Bone marrow was aspirated from the posterior iliac crest of three healthy adult volunteers. All procedures were performed with the approval of the ethics committee of SunYat-sen Memorial Hospital, Sun Yat-sen University (approval number no. 2014-57; Guangzhou, China) and following informed written consent by the volunteers. Nucleated cells were isolated with a density gradient (Lymphoprep, Stemcell Technologies, Inc., Vancouver, Canada) and resuspended in MSC culture medium (MesenCult Proliferation kit; Stemcell Technologies, Inc.), according to the manufacturer's instructions. Nucleated cells (1.2×10⁷) were plated in 20 ml culture medium in T75 tissue culture flasks (Corning, Inc., Corning, NY, USA) and incubated at 37°C with 5% CO₂ and 20% O₂. Following 24 h, nonadherent cells were discarded and adherent cells were thoroughly washed twice with PBS. The culture medium was changed every three days and following 10 days in culture, cells were 80% confluent. The cells were then incubated with 0.05% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 5 min at 37°C, and replated at 2,500 cells/cm² in T75 tissue culture flasks. Following 5 days incubation, the cultured cells were 80% confluent and were suspended by incubation in 0.05% trypsin-EDTA for 5 min at 37°C and rinsed with 5–7 ml culture medium, followed by collection in a 50-ml centrifuge tube. Cells were subsequently centrifuged at 300 × g (Sorvall™ ST 16R; Thermo Fisher Scientific, Inc.) for 5 min at room temperature. Pellets were washed with culture medium and centrifuged at 300 × g for 5 min at room temperature again, following which the cells were resuspended in 10% dimethyl sulfoxide and 90% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 2×10⁵ cells per freezing vial, and frozen in liquid nitrogen (passage-1 cells). To expand a culture, a frozen vial of MSCs was thawed, plated in a T75 tissue culture flask, and incubated for 5 days at 37°C with 5% CO₂ and 20% O₂ (passage-2 cells).

Passage-1 cells were plated in T75 tissue culture flasks after being thawed from liquid nitrogen, and incubated at 37°C with 5% CO₂ and 20% O₂. When the cultured cells reached 90% confluency (passage-2 cells), they were harvested, centrifuged at 300 × g for 5 min, and resuspended in PBS. Cells were then stained with mouse anti-human CD11b (cat. no. 557321; 1:100), CD14 (cat. no. 557154; 1:100), CD34 (cat. no. 550619; 1:100), CD45 (cat. no. 555482; 1:100), CD73 (cat. no. 561014; 1:100), CD90 (cat. no. 555596; 1:100) and CD105 (cat. no. 561443; 1:100) antibodies (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by flow cytometry using a BD FACSCalibur (BD Biosciences). An isotype control immunoglobulin (cat no. PE-R3-34; BD Biosciences) was used at the indicated concentration. BD FACSComp software (version no. 5.1) was used for the data analysis (BD Biosciences).

Passage-2 cells were plated into 6-well plates at 100 cells/well in complete Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS, 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin and incubated at 37°C with 5% CO₂ and 20% O₂ for 12 days. The colonies were stained with Giemsa (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). A cluster of >50 cells was counted as one colony, and images were acquired using a Leica Application Suite software (version no. 4.0; Leica Microsystems GmbH, Wetzlar, Germany).

The assay was performed with passage-2 cultured hMSCs according to a previously described protocol (10), for evaluation of their multipotent differentiation ability. The cells were split into three separate differentiation-specific media to induce osteoblast, adipocyte, or chondrocyte differentiation. The media were changed every 3–5 days for 3–4 weeks. For adipocyte differentiation, the cultures were incubated in DMEM containing 10% FBS, 10 µM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 20 µM indomethacin, 0.5 µM isobutylmethylxanthine (Sigma-Aldrich; Merck Millipore), and 1×10⁻⁷ M dexamethasone (Sigma-Aldrich; Merck Millipore). Following 28 days' incubation, the cells were fixed with 4% formalin and stained with 0.5% oil red O. For osteoblast differentiation, the cells were cultured in DMEM containing 10% FBS, 10 mM HEPES, 100 U/ml penicillin, 100 U/ml streptomycin, 50 µg/ml ascorbic acid (Sigma-Aldrich; Merck Millipore), 1×10⁻⁷ M dexamethasone and 10 mM glycerol phosphate. Following 21 days' incubation, the cells were fixed with 4% formalin and stained with 1% alizarin red S (pH 4.1). Chondrocyte differentiation was induced with OriCell Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Cyagen Biosciences, Guangzhou, China), according to the manufacturer's instructions. The chondrogenic pellets were harvested following 18 days in culture, fixed with 4% formalin, and embedded in paraffin for Alcian blue staining. Adipocyte and chondrocyte differentiation assays were carried out similarly for both adherent and nonadherent cultured cells at 72 h to evaluate their differentiation potential.

