The experimental protocol was approved by Jiangsu University ethics committee (Zhenjiang, China). Fresh umbilical cords were collected in March 2015, immediately after birth from healthy donors at the First People's Hospital of Zhenjiang (Zhenjiang, China). The umbilical cords were rinsed twice in phosphate-buffered saline (PBS) until the cord blood was cleared. The blood vessels were removed from each cord, then the remaining tissue was cut into 1-mm³ pieces, and suspended in low-glucose Dulbecco's modified Eagle's medium (L-DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 1% penicillin and 1% streptomycin (Beyotime Institute of Biotechnology, Haimen, China). All cultures were incubated at 37°C with an atmosphere of 5% CO₂ in a humidified chamber. The medium was changed every 3 days after initial plating. When well-developed colonies of fibroblast-like cells reached 70-80% confluence, the cells were trypsinized with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Inc.) and passaged into new culture flasks for further expansion. To establish a cell model of H₂O₂-induced MSC premature senescence, early passage (P4) hucMSCs were treated with H₂O₂ at different concentrations (0, 200, 400, 600 and 800 μM) for 2 h. The stimulus was then removed and cells were further incubated in fresh medium for 48 h.

To determine the phenotypes of hucMSCs, fluo-rescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled mouse monoclonal antibodies against human leukocyte antigen (HLA)-DR, cluster of differentiation (CD)105, CD34, CD29, CD90 and CD44 (1:10; BD Biosciences, Franklin Lakes, NJ, USA; cat. nos. 555811, 560839, 348053, 555443, 555596 and 555479, respectively) were used. Briefly, at P4, MSCs were trypsinized, washed twice with PBS and stained with the monoclonal antibodies, according to the manufacturer's protocol. Mouse monoclonal PE-immunoglobulin G1 (IgG1) and FITC-IgG1 (BD Biosciences; cat. nos. 555574 and 555573, respectively) were used as isotype controls. The stained cells were analyzed using the FACSAria flow cytometer(BD Biosciences).

The cell surface morphology of hucMSCs was analyzed using scanning electron microscopy (SEM; Hitachi S-3400N; Hitachi, Ltd., Tokyo, Japan) at P4, P11 and P17. Cells were seeded in 6-well plates and the media was removed following 3 days of culture. The cells were washed with PBS and fixed with 2.5% glutaraldehyde (Sangon Biotech Co., Ltd., Shanghai, China) for 1 h. The cells were then rinsed with distilled water and dehydrated with a series of ethanol gradients starting at 30% and increasing to 50, 70, 80, 90, 95 and 100% (v/v). Subsequently, the cells were air-dried overnight at room temperature in a fume hood. The cells were gold-coated and cell morphology was analyzed using SEM.

To image the cell nuclei, cells were cultured at a comparable density on coverslips in 6-well plates. They were washed with PBS, fixed for 15 min with 4% formaldehyde (Sangon Biotech Co., Ltd.) and washed with PBS 3 times. DNA was visualized with 4′6-diamidino-2-phenylindole (1 mg/ml) (Beyotime Institute of Biotechnology) by fluorescence microscopy (Molecular Devices, LLC, Sunnyvale, CA, USA).

Assessment of the proliferative ability of hucMSCs was performed using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT; AMRESCO LLC, Solon, OH, USA) assay at P4, P11 and P17. The cells were seeded in 96-well plates at a density of 1,000 cells per well. At days 1, 2, 3, 4, 5, 6 and 7, MTT (20 μl) was added to each well for 4 h. When the reaction was terminated, the medium was discarded and 150 μl dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Following uniform oscillation for 10 min to fully dissolve the purple formazan crystals, the absorbance values were determined at 490 nm with a spectrophotometer (FLX800; BioTek Instruments, Inc., Winooski, VT, USA).

SA-β-gal activity was analyzed in different passages (P4, P11 and P17) of hucMSCs using a SA-β-gal staining kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. In brief, the cells were cultured to comparable densities on coverslips in 24-well plates and washed with PBS, fixed for 15 min with 4% formaldehyde and washed with PBS. Subsequently, the cells were incubated overnight at 37°C in a CO₂-free chamber with freshly prepared SA-β-gal stain solution. SA-β-gal-positive cells exhibited a blue color; the number of positive cells was counted for every 200 cells in randomly selected fields of view using light microscopy (TE300; Nikon Corporation, Tokyo, Japan).

