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.
Graphical Abstract
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Data Availability
The data from the current study are available from the corresponding author on request.
Abbreviations
- 2D:
-
two-dimensional
- 3D:
-
three-dimensional
- SEM:
-
scanning electron microscopy
- TEM:
-
transmission electron microscopy
- qRT-PCR:
-
quantitative reverse transcription-polymerase chain reaction
- IF:
-
immunofluorescence staining
- MSC:
-
mesenchymal stem cell
- DPSCs:
-
dental-pulp derived mesenchymal stem cells
- ECM:
-
extracellular matrix
- PDLMSC:
-
periodontal ligament mesenchymal stem cell
- GMSC:
-
gingival mesenchymal stem cells
- ADMSC:
-
adipose mesenchymal stem cell
- MNP:
-
magnetic nanoparticle
- PFA:
-
paraformaldehyde
- VEGF:
-
vascular endothelial growth factor
- OCN:
-
osteocalcin
- DAPI:
-
4’,6-diamidino-2-phenylindole
- RIPA:
-
radioimmunoprecipitation assay
- MAPK:
-
mitogen-activated protein kinase
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Acknowledgements
The authors acknowledge the academic and science graphic illustration services provided by TMU Research Promotion Center.
Funding
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).
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YHC and YCL performed the whole research and data collection. CYH carried out the isolation of DPSCs and data analysis. PJY carried out the western blot. PCL helped in the critical discussion. SWF designed the experiments and performed the manuscript drafting. All authors read and approved the final manuscript.
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All experimental protocols were performed with ethics approval from the ethical committee of the Taipei Medical University, Taipei, Taiwan (approval no. N201904079). Consent to participate is not applicable.
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Chan, YH., Lee, YC., Hung, CY. et al. 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. Stem Cell Rev and Rep 17, 1810–1826 (2021). https://doi.org/10.1007/s12015-021-10172-4
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DOI: https://doi.org/10.1007/s12015-021-10172-4