Passage-2 cultures were plated in 20 ml complete DMEM containing 10% FBS, 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin in T75 tissue culture flasks. The cells were incubated at 37°C with 20% O₂ and 5% CO₂. The medium was changed every 5–7 days. When MSCs reached 90% confluency, they were suspended by incubation in 0.05% trypsin-EDTA for 5 min at 37°C and then reseeded at 2,500 cells/cm² in a T75 tissue culture flask (passage-3 cells). To test the effect of adherent vs. nonadherent culture conditions in MSCs, passage 3 cells were seeded at 2,500 cells/cm² in adherent culture plates (BD Biosciences) or in ultra-low-adherence tissue culture plates (Corning, Inc.) respectively, and allowed to grow for 24 or 72 h prior to experimental assays.

Adherent-cultured cells and nonadherent-cultured cells grown for 24 or 72 h were collected in 1.5 ml Eppendorf (EP) tubes and centrifuged at 300 × g for 5 min at room temperature. Pellets were washed twice with PBS, followed by resuspension in 400 µl binding buffer (as per the kit instructions; eBioscience, Inc., San Diego, CA, USA), and divided equally into two new tubes. One sample was used as the negative control, while 5 µl Annexin V-FITC (eBioscience, Inc.) was added to the other sample and incubated at room temperature for 15 min. Propidium iodide (10 µl) was added to both samples, and they were analyzed by flow cytometry on a FACSCalibur using CellQuest software (version no. 3.3; BD Biosciences).

Adherent-cultured cells and nonadherent-cultured cells grown for 24 or 72 h were collected in 50-ml centrifuge tubes and centrifuged at 300 × g for 5 min at room temperature. Pellets were resuspended with Dulbecco's PBS and transferred into 1.5 ml EP tubes. Total RNA was extracted using the High Pure RNA Isolation kit (Roche Diagnostics, Ltd., Basel, Switzerland), according to the manufacturer's instructions. cDNA was synthesized from 1 µg RNA using SuperScript III and Oligo primers (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Primers were as follows: caspase-3, forward 5-CTG GAC TGT GGC ATT GAG AC-3′ and reverse 5′-ACAAAGCGACTGGATGAACC-3′; caspase-7, forward 5′-CAAGATCCCAGTGGAAGCTGAC-3′ and reverse 5′-GGCTTGCACAAACCAGGAG-3′; caspase-8, forward 5′-GAAGGTGCTACCATCGTGAGAGTAA-3′ and reverse 5′-CCTGGAGTCTCTGGAATAACATCAA-3′; caspase-9, forward 5′-CGCAAACCAGAGGTTCTCAGAC-3′ and reverse 5′-AGGATGTAAGCCTGCCAGCAC-3′; and GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′. qPCR analysis was performed using the LightCycler 480 platform and the LightCycler 480 SYBR-Green I Master kit (Roche Diagnostics, Ltd.). Each reaction mixture contained 10 ml SYBR Green I master mix, 6 µl RNase-free H₂O, 1 µl 10 mM forward primer, 1 µl 10 mM reverse primer, and 2 µl cDNA (undiluted) in a final reaction volume of 20 ml. Gene expression was normalized to the internal reference gene β-actin. The 2^−∆∆Cq method (11) was used to determine relative fold changes using the LightCycler480 software (Roche Diagnostics, Ltd.). The experiment was performed three independent times in triplicate.

Adherent-cultured cells and nonadherent-cultured cells grown for 24 or 72 h were lysed on ice for 5 min using Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China) and protease inhibitors (Roche Diagnostics, Ltd.), then sonicated briefly and centrifuged for 10 min at 14,000 × g in a cold microfuge, according to the manufacturer' protocol. Protein concentrations were determined using the bicinchoninic acid method (Beyotime Institute of Biotechnology). Total protein samples (30 µg per lane) were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). Following blocking with 5% dried skim milk in TBS/0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated overnight at 4β with primary antibodies (1:1,000 dilution) against caspase-3 (cat. no. 9665; Cell Signaling Technology, Inc.), caspase-7 (cat. no. 12,827; Cell Signaling Technology, Inc.), caspase-8 (cat. no. AC056; Beyotime Institute of Biotechnology), caspase-9 (cat. no. 9508; Cell Signaling Technology, Inc.) and β-actin (cat. no. AA128; Beyotime Institute of Biotechnology) in primary antibody dilution buffer. Following 3 washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-linked anti-mouse immunoglobulin (Ig) G antibody (cat. no. 7076; 1:2,000 dilution; Cell Signaling Technology, Inc.) or HRP-linked anti-rabbit IgG antibody (cat. no. 7074; 1:2,000 dilution; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein expression signals were detected with BeyoECL Plus enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology) using the Intelligent imaging system (Syngene, Frederick, MD, USA) and ImageJ software (version no. 1.44) (12), according to the manufacturer's instructions.

GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform Student's t-test analysis in order to identify significant differences between culture conditions. P<0.05 was considered to indicate a statistically significant difference.

Growth and multipotency are key features of MSCs. To confirm the functional properties of the hMSCs isolated in the present study, their clonogenic and multipotent lineage potential were evaluated by CFU-F and in vitro differentiation assays respectively. CFUs were detected within the cell population, and cells within individual colonies exhibited fibroblast-like morphology (Fig. 1A), indicating good self-renewing properties of the isolated cell populations. The differentiation assays of passage-2 hMSCs demonstrated that the cells were able to generate adipocytes (Fig. 1B), osteoblasts (Fig. 1C), and chondrocytes (Fig. 1D) in vitro. These findings suggest that the cells exhibit the multipotent differentiation potential characteristic of MSCs. In addition, adherent and nonadherent-cultured cells following 72 h culture also demonstrated successful adipocyte (Fig. 1E) and chondrocyte (Fig. 1F) differentiation.

The expression of surface markers, including CD11b, CD14, CD34, CD45, CD73, CD90 and CD105, has been previously reported as a means to confirm the correct cell identity for MSCs (13). Therefore, expression of these surface markers was analyzed by flow cytometry in passage-2 cells. Greater than 99% of passage-2 cells expressed CD73, CD90, and CD105 (Fig. 2A-C). By contrast, less than 1% of cells expressed CD11b, CD14, CD34 and CD45 (Fig. 2D-G, respectively). These findings suggest that the cells isolated in the present study are enriched in phenotypically defined hMSCs.

hMSCs seeded in tissue culture plates (at 2,500 cells/well) were attached to the bottom of the plates and exhibited flat morphology following 24 (Fig. 3A) and 72 h (Fig. 3B) of culture. However, hMSCs seeded in the ultra-low-adherence culture plates appeared suspended and scattered throughout the medium, and exhibited a rounded morphology, following 24 (Fig. 3D) and 72 h (Fig. 3E) of culture. In order to test whether this morphology change was a transient or permanent feature, adherent and nonadherent cells were harvested at 72 h and then reseeded in tissue culture plates. Following incubation for 3 days in adherent culture conditions, hMSCs reseeded from the nonadherent cultures exhibited a similar flat morphology (Fig. 3F) to the ones reseeded from the adherent cultures (Fig. 3C), suggesting that nonadherent cells were capable of adherent growth and fibroblast-like morphology.

Apoptosis of passage-3 hMSCs was analyzed by flow cytometry in three independent repeats. At 24 h, the % of apoptotic cells in nonadherent-cultured cells was not significantly different compared with the control adherent-cultured cells (P>0.05; Fig. 4A). At 72 h, the % of apoptotic cells was significantly higher in the nonadherent-cultured cells compared with the adherent-cultured cells (P<0.05; Fig. 4B), indicating that nonadherent culture conditions result in increased apoptosis in hMSCs.

RT-qPCR was used to compare levels of caspase-3, −7, −8, and-9 mRNA expression in adherent and nonadherent-cultured cells at 24 and 72 h. At 24 h, mRNA expression levels of all the caspases examined were not significantly different in nonadherent-cultured cells compared with the adherent-cultured cells (P>0.05; Fig. 5A-D, respectively). At 72 h however, mRNA expression levels of caspase-3, −7, and −9 were significantly increased in nonadherent-cultured cells compared with the control adherent-cultured cells (P<0.05; Fig. 5A, B and D, respectively). Expression of caspase-8 did not differ significantly between the two groups at either time point (P>0.05; Fig. 5C).

Since caspase-3, −7, and −9 mRNA expression at 72 h differed between adherent and nonadherent-cultured cells, protein levels were also examined by western blot analysis. The results demonstrated that at 24 h, the protein expression levels of caspases 3, 7, 8, and 9 did not differ significantly between culture conditions (P>0.05; Fig. 6A). At 72 h, levels of caspase-3, caspase-7, and caspase-9 proteins were significantly lower in the nonadherent-cultured cells compared with the adherent-cultured cells (P<0.05; Fig. 6B), while the level of caspase-8 protein did not differ significantly between the two populations at 24 or 72 h (P>0.05; Fig. 6).