Ya-Hui Chan1, Yu-Chieh Lee2, Chia-Yi Hung3, Pi-Ju Yang4, Pin-Chuang Lai5, Sheng-Wei Feng6,7,8. 1. School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan. 2. Department of Obstetrics and Gynecology, Taipei Medical University Hospital, Taipei, Taiwan. 3. School of Dentistry, College of Oral Medicine, Taipei Medical University, No. 250, Wuxing St, Taipei, 11031, Taiwan. 4. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan. 5. Department of Diagnosis and Oral Health, School of Dentistry, University of Louisville, Louisville, KY, USA. 6. School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan. shengwei@tmu.edu.tw. 7. School of Dentistry, College of Oral Medicine, Taipei Medical University, No. 250, Wuxing St, Taipei, 11031, Taiwan. shengwei@tmu.edu.tw. 8. Division of Prosthodontics, Department of Dentistry, Taipei Medical University Hospital, Taipei, Taiwan. shengwei@tmu.edu.tw.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: This study confirmed that 3DM-spheroids efficiently enhanced the therapeutic efficiency of DPSCs.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: This study confirmed that 3DM-spheroids efficiently enhanced the therapeutic efficiency of DPSCs.
Authors: Y Moritani; M Usui; K Sano; K Nakazawa; T Hanatani; M Nakatomi; T Iwata; T Sato; W Ariyoshi; T Nishihara; K Nakashima Journal: J Periodontal Res Date: 2018-06-14 Impact factor: 4.419
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