Cells at P4, P11 and P17 were collected, washed twice with PBS and stained with propidium iodide (Sigma-Aldrich) for 30 min in dark conditions. The stained cells were analyzed by flow cytometry (FACSAria; BD Biosciences).

To prepare CM, 8×0⁴ hucMSCs of P4, P11 and P17 were plated on 6-well culture plates with 10% FBS L-DMEM and allowed to adhere overnight at 37°C with 5% CO₂ atmosphere. The following day, the media was removed, the cells were washed twice with PBS and then re-incubated with 1.5 ml serum-free culture media. After 12 h, the CM was collected, centrifuged for 5 min at 447 × g to remove cell debris and passed through a 0.45-μm filter (Sigma-Aldrich). CM aliquots were frozen at -20°C until analysis (not exceeding 2 weeks).

P4 MSCs (4×10⁴) in 200 μl serum-free L-DMEM were plated in the upper chambers and 600 μl undiluted CM with 10% FBS was added to the lower chambers. After 10 h of incubation, the cells were fixed with 4% formaldehyde for 30 min. The cells that remained on the membrane of the upper chamber were removed with cotton swabs and migrating cells were stained with crystal violet (Sigma-Aldrich). Four low-power fields (×100) were randomly selected in each chamber to observe the cells and the number of stained migrated cells on each image was counted.

To detect the accumulation of intracellular ROS in hucMSCs, a ROS assay kit (Beyotime Institute of Biotechnology) was used. Following culture to different passages (P4, P11 and P17), the cells were washed 3 times in serum-free medium and incubated in a final concentration of 10 mM dihydrodichlorofluorescein diacetate (H2DCFDA) at 37°C for 25 min. The media was then removed and cells were washed 3 times with serum-free medium. The cells were observed using a fluorescence microscope (TE300; Nikon Corporation).

To quantify the ROS level, the H2DCFDA fluorescence intensity of the cells was detected by flow cytometry. Briefly, 5×10⁴ cells were collected and resuspended in a final concentration of 10 mM H2DCFDA with serum-free medium. After 25 min incubation at 37°C, cells were washed with serum-free medium 3 times and resuspended in PBS, then placed on ice for immediate detection using a FACScan flow cytometer (BD Biosciences) with excitation at 488 nm and emission at 525 nm.

HucMSCs at P4, P11 and P17 were cultured in a medium containing either osteogenic (50 mM ascorbate-phosphate, 10 mM β-glycerophosphate and 0.1 mM dexamethasone) or adipogenic (1 mM dexamethasone, 10 mM insulin, 0.5 mM isobutyl-methylxanthine and 200 mM indomethacin) reagents (Sigma-Aldrich) at 5% CO₂ at 37°. After 2 weeks of culturing, osteogenic differentiation was assessed by the examination of neutrophil alkaline phosphatase with Alizarin Red dye (Sigma-Aldrich). After 3 weeks of culturing, adipogenic differentiation was detected via intracellular lipid vesicles; the cells were stained with Oil Red-O (Cyagen, Guangzhou, China) to detect lipids using an inverted microscope (TE300; Nikon Corporation).

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Reverse transcription was conducted using Moloney murine leukemia virus reverse transcriptase (Promega, USA) and the obtained cDNA was subjected to PCR. PCR was performed using 1 μg of cDNA sample with 0.3 U of Taq polymerase (CinnaGen Co., Tehran, Iran), 200 μM dNTPs, 10 pM of each primer, reaction buffer and MgCl2 (Takara Bio, Inc., Otsu, Japan) in a 25-μl volume. PCR amplification was performed for 35 cycles using an ABI 2720 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions were: 94°C for 30 sec, 60°C (primer) for 30 sec, 72°C for 30 sec and a final extension at 72°C for 10 min. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide (Thermo Fisher Scientific, Inc.) and visualized under UV light using the GeneGenius bio-imaging system (Syngene, MD, USA). Primers were as follows: APE1/Ref-1, F 5′-GCTTCGAGCCTG GATTAAGA-3′ and R 5′-TCATCGCCTATGCCGTAAGA-3′; P21, F 5′-CTACCTCAGGCAGCTCAAG-3′ and R, 5′-AGCCTCTACTGCCACCATC-3′; P53, F 5′-TCTGTGACTTGCACGTACTC-3′ and R 5′-TGTAGTGGATGGTGGTACAG-3′; β-actin, F 5′-CACGAAACTACCTTCAACTC-3′ and R 5′-CATACTCCTGCTTGCTGATC-3′. The primers were produced by Shanghai Bio-Engineering Company (Shanghai, China).

