Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Abstract

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10⁵ (group A) or 8×10⁵ (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Keywords: cell culture techniques; cell survival; cellular spheroids; stem cell research; stem cells.

Figure 1.

Schematic illustration of generation of the spheroids with gingival-derived stem cells.

Figure 2.

The morphology of the stem-cell spheroids at days 1 and 3. The scale bar indicates 200 µm (original magnification, ×200). (A) Group A at day 1; (B) group B at day 1; (C) group A at day 3; (D) group B at day 3.

Figure 3.

The mean diameter (± standard deviation) of spheroids at days 1 and 3. *P<0.05 vs. group B at day 1.

Figure 4.

Live/dead cell imaging of spheroids at days 6 and 12. The scale bar indicates 200 µm (original magnification, ×200). (A-C) Group A at day 6; (D-F) group B at day 6; (G-I) group A at day 12; (J-L) group B at day 12.

Figure 5.

Mean absorbance (± standard deviation), indicative of cellular viability on days 1, 3, 6, and 12. *P<0.05 vs. group A at day 1.

Figure 6.

Evaluation of the maintenance of stemness with immunocytochemical staining at day 6. Spheroids were stained with NL493-conjugated SSEA-4 (green), NL557-conjugated TRA-1-60(R) (red) and NL557-conjugated SSEA-1 (red) antibodies. SSEA-4 and TRA-1-60(R) served as positive markers for stem cells, and SSEA-1 was used as a negative marker. The scale bar indicates 200 µm (original magnification, ×200). (A-F) Group A at day 6; (G-L) group B at day 6.

Figure 7.

Osteogenic differentiation at days 14 and 21. The scale bar indicates 200 µm (original magnification, ×100). (A) Group A at day 14; (B) group B at day 14; (C) group A at day 21; (D) group B at day 21.

Figure 8.

Quantitative results of osteogenic differentiation at days 14 and 21 (mean absorbance ± standard deviation). *P<0.05; ^#P<0.05 vs. group A at day 14; ^##P<0.05 vs. group B at day 14.

Figure 9.

Adipogenic differentiation at days 6 and 18. The scale bar indicates 200 µm. (A) Group A at day 6 (original magnification, ×100); (B) group B at day 6 (original magnification, ×100); (C) group B at day 18 (original magnification, ×50); (D) group B at day 18 (original magnification, ×100).

Figure 10.

Chondrogenic differentiation at days 6 and 18. The scale bar indicates 200 µm. (A) Group A at day 6 (original magnification, ×100); (B) group B at day 6 (original magnification, ×100); (C) group A at day 18 (original magnification, ×100); (D) group A at day 18 (original magnification, ×200).