Cell Contact Accelerates Replicative Senescence of Human Mesenchymal Stem Cells Independent of Telomere Shortening and p53 Activation: Roles of Ras and Oxidative Stress

Abstract

Mesenchymal stem cells (MSCs) are of great therapeutic potentials due to their multilineage differentiation capabilities. Before transplantation, in vitro culture expansion of MSCs is necessary to get desired cell number. We observed that cell contact accelerated replicative senescence during such process. To confirm the finding as well as to investigate the underlying mechanisms, we cultured both human bone marrow- and umbilical cord blood-derived MSCs under noncontact culture (subculture performed at 60–70% of confluence), or contact culture (cell passage performed at 100% of confluence). It was found that MSCs reached cellular senescence earlier in contact culture, and the doubling time was significantly prolonged. Marked increase of senescence-associated β-galactosidase-positive staining was also observed as a result of cell contact. Cell cycle analysis revealed increased frequency of cell cycle arrest after contact culture. It was noted, however, that the telomere length was not altered during contact-induced acceleration of senescence. Moreover, cell cycle checkpoint regulator P53 expression was not affected by cell contact. Marked increase in intracellular reactive oxygen species (ROS) and a concomitant decrease in the activities of antioxidative enzymes were also observed during contact-induced senescence. Importantly, increased p16^INK4a following Ras upregulation was found after contact culture. Taken together, cell contact induced accelerated senescence of MSCs, which is telomere shortening and p53 independent. ROS accumulation due to defective ROS clearance function together with Ras and p16^INK4a upregulation play an important role in contact-induced senescence of MSCs. Overconfluence should therefore be avoided during in vitro culture expansion of MSCs in order to maintain their qualities for clinical application purposes. The contact-induced senescence model reported in this study will serve as a useful model system that allows further study of the molecular mechanisms of senescence in MSCs.

Keywords

Mesenchymal stem cells Senescence Ras Oxidative stress

Introduction

Aging affects all somatic cells including tissue stem cells (43). The effects of aging on tissue stem cells include a reduction of self-renewal as well as differentiation potentials (32,44,50). Replicative senescence is the ultimate consequence of aging and it also occurs to tissue stem cells (4). Mesenchymal stem cells (MSCs), which can be isolated from a variety of somatic tissues and can be culture expanded (12,29,42,45,57), possess great potentials of clinical transplantation. Before transplantation, in vitro culture expansion is necessary to get adequate numbers of cells. However, there are a variety of factors that affect the in vitro proliferation capacity of MSCs, including source (17,20,21), donor age (26, 27), and disease status of the donors (19,31). Besides, different culture conditions such as medium component (54), plating density (22,37,54), and plate materials (48,54) can affect the quality of the products. For therapeutic purposes, it is therefore imperative to optimize the culture conditions and methods to avoid the occurrence of early senescence.

From the cellular point of view, human diploid cell lines, following Hayflick limit, have a finite lifetime in vitro due to telomere shortening during cell division (14,25,38). For human MSCs, cells reach replicative senescence after long-term culture expansion (20,24) and cell passage affects differentiation potential of MSCs (24,27). During in vitro culture expansion, loss of telomere length reflects the aging of MSCs (2,3). Besides telomere shortening, free radical also plays an important role in cellular senescence (1,13). Reactive oxygen species (ROS), which is generated from respiratory chain in mitochondria (35) and attacks nuclear DNA, mitochondrial DNA, cell membrane, as well as other cellular components, is the primary determinant of aging (5,40, 41). Moreover, expression of oncogenic as well as tumor suppressor genes also results in cellular senescence (6, 33,51).

