With institutional review board approval, tonsils were obtained after informed consent from patients undergoing tonsillectomy as a result of recurrent episodes of tonsillitis. To isolate tonsil stem cell, tonsil was washed extensively with equal volumes of phosphate-buffered saline (PBS), and tissue were digested at 37℃ for 30 minutes with 0.075% collagenase (type I; Sigma, St. Louis, MO, USA). Enzyme activity was neutralized with α-modified Eagle's medium (α-MEM), containing 10% fetal bovine serum (FBS) and centrifuged at 1,200 g for 10 minutes to obtain a pellet. The pellet was filtered through a 100-µm nylon mesh to remove cellular debris and incubated overnight at 37℃/5% CO₂ in control medium (α-MEM, 10% FBS, 100 unit/mL of penicillin, 100 µg/mL of streptomycin). Following incubation, the plate was washed extensively with PBS to remove residual nonadherent cells. The resulting cell population was maintained at 37℃/5% CO₂ in control media. One-week later, when the monolayer of adherent cells has reached confluence, cells were trypsinized (0.05% trysin-EDTA; Sigma) resuspended in α-MEM containing 10% FBS, and subcultured at a concentration of 2,000 cells/cm².

Adipogenic differentiation was induced by culturing tonsil stem cell for 2 weeks in adipogenic media (1 µM dexamethasone, 100 µg/mL 3-isobutyl-1 methylxanthine, 5 µg/mL insulin, and 60 µM indomethacine, 10% FBS in α-MEM) and assessed using an oil red O (Sigma) stain as indicator of intracellular lipid accumulation. Prior to staining, the cells were fixed 15 minutes at room temperature in 70% ethanol. The cells were incubated in 2% oil red O reagent for 1 hour at room temperature. Excess stain was removed by washing with 70% ethanol, followed by several changes of distilled water.

Osteogenic differentiation was induced by culturing ADSC for 2 weeks in osteogenic media (0.1 mM dexamethasone, 10 µM β-glycerophosphate, and 50 µg/mL ascorbic acid, 10% FBS in α-MEM) and examined for extracellular matrix calcification by alizarin red S (Sigma) staining. For alizarin red S staining, the cells fix with 70% ethanol and washed with distilled water. The cells were incubated in 2% arizarin red solution for 15 minutes at room temperature. The cells were washed several times with distilled water.

For chondrogenic differentiation, cells were stimulated in chondrogenesis induction media for 3 weeks. Cells were then rinsed twice with PBS and fixed in 10% formalin for 15 minutes at room temperature. Alcian Blue (Sigma) was used to stain chondrogenic pellets. Excess dye was removed, and cells were visualized by light microscopy.

Flow cytometry analysis was used to characterize the phenotypes of the ADSC. At least 50,000 cells (in 100 µL PBS/0.5% BSA/2 mmol/L EDTA) were incubated with FITC-labeled monoclonal antibodies against human CD73, CD90, CD105, CD45, CD31, CD117, HLA-DR (BD Biosciences Clontech, Palo Alto, CA, USA) or with the respective isotype control. After washing the labeled cells were analyzed by flow cytometry using FACS-Calibur flow cytometer and the CellQuest Pro software (BD Biosciences).

The laryngeal carcinoma cell line (SNU-899) was purchased from the Korean Cell Line Bank (28). PNUH-12 cells originating from hypopharyngeal squamous cell carcinoma were established at the Department of Otolaryngology, Pusan National University College of Medicine (29).

Human tonsil stem cells were isolated and treated with mitomycin C (MMC) (50 µg/mL, Sigma) at 37℃ for one hour to block the proliferation. Then cells were washed twice with PBS and plated 0.1 mL onto 96-well plates at different number of cells. Tumor cells were suspended in RPMI-1640 medium and added into wells with or without stem cells. The ratios of stem cells to tumor cells were 1:20, 1:10, 1:5, 1:2, and cells were cocultured for 48 hours. Then the culture was analyzed by methylthiazolyl-diphenyl-tetrazolium (MTT) assay.

In MTT assay, MTT (0.5 mg/mL, pH 4.7; Sigma) was added 4 hours before the end of the culture time. The supernatant was removed, 150 µL DMSO was added and shaken for 5 minutes at room temperature. The absorbance at 570 nm was measured with Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Inhibitory effect of stem cells on viability of tumor cells was expressed as follows: inhibitory rate (%)=(OD₅₇₀ of tumor cells-OD₅₇₀ of tumor with stem cells+OD₅₇₀ of stem cells control)/OD₅₇₀ of tumor cells×100%. The tumor cells originated from head and neck squamous cell carcinoma were cocultured with TD-MSCs at different cell number ratios for 48 hours. In the time-dependent experiment, the ratio of TD-MSCs to tumor cells was 1:20. TD-MSCs and tumor cells were cocultured for 24, 48, and 72 hours, respectively. The fibroblasts were purchased from the Korean Cell Line Bank (28) and treated by MMC at the same concentration. Then they were cocultured with tumor cells as the control group so that the influence of the portion of MMC treated TD-MSCs mixed with tumor cells should be evaluated.

