The Institutional Review Board examined and approved the present study (grant no. KC20SISE0703). Stem cell spheroids were fabricated in silicone elastomer-based microwells that were concave in shape with a 600 µm diameter (cat. no. H389600; StemFIT 3D; MicroFIT). A total of 1x10⁶ gingiva-derived stem cells and/or bone marrow-derived stem cells were seeded in per well. Gingiva-derived stem cells were obtained as described previously (14). Human bone marrow-derived mesenchymal stem cells (Catholic MASTER Cells) were obtained from the Catholic Institute of Cell Therapy (15), and informed consent was obtained from all participants. The experiments were performed in accordance with the relevant guidelines and regulations specified in the Declaration of Helsinki (16). The ratios between human bone marrow-derived mesenchymal stem cells and gingiva-derived stem cells were: 6:0, Group 1; 4:2, Group 2; 3:3, Group 3; 2:4, Group 4; and 0:6, Group 5(13). Cell aggregation and cell-spheroid formation were observed using an inverted microscope.

The viability of spheroids was qualitatively analyzed using a Live/Dead kit assay (Molecular Probes) on day 1(17). Stem cell spheroids were cultured in α-minimal essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) containing 15% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), 200 mM L-Glutamine (Sigma-Aldrich; Merck KGaA) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich; Merck KGaA). These spheroids were washed twice with growth media. A suspension containing calcein acetoxymethyl ester working solution ethidium homodimer-1 was added and incubated at room temperature for 30 min. The spheroids were observed under a fluorescence microscope on days 3 and 5 (magnification, x100).

Qualitative cellular viability analysis was performed on days 1, 3, 5 and 7 using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.) (18). The spheroids were incubated for 45 min at 37˚C and the spectrophotometric absorbance was measured at 450 nm.

Cells were harvested on days 7 and 10. The total RNA was isolated using a GeneJET RNA Purification kit according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.); 1 ng total RNA was used as a template for reverse transcription using SuperScript II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.), and quantities were determined by spectrophotometry on a NanoDrop 2000 (Thermo Fisher Scientific, Inc.) at 260 and 280 nm on days 7 and 10.

mRNA expression was detected using qPCR with a SYBR-Green Real-Time PCR MasterMix (Enzynomics) according to the manufacturer's protocol (19). The sense and antisense primers were designed based on GenBank. The primer sequences were as follows: Nanog (accession no. NM_001297698.2) forward, 5'-AGTCCCAAAGGCAAACAACCCACTTC-3' and reverse, 5'-TGCTGGAGGCTGAGGTATTTCTGTCTC-3'; and β-actin (accession no. NM_001101.5) forward, 5'-TGGCACCCAGCACAATGAA-3' and reverse, 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'; the mRNA levels were normalized to β-actin and expressed as the fold change (20). The mRNA expression was detected by qPCR using SYBR Green Real-Time PCR MasterMix (Enzynomics, Daejeon, South Korea) according to the manufacturer's protocol.

Statistical analysis was performed using SPSS version 12 (SPSS, Inc.) Data are presented as the mean ± standard deviation. A test of normality was performed, and a one-way ANOVA with a post-hoc Tukey's test, or a Kruskal Wallis test with Bonferroni corrected Mann-Whitney-U test was performed to determine differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

Stem cell spheroids were well formed in the silicone elastomer-based concave microwells with only bone marrow-derived stem cells on days 1, 3, 5 and 7 (Fig. 1A). There were no significant changes in the morphology with the different ratios of bone marrow and gingiva-derived stem cells on days 1 and 3. In general, the shapes of the cells on day 5 were similar to the shapes in each group on day 1. There were no significant changes in the morphology with the longer incubation time of 7 days.

The average spheroid diameter in each group on days 1, 3, 5 and 7 are shown in Fig. 1B. The average spheroid diameter for Groups 1, 2, 3, 4 and 5 on day 1 were 152.7±2.1, 187.4±27.6, 168.9±7.4, 207.1±10.2 and 224.9±5.1 µm, respectively (P<0.05). There were statistically significant increases in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. The average spheroid diameters for Groups 1, 2, 3, 4 and 5 on day 3 were 151.6±9.3, 169.9±7.7, 152.5±4.9, 179.7±10.9 and 185.1±12.9 µm, respectively. The diameters of the spheroids appeared to increase the ratio of gingiva-derived stem cells increased.

The cellular viability was determined using a Live/Dead kit assay using a fluorescent microscope and is shown in Fig. 2. Most of the cells in the spheroids emitted green fluorescence, and the morphology was round without significant changes at day 1.

The results of cell viability on days 1, 3, 5 and 7 are shown in Fig. 3. The relative viability value of Groups 1, 2, 3, 4 and 5 on day 1 were 100.0±10.7%, 115.5±3.3%, 119.1±7.5%, 115.4±3.4% and 116.8±5.4%, respectively (P<0.05). There were significant increases in the values seen in Groups 3 when compared with Group 1 on day 1.

RT-qPCR was performed to assess the mRNA expression levels of Nanog and β-actin on days 7 and 10 (Fig. 4). The mRNA levels were normalized to β-actin levels and expressed as a fold change. The relative expression of Nanog in Groups 1, 2, 3, 4 and 5 on day 7 were 100.0±8.3%, 77.6±22.1%, 98.6±19.6%, 65.7±33.9% and 64.8±32.7%, respectively (Fig. 4A). The relative expression of Nanog in Groups 1, 2, 3, 4 and 5 on day 10 were 100.0±1.2, 102.1±16.0, 126.7±12.0, 101.2±14.2 and 124.1±21.3%, respectively, with the highest value observed in Group 3 (Fig. 4B). However, no significant differences in Nanog expression were observed in Groups 2-5 when compared with Group 1 (P>0.05; Fig. 4A and B).