In cultured cells, it is known that cell contact inhibits cell movement and proliferation. Such process involves transmembrane signaling and cytoskeletal reorganization in addition to transcriptional regulation (10,36,52). MSCs, which are fibroblast-like adherent cells, may also encounter cell–cell contact in culture. However, the effect of contact inhibition and its relationship with senescence remains elusive in MSCs. The purpose of this study is to elucidate the relationship between cell contact and replicative senescence as well as the underlying mechanisms during culture expansion of MSCs. In this study we compared the growth kinetics, cell cycle status, intracellular oxidative stress levels of MSCs with and without cell contact, as well as to elucidate the alterations of the molecular mechanisms that are involved in cellular senescence. This information will be critical for understanding the roles that cell contact play to trigger senescence of MSCs.

Materials and Methods

Isolation and Culture Expansion of MSCs

Human bone marrow-derived MSCs (BM-MSCs) and umbilical cord blood-derived MSCs (U-MSCs) were isolated and characterized as previously described (28). Bone marrow was obtained from five healthy donors with the average age of 37.2 years (range: 17–49 years) during fracture surgeries. Three term umbilical cord blood samples were collected during delivery. An approval from the Institutional Review Board was obtained prior to the commencement of the study and all the samples were collected with informed consent. Briefly, MSCs were isolated from the mononuclear fraction by immune depletion of CD3⁺, CD14⁺, CD19⁺, CD38⁺, CD66b⁺, and glycophorin A⁺ cells, followed by limiting dilution with the final seeding density of 0.3 cell per well. Colonies obtained from single cells were culture expanded in T75 flasks with MesenPro medium (Invitrogen, Carlsbad, CA, USA). When the density reached 60–70% confluence, cells detached from one flask would be reseeded to three new flasks (seeding density: 2.7 × 10³ cells/cm²) after two gentle phosphate-buffered saline (PBS, Gibco BRL, Grand Island, NY, USA) washes. Cell numbers as well as cumulative population doublings (PDs) and cumulative time were estimated during each passage. In this study, all the experiments were performed in triplicate.

Contact Culture

The experiments started when MSCs reached 29 cumulative PDs. In the experimental group in which cell contact was purposely allowed, MSCs were cultured in MesenPro medium and subcultures were not carried out until the cell density reached 100% confluence and cell-cell contact was evident under light microscopy. Cells detached from one flask would be reseeded to three new flasks (seeding density: 4 × 10³ cells/cm²) after two gentle PBS washes, and cell numbers, cumulative PDs, and cumulative time were measured. The noncontact culture group, in which subculture was performed at 60–70% cell density, served as the controls.

Senescence-Associated β-Gal (SA-β-Gal) Staining

SA-β-gal activity was qualitatively assessed using a senescence cell histochemical staining kit (Sigma-Aldrich) as per the manufacturer's instructions. Briefly, MSCs were covered and fixed with 1× fixation buffer for 6–7 min after two gentle PBS washes, followed by quiet stain by incubation with staining mixture containing X-gal solution. After three times of gentle PBS rinse, cells were examined under light microscopy.

Cell Cycle Analysis

Cell cycle analysis was performed using the Cycle-TEST^™ PLUS DNA Reagent Kit (Becton Dickinson, San Jose, CA, USA) per the manufacturer's instructions. Briefly, cells were detached and suspended in an Eppendorf tube. After three cycles of centrifugation at 300 × g at room temperature for 5 min, removal of supernatant followed by addition of 1 ml buffer solution containing sodium citrate, sucrose, and dimethyl sulfoxide (DMSO) was done, followed by resuspension of the cells by gentle shaking at low speed. Cells were then fixed with dry ice and 99% ethanol for 30 min. After centrifugation of the suspensions at 400 × g for 5 min at room temperature and removal of the supernatant, 250 μl of solution A containing trypsin buffer was added into the tube. Ten minutes later, 200 μl of solution B containing trypsin inhibitor and RNase buffer was added for another 10-min reaction. Cells were then incubated with 200 μl of cold solution C containing propidium iodide for 10 min, and cells were analyzed with FACSCalibur (Becton Dickinson).

