Lee Sung-Il, Ko Youngkyung, Park Jun-Beom
Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Exp Ther Med. 2017 May;13(5):1757-1764. doi: 10.3892/etm.2017.4194. Epub 2017 Mar 8.
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 4×10 (group A) or 8×10 (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.
牙龈来源的干细胞已被应用于组织工程目的，并且由于在局部麻醉下可以轻松地从下颌骨或上颌骨采集干细胞，因此可被视为间充质干细胞的良好来源。本研究旨在使用凹形微孔制造干细胞球状体，并评估干性、活力和分化潜能的维持情况。分离牙龈来源的干细胞，将4×10个（A组）或8×10个（B组）细胞接种到直径为600 µm的聚二甲基硅氧烷基凹形微模具中。评估微球的形态和球状体直径的变化。通过活/死试剂盒检测对球状体的活力进行定性分析。使用细胞计数试剂盒-8在第1、3、6和12天进行细胞活力分析。使用SSEA-4、TRA-1-60（R）（阳性标志物）和SSEA-1（阴性标志物）通过免疫细胞化学染色评估干性的维持情况。分别将球状体在成骨、成脂和成软骨诱导培养基中孵育，评估其成骨、成脂和成软骨分化潜能。牙龈来源的干细胞在凹形微孔中形成球状体。A组球状体的直径大于B组。球状体中的大多数细胞发出绿色荧光，表明在第6天存在活细胞。在第12天，球状体中的大多数细胞发出绿色荧光，还观察到一小部分红色荧光，这表明存在死细胞。球状体对干细胞标志物SSEA-4和TRA-1-60（R）呈阳性，对SSEA-1呈阴性，表明这些球状体主要包含未分化的人类干细胞。随着孵育时间的增加，成骨、成脂和成软骨分化更加明显：在第14天和21天茜素红S染色后观察到矿化的细胞外沉积物；与第6天相比，第18天油滴增加；与第6天相比，第18天阿尔新蓝染色更明显。在本研究的范围内，牙龈细胞来源的干细胞球状体在实验期间维持了干性、活力和分化潜能。该方法可作为一种有前景的干细胞治疗策略。
