TY - JOUR
T1 - Three-dimensional Spheroid Culture Enhances Multipotent Differentiation and Stemness Capacities of Human Dental Pulp‐derived Mesenchymal Stem Cells by Modulating MAPK and NF-kB Signaling Pathways
AU - Chan, Ya Hui
AU - Lee, Yu Chieh
AU - Hung, Chia Yi
AU - Yang, Pi Ju
AU - Lai, Pin Chuang
AU - Feng, Sheng Wei
N1 - Funding Information:
This study was supported by the Ministry of Science and Technology, Taiwan (Grants MOST 107-2314-B-038-069 and 108-2314-B-038-032). Acknowledgements
Funding Information:
This study was supported by the Ministry of Science and Technology, Taiwan (Grants MOST 107-2314-B-038-069 and 108-2314-B-038-032).
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - 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. Graphical Abstract: [Figure not available: see fulltext.]
AB - 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. Graphical Abstract: [Figure not available: see fulltext.]
KW - 3DM-spheroid culture
KW - Cell differentiation
KW - Gene expression
KW - MAPK
KW - Mesenchymal stem cells
KW - NF-kB
KW - Signaling
UR - http://www.scopus.com/inward/record.url?scp=85105121439&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85105121439&partnerID=8YFLogxK
U2 - 10.1007/s12015-021-10172-4
DO - 10.1007/s12015-021-10172-4
M3 - Article
C2 - 33893620
AN - SCOPUS:85105121439
SN - 2629-3269
VL - 17
SP - 1810
EP - 1826
JO - Stem Cell Reviews and Reports
JF - Stem Cell Reviews and Reports
IS - 5
ER -