Telomere Length Measurement

Telomere length measurement was carried out by Southern blot analysis. DNA was extracted from 5 × 10⁵ MSCs using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) per the manufacturer's instructions. Telomere restriction fragment length was measured by Southern blot hybridization following the protocol outlined in the Telo TAGGG TL Assay Kit (Roche Applied Science, Mannheim, Germany). Briefly, 1–2 μg of purified genomic DNA was diluted with nuclease-free water to a final volume of 17 μl in a reaction vial. Following the addition of 2 μl of 10× digestion buffer and 1 μl of Hinf1/Rsa1 enzyme mixture, the solution was incubated for 2 h at 37°C. The reaction was stopped by the addition of 5 μl of 5× gel electrophoresis loading buffer. After this, the digested DNA solution was mixed with 4 μl DIG molecular weight marker, 12 μl nuclease-free water, and 4 μl 5× loading buffer. Each digested DNA (1–2 μg) was loaded onto a lane of 0.8% agarose gel, and the gel was run at 5 V/cm. Southern transfer of the digested DNA was then performed, and the transferred DNA was fixed by UV cross-linking (120 mJ). For hybridization, 6.5 ml of hybridization solution containing 1 μl telomere probe per 5 mp fresh prewarmed DIG Easy Hyb was incubated with 200 cm² blotting membrane for 3 h at 42°C. After two washes, the membrane was rewarmed in a heated water bath for 15–20 min at 50°C. Finally, the membrane was incubated in 50–100 ml anti-DIG-AP working solution for 30 min, followed by 100 ml of detection buffer for 2–5 min at room temperature. With dropped substrate solution, the membrane was exposed to X-ray film for 20 min.

For quantitative measurements of mean telomere restriction fragment (TRF) length, the chemiluminescence signals were scanned on a GS-800 densitometer using Quantity One software (Bio-Rad, Hercules, CA, USA). The mean TRF length has been defined according to the following formula:

\overline{TRF} = \frac{Σ ({OD}_{i})}{Σ ({OD}_{i} / L_{i})}

where OD_i is the chemiluminescent signal and L_i is the length of the TRF at position i. The calculation takes into account the higher signal intensity from larger TRFs due to multiple hybridizations of the telomere-specific hybridization probe.

Phosphorylated H2A.X Immunofluorescence Staining

To measure DNA damage, MSCs after contact culture were treated with UVB 0 or 200 J/m², respectively. Cells were washed by TBS, followed by treatment with 95% ethanol plus 5% acetic acid for 10 min and methanol for another 5 min. After two gentle TBS washes, blocking buffer (3% BSA/TBS) was added for 30 min. Phosphorylated H2A.X was stained by 4 μg/ml of FITC-conjugated anti-phospho-Histone H2A.X (Ser139) (Millipore, Billerica, MA, USA) in blocking buffer for 1 h at room temperature. After five times TBS washes, cells were assessed under a fluorescence microscope (Leitz, Germany) and imaging was performed with a SPOT RT Imaging system (Diagnostic Instruments, Sterling Heights, MI, USA).

Determination of Intracellular ROS Levels

To measure the intracellular ROS level, 2′,7′-dichlor-odihydrofluorescein diacetate (H₂DCFH-DA, Molecular Probes, Eugene, OR, USA), a fluorogenic substrate, and hydroethidine (HE, Molecular Probes), a fluorescent probe, were used. Cells were incubated with 40 μM H₂DCFH-DA at 37°C in the dark for 30 min or with 5 μg/ml HE at 37°C in the dark for 10 min. Cells were then harvested and resuspended in 50 mM HEPES buffer (KCl 5 mM, NaCl 140 mM, CaCl₂ 2 mM, MgCl₂ 1 mM, glucose 10 mM, HEPES 5 mM, pH 7.4), and were used for flow cytometric analysis. Fluorescence intensity of dihydrofluorescein (DCF) and ethidine of 10⁴ cells were recorded in each experiment. Vitamin C is known to lower intracellular ROS levels and 200 mM of vitamine C (Sigma-Aldrich) was used as antioxidant to evaluate the role of ROS in this study.

