Bu Nam-Ung, Lee Hyo-Seol, Lee Bin-Na, Hwang Yun-Chan, Kim Sun-Young, Chang Seok Woo, Choi Kyoung-Kyu, Kim Duck-Su, Jang Ji-Hyun
Department of Conservative Dentistry, Graduate School, Kyung Hee University, Seoul 02447, Korea.
Department of Pediatric Dentistry, School of Dentistry, Kyung Hee University, Seoul 02447, Korea.
J Clin Med. 2020 Jan 16;9(1):242. doi: 10.3390/jcm9010242.
Various three-dimensional (3D) culture methods have been introduced to overcome the limitations of in vitro culture and mimic in vivo conditions. This study aimed to evaluate two microsphere-forming culture methods and a monolayer culture method. We evaluated cell morphology, viability, osteo-, adipo-, and chondrogenic differentiation potential of dental pulp stem cells (DPSCs) cultured in 3D culture plates: ultra-low attachment (ULA) and U-bottomed StemFit 3D (SF) plates, and a two-dimensional (2D) monolayer plate. RNA sequencing (RNA-seq) revealed differentially expressed gene (DEG) profiles of the DPSCs. In contrast to an increasing pattern in the 2D group, cell viability in 3D groups (ULA and SF) showed a decreasing pattern; however, high multilineage differentiation was observed in both the 3D groups. RNA-seq showed significantly overexpressed gene ontology categories including angiogenesis, cell migration, differentiation, and proliferation in the 3D groups. Hierarchical clustering analysis revealed a similar DEG regulation pattern between the 3D groups; however, a comparatively different DEG was observed between the 2D and 3D groups. Taken together, this study shows that DPSCs cultured in microsphere-forming plates present superior multilineage differentiation capacities and demonstrate higher DEG expression in regeneration-related gene categories compared to that in DPSCs cultured in a conventional monolayer plate.
为克服体外培养的局限性并模拟体内条件，已引入了各种三维（3D）培养方法。本研究旨在评估两种微球形成培养方法和一种单层培养方法。我们评估了在3D培养板中培养的牙髓干细胞（DPSC）的细胞形态、活力、成骨、成脂和成软骨分化潜能：超低附着（ULA）和U型底StemFit 3D（SF）培养板，以及二维（2D）单层培养板。RNA测序（RNA-seq）揭示了DPSC的差异表达基因（DEG）谱。与2D组的增加模式相反，3D组（ULA和SF）的细胞活力呈下降模式；然而，在两个3D组中均观察到高多向分化。RNA-seq显示3D组中包括血管生成、细胞迁移、分化和增殖在内的基因本体类别显著过表达。层次聚类分析显示3D组之间存在相似的DEG调控模式；然而，在2D组和3D组之间观察到相对不同的DEG。综上所述，本研究表明，与在传统单层培养板中培养的DPSC相比，在微球形成培养板中培养的DPSC具有卓越的多向分化能力，并且在再生相关基因类别中表现出更高的DEG表达。