All data are expressed as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA). All experiments were replicated 3 times. Analysis of variance was used to analyze variance among all groups, and Student's t-test was performed to compare experimental and relative control groups. P<0.05 was considered to indicate a statistically significant difference.

Following the initial 7 days of primary culture, hucMSCs adhered to the plastic surface of the culture flasks and presented as a small population of single cells with spindle-like shape. On day 10 after initial plating, the cells exhibited long spindle-shaped fibroblastic cells, they had began to form colonies and were confluent (Fig. 1A). At P4, hucMSCs were positively stained with CD29, CD90, CD44 and CD105, but were negative for the hematopoietic lineage markers CD34 and HLA-DR, as measured by fluorescence-activated cell sorting (FACS) analysis (Fig. 1B).

HucMSCs from the same donors were characterized by their morphology, growth, SA-β-gal activity and differentiation capacity. The present study observed morphological changes during long-term culture of hucMSCs by analyzing the cell membrane and nucleus. The ultrastructure of the cell membrane was observed under SEM (Fig. 2A). Cells at P4 were spindle-like, plump, stereoscopic and exhibited a large population of uniformly distributed vimineous microvilli. However, at the middle and late phases of the culture (P11 and P17, respectively), hucMSCs became flatter, broader and had numerous large pseudopods, often with multiple secondary bifurcations. Furthermore, the number and length of microvilli was decreased during the late phases, particularly at P17. The current study also used immunofluorescence to observe changes to the cell nuclei (Fig. 2B). At P4, the chromatin distribution in the nuclei was homogeneous and the nucleus size was uniform. Compared with P4, the nuclei at P11 and P17 were swollen, and chromatin was localized into a small area termed the senescence-associated heterochromatic foci (SAHF). It was concluded that both the nuclei and the cell membranes of hucMSCs had undergone changes during the long-term in vitro culture.

Subsequently, the current study analyzed the growth characteristics of hucMSCs during long-term in vitro culture. The proliferation rate of hucMSCs was measured at P4, P11 and P17 by MTT assay (Fig. 2C). The hucMSC proliferation rate in the early and middle phases exhibited an S-shaped growth curve, with a minor decrease in proliferation rate in the middle phase compared with the early phase. However, when reaching P17, the hucMSC proliferation rate was significantly decreased compared with the early phase cells (P<0.05). The shape of long-term growth curves differed considerably between passages, with almost a straight line exhibited during the late phase of hucMSC culture.

Additionally, the current study detected SA-β-gal activity at the 3 passages (Fig. 2D). The number of senescent cells were increased during the middle and late phase, compared with the early phase (P<0.05; Fig. 2E). The results of the current study indicate that hucMSCs undergo replicative senescence during long-term in vitro culture.

Considering the functional implications of changes to growth characteristics during multiple passages, the present study analyzed the cell cycle status of hucMSCs. The cell cycle distribution at different passages was determined by FACS analysis. Single-variable histograms of DNA provided data measuring the percentages of cells in the G0/G1, S and G2/M phases of the cell cycle. As presented in Fig. 3A, hucMSCs demonstrated a progressive increase in the frequency of cells in G0/G1 phase during long-term in vitro culture. Cell cycle analysis of P17 revealed an obvious increase in the number of cells in the G0/G1 phase and a reduction of S-phase cells compared with P4 and P11. The current study did not observe the development of polyploidy in hucMSCs throughout the long-term culture.

CM was collected from P4, P11 and P17 cells, and used as the medium in the lower chamber during a cell migration assay. In the P4 and P11 CM groups, obvious migration of cells was observed, whereas few migrated cells were observed in the P17 CM group. Cell counting demonstrated that the migration of cells in the P17 CM group (19±3.4 cells/100 field) was significantly reduced compared with the P4 and P11 groups (77±3.9 and 62±4.3 cells/field, respectively; P<0.01; Fig. 3B and C). The results indicated that senescent hucMSCs may secrete toxic factors into the microenvironment that inhibit hucMSC migration and are harmful to neighboring cells.