Measurement of Intracellular ATP Levels

Intracellular ATP was measured using Bioluminescent Somatic Cell Assay Kit (Sigma-Aldrich) according to the manufacturer's instruction. Briefly, cells cultured in six-well plate were trypsinized and resuspended (without centrifugation) in fresh medium. Then 50 μl of the cell suspension was added to 150 ml Somatic Cell Releasing Reagent to release the intracellular ATP. One hundred microliters of the mixture was then transferred into a black OptiPlate^™-96F 96-well plate (Packard Biosciences, Groningen, The Netherlands) containing 100 μl ATP Assay Mix and was measured by Victor2^™ 1420 multiabel counter (PerkinElmer Life Sciences, Inc., Boston, MA, USA).

Detection of Antioxidative Enzyme Activities

To determine the enzyme activities of superoxide dismutase (SOD) and catalase, cells were trypsinized, washed with PBS, and centrifuged. Pellets were resuspended in 50 μl lysis buffer (1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, and protease inhibitors) and incubated on ice for 30 min and vortex mixed every 10 min. SOD activity was assayed by monitoring nitroblue tetrazolium (NBT) reduction according to Spitz and Oberley (49) with some modifications. SOD inhibits NBT reduction caused by O₂^•- in the aerobic xanthine/xanthine oxidase system, and changes of absorbance at 560 nm was recorded. One unit of SOD is defined as the amount of enzyme that causes 45% inhibition of NBT reduction under the assay condition described. Catalase activity was determined by monitoring the rate of decomposition of H₂O₂ from the decrease in absorbance at 240 nm (8).

Western Blot Analysis

For Western blot analysis, cells were pelletized and washed once with PBS and then resuspended in 50 μl lysis buffer (4 mM EDTA, 2 mM EGTA, 1% Triton X-100, and an aliquot of complete protease inhibitors mixture, ph 7.4; Roche Diagnostics, Mannheim, Germany). Cell suspension was incubated on ice for 30 min and then centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected and protein concentration was measures by Bradford assay (Bio-Rad). Protein (25 μg) was separated on 12% SDS-PAGE and blotted onto PVDF membrane (Amersham Biosciences, Uppsala, Sweden). Nonspecific bindings were blocked by 3% skim milk in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4) at room temperature for 1 h. The membrane was then blotted with indicated primary antibodies: manganese-superoxide dismutase (Mn-SOD) and copper/zinc-superoxide dismutase (Cu/Zn-SOD) (1:5000, Upstate Biotechnology, Charlottesville, VA, USA), catalase (1:3000, Calbiochem, San Diego, CA, USA), p53 (1:1000, epitomic Inc., Burlingame, CA, USA), p16^INK4a (1:1000, abcam Inc., Cambridge, MA, USA), Bmi-1 (1:1000, Millipore), Ras (1:500,000, epitomic Inc.), connexin 43 (1:2000, Becton Dickinson), and β-actin (1:5000, Sigma-Aldrich). After three washes with TBST, 5 min each wash, the membrane was incubated with the HRP-conjugated secondary antibody (1:5000, Amersham Biosciences) at room temperature for 1 h. After another three times of washes, the protein intensity was determined by ECL chemiluminescence reagent (PerkinElmer Life Sciences, Inc.), and their intensities were quantitatively measured by a densitometry (LabWorks, UVP Inc., Upland, CA, USA).

Statistical Analysis

Statistical analysis was performed using the Statistical Package for Social Science-10 software (SPSS Inc., Chicago, IL, USA). Results of intracellular ROS level, ATP, and quantitative Western blot analysis were analyzed by two-tail, nonpaired t -tests, and values of p < 0.05 were considered statistically significant. Comparison of antioxidative enzyme activities was analyzed by one-way ANOVA with Tukey's post hoc tests at 95% confidence intervals. Different characters represented different levels of significance when values are p < 0.05.

