School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan.
Department of Obstetrics and Gynecology, Taipei Medical University Hospital, Taipei, Taiwan.
Stem Cell Rev Rep. 2021 Oct;17(5):1810-1826. doi: 10.1007/s12015-021-10172-4. Epub 2021 Apr 24.
Three-dimensional (3D) culture of mesenchymal stem cells has become an important research and development topic. However, comprehensive analysis of human dental pulp-derived mesenchymal stem cells (DPSCs) in 3D-spheroid culture remains unexplored. Thus, we evaluated the cellular characteristics, multipotent differentiation, gene expression, and related-signal transduction pathways of DPSCs in 3D-spheroid culture via magnetic levitation (3DM), compared with 2D-monolayer (2D) and 3D-aggregate (3D) cultures.
The gross morphology and cellular ultrastructure were observed in the 2D, 3D, and 3DM experimental groups using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Surface markers and trilineage differentiation were evaluated using flow cytometry and staining analysis. Quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining (IF) were performed to investigate the expression of differentiation and stemness markers. Signaling transduction pathways were evaluated using western blot analysis.
The morphology of cell aggregates and spheroids was largely influenced by the types of cell culture plates and initial cell seeding density. SEM and TEM experiments confirmed that the solid and firm structure of spheroids was quickly formed in the 3DM-medium without damaging cells. In addition, these three groups all expressed multilineage differentiation capabilities and surface marker expression. The trilineage differentiation capacities of the 3DM-group were significantly superior to the 2D and 3D-groups. The osteogenesis, angiogenesis, adipogenesis, and stemness-related genes were significantly enhanced in the 3D and 3DM-groups. The IF analysis showed that the extracellular matrix expression, osteogenesis, and angiogenesis proteins of the 3DM-group were significantly higher than those in the 2D and 3D-groups. Finally, 3DM-culture significantly activated the MAPK and NF-kB signaling transduction pathways and ameliorated the apoptosis effects of 3D-culture.
This study confirmed that 3DM-spheroids efficiently enhanced the therapeutic efficiency of DPSCs.
三维（3D）培养的间充质干细胞已成为一个重要的研究和开发课题。然而，关于牙髓间充质干细胞（DPSCs）在 3D 球体培养中的全面分析尚未得到探索。因此，我们通过磁悬浮（3DM）比较了 2D 单层（2D）和 3D 聚集（3D）培养中的 DPSCs 的细胞特征、多能分化、基因表达和相关信号转导途径。
通过扫描电子显微镜（SEM）和透射电子显微镜（TEM）观察 2D、3D 和 3DM 实验组的大体形态和细胞超微结构。使用流式细胞术和染色分析评估表面标志物和三系分化。采用定量逆转录-聚合酶链反应和免疫荧光染色（IF）检测分化和干性标志物的表达。采用 Western blot 分析评估信号转导途径。
细胞聚集体和球体的形态主要受细胞培养板类型和初始细胞接种密度的影响。SEM 和 TEM 实验证实，在 3DM 培养基中，无需破坏细胞即可快速形成坚实的球体结构。此外，这三组均表达多系分化能力和表面标志物表达。3DM 组的三系分化能力明显优于 2D 和 3D 组。成骨、血管生成、脂肪生成和干性相关基因在 3D 和 3DM 组中显著增强。IF 分析表明，3DM 组的细胞外基质表达、成骨和血管生成蛋白明显高于 2D 和 3D 组。最后，3DM 培养显著激活了 MAPK 和 NF-kB 信号转导途径，并改善了 3D 培养的细胞凋亡作用。
本研究证实，3DM 球体有效地提高了 DPSCs 的治疗效果。