In order to observe the differentiation potential of hucMSCs at P4, P11 and P17, the cells were induced to differentiate into adipocytes or osteocytes, as measured by positive staining of Oil Red-O (Fig. 4A) and Alizarin Red (Fig. 4B), respectively. Based on visual assessment of the extent of Oil Red O-positive lipid inclusions, it was determined that the adipogenic differentiation capacity of hucMSCs was decreased at P11 and P17 compared with at P4. Similarly, the capability of osteogenic differentiation of hucMSCs was decreased at P11 and P17 compared with at P4.

To investigate the levels of intracellular ROS during long-term culture, a fluorescence protocol using H2DCFDA was performed on P4, P11 and P17 hucMSCs. As presented in Fig. 5A, the green fluorescence was more intense in P11 and P17 hucMSCs compared with P4 hucMSCs. This suggests that hucMSCs exhibit low intracellular ROS levels during the early phases; but the levels increase in the middle and late phases. To quantify the ROS levels, fluorescence intensity was detected by flow cytometry (Fig. 5B and C). The mean fluorescence intensity (MFI) of P11 and P17 were increased compared with at P4. The MFI was increased by 3-fold in P17 hucMSCs and by 2-fold in P11 hucMSCs compared with P4 hucMSCs. Thus, hucMSCs exhibited higher intracellular ROS levels at later passages during the long-term culture (P<0.05; Fig. 5C).

To further investigate the association between senescence and oxidative stress, the current study analyzed the mRNA expression of P53, P21 and APE1/Ref-1 in different phases of hucMSC culture. In agreement with a previous study (19), telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. As demonstrated in Fig. 5D, compared with the early passages, P21, P53 and APE1/Ref-1 mRNA levels were significantly increased in the middle and late phases.

Early passage hucMSCs were treated with H₂O₂ at different concentrations (0, 200, 400, 600 and 800 μM) for 2 h. The stimulus was then removed and cells were further incubated in fresh medium for 48 h. The effect of H₂O₂ treatment on the cell cycle in P4 hucMSCs was analyzed (Fig. 6A). FACS analysis demonstrated that, with increasing concentrations of H₂O₂, the number of hucMSCs in the G0/G1 fraction progressively increased, and treatment with 600 μM H₂O₂ significantly promoted cell cycle arrest in the G0/G1 phase (Fig. 6; P=0.036).

Following H₂O₂ treatment, the number of apoptotic cells did not markedly increase, however, the hucMSCs became larger and flatter. Staining of hucMSCs with SA-β-gal indicated that hucMSCs were subjected to senescence following H₂O₂ stimulation (Fig. 6B). In the five H₂O₂-treated groups, the proportion of premature senescence cells, as measured by SA-β-gal staining, increased progressively with increasing concentrations of H₂O₂ (8.17, 13.33, 22.33, 52.17 and 50%; Fig. 6C). This indicates that H₂O₂ promotes senescence of early passage hucMSCs in a concentration-dependent manner. However, the dose of 800 μM H₂O₂ exhibited a slight cytotoxic effect, with viable cells exhibiting poor adherence when washed with PBS. Hence, we propose that a dose of 600 μM H₂O₂ is suitable to establish a model of aging in hucMSCs. Additionally, treatment of P4 hucMSCs with 600 μM H₂O₂ caused them to become larger and flatter, and the cells appeared to be morphologically indistinguishable from replicative senescent cells (P11 or P17).

The expression levels of P53, P21 and APE1/Ref-1 are upregulated in replicative senescence and premature senescence. P53 (P>0.05) and P21 (P<0.05) mRNA expression levels were also increased following H₂O₂ treatment (Fig. 6D). However, at the higher concentrations of H₂O₂ (600 and 800 μM), the expression level of P53 was reduced. The current study interpreted that excessive stimulation of oxidation may decrease the activities of P53 or even inactivate it. Additionally, exposure to H₂O₂ for 2 h resulted in dose-dependent increased expression levels of APE1/Ref-1 and P21 mRNA in the premature senescence hucMSCs (P<0.05; Fig. 6D). This indicates that oxidative stress is important in regulating MSC senescence.