Results

Contact Culture Induced Early Replicative Senescence of MSCs In Vitro

In the control group, MSCs were subcultivated when cell density reached 60–70% confluence (Fig. 1). In the experimental group, subcultivation was not carried out until cell density reached 100% confluence (Fig. 1B). BM-MSCs reached replicative senescence at 47 cumulative PDs in the control group and cell contact induced early senescence at 38 cumulative PDs (Fig. 1C); U-MSCs reached replicative senescence at 65 cumulative PDs and cell contact also induced early senescence at around 40 cumulative PDs (Fig. 1D). In BM-MSCs, cell contact induced morphological changes from short, spindle-shaped cells into large, flattened cells. Many BM-MSCs stained positive for SA-β-gal after cell contact (Fig. 1E).

Figure 1.

Early replicative senescence of mesesnchymal stem cells (MSCs) occurred as a consequence of cell contact. (A) Under noncontact culture, MSCs were subcultivated at the density of 60–70% confluence. (B) Under contact culture, MSCs were not subcultivated until the density reached 100% confluence, which allowed the occurrence of cell contact. (C) Growth kinetics demonstrated the contact-induced early replicative senescence of bone marrow-derived MSCs (BM-MSCs). (D) Growth kinetics demonstrated the contact-induced early replicative senescence of umbilical cord blood-derived MSCs (U-MSCs). (E) Comparison of MSCs at the same population doubling (PD), cell contact induced early senescence evidenced by strongly positive senescence-associated β-gal (SA-β-gal) stain. Scale bars: 50 μm.

Cell Contact Increased the Frequency of G₀/G₁ in BM-MSCs

To further validate the changes of growth kinetic curves resulting from cell contact, cell cycle analysis was performed to quantitatively measure the alteration of growth kinetics. As shown in Figure 2, in subconfluent culture of BM-MSCs without contact, the frequency of G₀/G₁ phases gradually increased from 74.19% at PD29 (Fig. 2A) to 79.68% at PD33 (Fig. 2B) and then 81.63% at PD35 (Fig. 2C). As expected, cell contact markedly increased the frequency of G₀/G₁ phases and it was associated with decrease in the frequency of S and G₂/M phases (Fig. 2D–F). Under contact culture, the frequency of G₀/G₁ phases increased from 83.20% at PD29 (Fig. 2D) to 94.01% at PD33 (Fig. 2E) and 94.15% at PD35 (Fig. 2F). Besides, insignificant changes of sub-G1 phase (Fig. 2D–F), as well as undetectable level of active caspase-3 expression (data not shown) during contact culture, have ruled out the possibility of contact-induced apoptosis.

Figure 2.

Cell contact increased the frequency of cell cycle arrest in MSCs. (A–C) Under noncontact culture, the frequency of G₀/G₁ phases in BM-MSCs gradually increased from 74.19% at PD29 to 79.68% at PD33 and then 81.63% at PD35. (D–F) Under contact culture, dramatic increase in the frequency of G₀/G₁ phases in BM-MSCs accompanied by a decrease in S and G₂/M population was found compared to the control group at the same PD.

Cell Contact Affected Doubling Time but not Telomere Length of MSCs

To investigate whether cell contact results in replicative senescence through accelerated telomere shortening, measurement of the rate telomere shortening was performed. As shown in Figure 3B, doubling time of both BM-MSCs and U-MSCs was prolonged. However, cell contact did not result in significant acceleration of telomere shortening (Fig. 3A, B). Also, cell contact did not induce increased DNA damage, evidenced by negative H2AX staining (Fig. 3C).

Figure 3.

Cell contact did not induce significant telomere length shortening and DNA damage. (A) Cell contact did not result in acceleration of telomere shortening in BM-MSCs. (B) In both BM-MSCs and in U-MSCs, reduced cumulative PDs and prolonged doubling time induced by cell contact was not associated with telomere length, which did not significantly alter by cell contact. (C) Cell contact did not induce increased DNA damage, evidenced by negative H2AX staining. MSCs stained positive for H2AX after UV treatment, which served as positive control. Scale bars: 50 μm.