To determine the effect of tonsil stem cells on cell death and cell cycle distribution of tumor cells, 5×10⁴ stem cells were seeded onto 6-well plate and incubated for 18 hours. Then culture medium was removed, the ratios of stem cells to tumor cells were 1:20, 1:10, 1:5, 1:2 and cocultured for 48 hours. Cells were harvested, washed twice with PBS and measured by flow cytometry analysis, reverse transcriptase (RT)-PCR, and real-time PCR assay. In flow cytometry analysis, 0.5 mL of PBS containing propidium iodide (5 mg/mL, BD Biosciences) was added to the culture before flow cytometry analysis (BD Biosciences). In RT-PCR analysis, total RNA was extracted from cucultured tumor cells using TRIzol regent. RNA (1 µg) was reverse transcribed into cDNA using a PCR cDNA synthesis kit (Clontech Laboratories Inc., Palo Alto, CA, USA). cDNA was used in the PCR normalized to β-actin gene. RNA (200 ng) was reverse transcribed and amplified using TaKaRa One-Step RT-PCR Kit (Takara Shuzo Co., Shiga, Japan). PCR was performed using the following primers: p21 sense, (5'-CCCGAGAACGGT GGAACT-3') and antisense (5'-AGAGGGCAGGCAGCGTAT-3'), product size: 493 bp; caspase 3 sense (5'-ATGTCATCTCGCTCTGGT-3') and antisense (5'-TCTGTTTCTTTGCGTGGA-3'), product size: 604 bp; and β-actin sense (5'-GCCATGTACGTAGCCATCCA-3') and antisense (5'-AACCGCTCATTGCCGATAGT-3'), product size: 373 bp. After 32 cycles of amplification, 10 µL of the reaction mix was removed, analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.

Real time PCR amplification of the cDNAs was carried out with the reverse-transecribed RNA, primers p21, product size: 493 bp; caspase 3, product size: 604 bp; and β-actin, product size: 373 bp, using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were run on a LightCycler detection system (Roche Molecular Systems, Alameda, CA, USA).

All experimental data were expressed as mean±SD of at least three independent experiments performed in duplicate. The statistical analysis was performed with the Student's t-test or covariance analysis for statistical significance. Statistical significance was set at a P-value of less than 0.05. All analyses were carried out using SPSS ver. 15.0 (SPSS Inc., Chicago, IL, USA).

To characterize the TD-MSC population, the isolated cells were analyzed using flow cytometry analysis. Cells were analyzed for the expression of cell membrane protein markers: positive for CD73, CD90, and CD105, which are generally considered as markers of MSCs, but negative for the expression of hematopoietic markers such as CD45, CD31, and CD117 (Fig. 1A).

TD-MSCs showed a fibroblast-like morphology and were plastic adherent under standard culture conditions (Fig. 1B). The differentiation potential for mesenchymal lineage was assessed by oil red O, alizarin red S, and alcian blue staining. TD-MSCs exhibited a characteristic of differentiation morphology in induction medium but not control medium (Fig. 1B). Two weeks after adipogenic induction, morphology of lipid droplets were observed and stained by oil red O. Under osteogenic culture conditions after 3 weeks, calcium deposits were detected by alizarin red S staining. Chondrogenic differentiation was revealed by alcian blue staining after 3 weeks of chondrocyte induction (Fig. 1B).

TD-MSCs exhibited a number-dependent growth inhibitory effect on different tumor cell lines (Fig. 2), but not the time-dependent effect (data not shown). However, fibroblast treated control showed neither a number-dependent nor the time-dependent growth inhibitory effect. In addition, TD-MSCs exhibited significant growth inhibitory effect on each tumor cell line more than fibroblasts at the cell ratio of 1:5 or 1:2 (Fig. 2).

We characterized the effect of TD-MSCs on the cell cycle of tumor cells to explain the mechanism of growth inhibition of MSCs. Cell cycle analysis showed an accumulation of tumor cells increased in G0/G1 phase with an increase in concentration of TD-MSCs, which was statistically significant (PNUH-12, 33.1 vs. 50.6; SNUH-899, 36.6% vs. 58.5%; P<0.05) on each tumor cell line (Fig. 3A, B). On the contrary, control showed the increased tumor cells in G0/G1 phase with an increase in concentration of TD-MSCs which was not statistically significant. Furthermore, the increase of G0/G1 phase was more in MSC treated SNU-899 cell line than in fibroblast treated control at the cell ratio of 1:5 and 1:2 (Fig. 3B). This phenomenon was confirmed by mRNA expression of cell cycle negative regulator p21. The results of RT-PCR demonstrated that TD-MSCs could upregulate mRNA expression of p21 in tumor cells significantly as concentration of TD-MSCs increased (Fig. 3C).

To address this issue, HNSCC cells were cocultured with TD-MSCs, and tumor cells were harvested for apoptotic cell death and cell cycle distribution analysis. Apoptosis of tumor cells increased significantly as concentration of cocultured TD-MSCs increased on each tumor cell line (Fig. 4), and TD-MSCs exhibited significantly increased apoptosis of tumor cells more than fibroblasts at the cell ratio of 1:10, 1:5, and 1:2. Additionally, upregulated mRNA expression of caspase 3 was demonstrated with increased concentration of TD-MSCs (Fig. 5). TD-MSCs treated PNUH-12 cell line exhibited significantly increased mRNA expression of caspase 3 more than fibroblast treated control at the cell ratio of 1:2 (Fig. 5B).