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.

The effects of doxorubicin‑loaded liposomes on viability, stem cell surface marker expression and secretion of vascular endothelial growth factor of three‑dimensional stem cell spheroids

“…Various materials can be used for the surface of microwells, including polyethylene glycol, polydimethylsiloxane, and polyurethane ( 20 , 21 ). The morphology can be modified by the shape of the microwells ( 10 ), and the size of the spheroids can also be controlled by the number of applied cells ( 5 ). Three-dimensional cultures can be made with the aid of scaffolds including alginate ( 6 ).…”

“…Three-dimensional spherical spatial boundary conditions have been reported to regulate the differentiation of mesenchymal stem cells ( 8 ). Moreover, three-dimensional culture system can be used for the evaluation of intercellular interactions in the regulation of cell proliferation and differentiation ( 9 , 10 ). Doxorubicin is reported to suppress bone marrow stem cells while expanding cancer stem cells ( 11 ).…”

Morphological Comparison of Stem Cells Using Two- Dimensional Culture and Spheroid Culture

“…In more recent years, three-dimensional cell culture methods have been widely applied and are regarded to have high importance in evaluating the biological processes [12]. Three-dimensional culture systems may simulate the intercellular interactions in regulation of stem cell self-renewal and differentiation [13]. It was shown that a three-dimensional culture enhanced the production of extracellular matrix-related genes when compared with two-dimensional monolayer culture [12].…”

The effects of simvastatin on cellular viability, stemness and osteogenic differentiation using 3-dimensional cultures of stem cells and osteoblast-like cells

“…10 These stem-cell spheroids maintained the viability and stemcell characteristics of stem-cell surface-marker expression and differentiation potential. 11 In a previous study, cell spheroids from gingival cells and osteoprecursor cells were shown to be a promising strategy for stem-cell therapy. 12 The current study was therefore performed to evaluate the effects of simvastatin on cellular viability, stemness and osteogenic differentiation using 3-dimensional cell spheroids of stem cells and osteoblast-like cells.…”