Cell Contact Induced Increased Intracellular ROS Levels Due to Increased ATP Production and Defective Antioxidative Enzyme Functions

Since ROS are well known inducers of cellular senescence (30), to further evaluate the mechanisms of accelerated cellular senescence caused by cell contact, intracellular ROS levels were measured in BM-MSCs and U-MSCs (data not shown) at different population doublings. Two fluorogenic probes, H₂DCFH-DA and HE, were used to determine the alteration of intracellular ROS levels after cell contact. It was found intracellular ROS levels were not significantly altered during culture expansion in the control group. However, cell contact significantly increased intracellular levels (Fig. 4A, B). Since ROS is the inevitable deleterious by-product during oxidative phosphorylation of ATP production (7), we measured the levels of intracellular ATP. As expected, cell contact induced significant elevation of intracellular ATP levels (Fig. 4C).

Figure 4.

Cell contact induced increased intracellular ROS levels due increased ATP production and defective antioxidative enzyme functions. Increased intracellular ROS levels, which were measured by DCF (A) and ethidine (B) intensities, indicated the accumulation of intracellular ROS in BM-MSCs after cell contact. (C) Cell contact induced significant elevation of intracellular ATP levels in BM-MSCs (^*p < 0.05, n = 3). (D) Protein level of antioxidative enzymes including catalase, manganese-superoxide dismutase (Mn-SOD), and copper/zinc-superoxide dismutase (Cu/Zn-SOD) were upregulated after cell contact. However, the overall activities of SOD (E) and catalase (F) were significantly reduced as a consequence of cell contact (^*p < 0.05, n = 3).

To further elucidate whether the antioxidative enzymes were also affected and contributed to the elevation of intracellular ROS levels, the amount and the activities of these enzymes including catalase, Mn-SOD, and Cu/Zn-SOD were measured. It was found that all three antioxidative enzymes were upregulated after cell contact (Fig. 4D); nevertheless, the overall activities of SOD and catalase were significantly reduced (Fig. 4E, F, respectively) as a consequence of cell contact in both BM-MSCs and U-MSCs.

Cell Contact-Induced Senescence Was Associated with Upregulation of Ras and p16^INK4a

After evaluation of the roles of ROS antioxidative enzymes in cell contact-induced senescence in MSCs, we further investigated the alterations of cell cycle check point regulators after cell contact, including p53, p16^INK4a, Bmi-1, and Ras. It was found that expression of p16^INK4a and Ras was markedly increased as a consequence of cell contact by Western blot analysis (Fig. 5A). Quantitative analysis also confirmed that there was no significant change of p53 and Bmi-1 after cell contact (Fig. 5B, D, respectively); however, expression of p16^INK4a and Ras was significantly increased after cell contact (Fig. 5C, E, respectively). Notably, Ras upregulation occurred immediately after cell contact at PD29 (Fig. 5E), and was prior to the upregulation of p16^INK4a, which was not significantly increased until PD33 (Fig. 5C).

Figure 5.

Upregulation of Ras-p16^INK4a in cell contact-induced senescence. (A) Expression of cell cycle check point regulators, including p53, p16^INK4a, Bmi-1, and Ras, was measured by Western blot analysis. Only p16^INK4a and Ras were markedly increased as a consequence of cell contact. Quantitative analysis confirmed that there was no significant change of p53 after cell contact (B). However, upregulation of p16^INK4a was noted at PD33 (C), which was not related to Bmi-1 (D) but was preceded by Ras upregulation at PD29 (E) (t-test, ^*p < 0.05, n = 3).

ROS Accumulation Was the Consequence of Ras Expression After Cell Contact

To determine whether ROS scavenger can rescue contact-induced senescence, vitamin C was added into the culture medium during contact culture. It was found that vitamin C lowered the intracellular ROS level (Fig. 6A); however, it failed to reverse the reduced SOD (Fig. 6B) and catalase (Fig. 6C) activities. Besides, vitamin C did not alter the increased Ras protein expression both in BM-MSCs and U-MSCs induced cell contact (Fig. 6D).

Figure 6.

