Abstract
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.
Highlights
Dental pulp stem cells (DPSCs) extracted from the pulp of human molars are considered a superior source of multipotent mesenchymal stem cells (MSCs) for use in tissue engineering [1]
Our study demonstrated that DPSCs cultured in 2D and microsphere-forming plate culture methods presented different proliferation and differentiation properties
DPSCs cultured in the microsphere-forming plate showed increased multilineage differentiation capacities, which includes osteogenic, adipogenic, and chondrogenic differentiation
Summary
Dental pulp stem cells (DPSCs) extracted from the pulp of human molars are considered a superior source of multipotent mesenchymal stem cells (MSCs) for use in tissue engineering [1]. DPSCs typically express the STRO-1 and CD146 antigens and are able to differentiate into neurons, cardiomyocytes, chondrocytes, osteoblasts, liver cells, and β cells of islet of pancreas [2,3]. As DPSCs can be obtained, they have attracted considerable interest on account of their wide potential for use in regenerative endodontics [4]. Much of our understanding of the biological mechanisms underlying cellular functions of DPSCs, such as differentiation and multipotency, has been shaped from studying cells cultured on two-dimensional (2D) monolayer dish surfaces [5]. Another study reported that the 2D culture method has limitations because it does not replicate the cell–cell and cell–extracellular matrix (ECM) interactions that tissues possess [7]
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