFN) program established by the BMBF. We thank Marzena Princ for excellent technical assistance, Marion Herrmann for 2DE, and Silke Becker for technical assistance in mass spectrometric protein identification. They also thank Dr. C. Wilson for critical reading of the manuscript.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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