Role of ROS scavenger and cell–cell communication in contact-induced senescence. (A) Vitamin C (Vit C) decreased the intracellular ROS level in BM-MSCs at PD38 after contact culture. However, vitamin C failed to rescue the activity of SOD (B) and catalase (C) (ANOVA, each letter represents the different level of significance, p < 0.05, n = 3). (D) Vitamin C did not alter Ras expression in both BM-MSCs and U-MSCs with or without contact culture. (E) Markedly increased expression of connexin 43 was found as the consequence of cell contact.

Cell Contact Upregulated Connexin 43 Expression

Connexin 43, a gap junction subunit, has been demonstrated that the expression changed as a function of cell contact (11,39,53). Indeed, during noncontact culture, the expression of connexin 43 of MSCs was unchanged. However, the expression of connexin 43 was immediately upregulated and accumulated after cell contact (Fig. 6E), which was similar to what we found in Ras expression (Fig. 5A, E).

Discussion

Despite their multilineage differentiation potentials, the major drawback of MSCs is that, unlike embryonic stem cells, these cells undergo replicative senescence during in vitro culture expansion (15). Various lines of evidence point out that the in vitro proliferation capabilities of MSCs are still subject to Hayflick limit (15,16, 27), although the underlying mechanisms still remain elusive (55). It has been found that during in vitro culture expansion of MSCs, low plating density is favorable for proliferation of MSCs (37,55). However, the detrimental effects induced by cell contact were not reported. For the first time, we demonstrated that cell contact resulting in accelerated replicative senescence of human MSCs was independent of telomere shortening and was associated with increased intracellular oxidative stress and defective antioxidative enzyme functions. Cell contact also triggered cell cycle arrest through selective activation of Ras and p16^INK4a. For clinical application purposes, it is imperative to take into consideration the above-mentioned findings and avoid cell contact by performing early subplating when 60–70% confluence is reached, so that the MSCs of better qualities can be obtained after culture expansion.

It has been reported previously that p16^INK4a is closely associated with senescence of human MSCs (47). In the current study we found that cell contact induced upregulation of not only p16^INK4a but also Ras (Fig. 5A, C, E). It has been demonstrated that Ras induces irreversible growth arrest known as oncogene-induced senescence, and the activation of p16^INK4a occurs downstream of Ras activation (9,46). According to our data, it is likely that Ras plays a major role and works cooperatively with p16^INK4a to trigger cell contact-induced senescence in human MSCs as Ras activation occurred prior to the activation of p16^INK4a. It is also likely that the activation of Ras is related to the reorganization of cytoskeleton caused by cell contact; in other words, the consequence of mechanotransduction (56). How alterations of the mechanical environment caused by cell contact regulate the machinery of replicative senescence in MSCs requires further investigation. Since it has recently been reported that MSCs in hypoxia culture could escape from senescence by downregulating p16, hypoxic culture may serve as an adjunct in addition to subconfluent culture to further ensure the quality of MSCs after culture expansion (23).

ROS play an important role in triggering cellular senescence (41). In this study, it was found that intracellular ROS levels increased (Fig. 4A, B) as a consequence of cell contact-induced senescence since ROS scavenger could lower the intracellular ROS level (Fig. 6A) but failed to rescue the contact-induced senescence (Fig. 6B–D). Besides increased ATP production (Fig. 4C) and functionally defective antioxidative enzymes (Fig. 4E, F), Ras has also been reported to increase ROS production (18). Notably, it has been reported that the increase of ATP production is a mechanism to resist ROS-induced cell death (35), and this may explain why intracellular ATP levels sharply increased after cell contact (Fig. 4C). However, during aerobic respiration of ATP production, more ROS will be produced (7) and thus may further aggravate replicative senescence induced by cell contact. Regarding the reduced functions of the antioxidative enzymes, the exact mechanism and its relationship with Ras remains to be further elucidated. Nevertheless, increased intracellular ROS production caused by Ras and increased ATP production has comprised a vicious cycle in conjunction with functionally compromised antioxidative enzymes during cell contact-induced senescence.

It has been demonstrated by many studies that change of cell morphology after cell–cell contact was due to increased cell–cell communication mediated through increased expression of gap junction molecules (11,39,53). Similar to what we found in Ras expression, we first demonstrated the expression of connexin 43 in MSCs was significantly increased after contact (Fig. 6E), indicating that increased cell–cell communication was involved in contact-induced senescence of MSCs. However, the role of increased connexin 43 in contact-induced senescence remains elusive, and the relationship between connexin 43 and Ras in contact-induced senescence of MSCs warrants further elucidation. Importantly, connexin 43 can be used as a quality assurance bio-marker in future culture expansion of MSCs.

Recently, there are several reports exploring the mechanisms of senescence in human MSCs (16,30,47). It is clear that donor age, or the intrinsic properties of the biologic clock, affects the in vitro replicative senescence rate of human MSCs (26,50). In order to control such confounding factor, we used U-MSCs as well as BM-MSCs from relatively young donors. It has become clear from the results of this study that cell contact significantly accelerates replicative senescence in a telomere-independent manner as cell contact did not significantly accelerate telomere shortening (Fig. 3A, B). How signals caused by cell contact interact with the intrinsic clock of aging requires further investigation.

Conclusion

In conclusion, cell contact significantly accelerates the occurrence of replicative senescence of MSCs, which are independent of telomere shortening, DNA damage or p53 activation. Instead, increased intracellular oxidative stress and the activation of Ras, p16^INK4a play an important role in contact-induced senescence of MSCs. For therapeutic purposes, cell contact should be avoided during in vitro culture expansion of MSCs from the perspective of quality controls of the cell products. For the same reason, early senescence caused by cell contact may confound the experimental results and should thus be controlled. Nevertheless, contact culture model reported here may serve as a useful platform in future studies regarding senescence of MSCs.

Footnotes

Acknowledgments

The authors acknowledge financial support from the Taipei Medical University (TMU97-AE1-B22, to J.H.H.). The authors also acknowledge the support of research grants from the Taipei Veterans General Hospital (VGH98E1-001 and VGH98C1-023, to O.K.L.), and the National Science Council (NSC98-2314-B-010-001, NSC98-2627-B-010-004, and NSC98-3111-B-010-003, to O.K.L.; and NSC98-2314-B-038-010-MY3, to J.H.H.). This study was also supported by a grant from the Ministry of Education, Aim for the Top University Plan.

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Cell Contact Accelerates Replicative Senescence of Human Mesenchymal Stem Cells Independent of Telomere Shortening and p53 Activation: Roles of Ras and Oxidative Stress

Abstract

Keywords

Introduction

Materials and Methods

Isolation and Culture Expansion of MSCs

Contact Culture

Senescence-Associated β-Gal (SA-β-Gal) Staining

Cell Cycle Analysis

Telomere Length Measurement

Phosphorylated H2A.X Immunofluorescence Staining

Determination of Intracellular ROS Levels

Measurement of Intracellular ATP Levels

Detection of Antioxidative Enzyme Activities

Western Blot Analysis

Statistical Analysis

Results

Contact Culture Induced Early Replicative Senescence of MSCs In Vitro

Cell Contact Increased the Frequency of G0/G1 in BM-MSCs

Cell Contact Affected Doubling Time but not Telomere Length of MSCs

Cell Contact Induced Increased Intracellular ROS Levels Due to Increased ATP Production and Defective Antioxidative Enzyme Functions

Cell Contact-Induced Senescence Was Associated with Upregulation of Ras and p16INK4a

ROS Accumulation Was the Consequence of Ras Expression After Cell Contact

Cell Contact Upregulated Connexin 43 Expression

Discussion

Conclusion

Footnotes

Acknowledgments

References

Cell Contact Increased the Frequency of G₀/G₁ in BM-MSCs

Cell Contact-Induced Senescence Was Associated with Upregulation of Ras and p16^INK4a