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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

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

References

  1. Mo, M., Zhou, Y., Li, S., & Wu, Y. (2018). Three-dimensional culture reduces cell size by increasing vesicle excretion. Stem Cells, 36, 286–292.

    CAS  PubMed  Article  Google Scholar 

  2. Bartold, M., Gronthos, S., Haynes, D., & Ivanovski, S. (2019). Mesenchymal stem cells and biologic factors leading to bone formation. Journal of Clinical Periodontology, 46, 12–32.

    PubMed  Article  Google Scholar 

  3. Moritani, Y., Usui, M., Sano, K., Nakazawa, K., Hanatani, T., Nakatomi, M., Iwata, T., Sato, T., Ariyoshi, W., Nishihara, T., & Nakashima, K. (2018). Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells. Journal of Periodontal Research, 53, 870–882.

    CAS  PubMed  Article  Google Scholar 

  4. Subbarayan, R., Girija, M. D., & Ranga Rao, S. (2018). Gingival spheroids possess multilineage differentiation potential. Journal of Cellular Physiology, 233, 1952–1958.

    CAS  PubMed  Article  Google Scholar 

  5. Kim, H. J., Sung, I. Y., Cho, Y. C., Kang, M. S., Rho, G. J., Byun, J. H., Park, W. U., Son, M. G., Park, B. W., Lee, H. J., & Kang, Y. H. (2019). Three-dimensional spheroid formation of cryopreserved human dental follicle-derived stem cells enhances pluripotency and osteogenic induction properties. Tissue Engineering and Regenerative Medicine, 16, 513–523.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Li, N., Li, X., Chen, K., Dong, H., & Kagami, H. (2019). Characterization of spontaneous spheroids from oral mucosa-derived cells and their direct comparison with spheroids from skin-derived cells. Stem Cell Research & Therapy, 10, 184.

    Article  CAS  Google Scholar 

  7. Lee, Y. C., Chan, Y. H., Hsieh, S. C., Lew, W. Z., & Feng, S. W. (2019). Comparing the osteogenic potentials and bone regeneration capacities of bone marrow and dental pulp mesenchymal stem cells in a rabbit calvarial bone defect model. International Journal of Molecular Sciences, 20, 5015.

    CAS  PubMed Central  Article  Google Scholar 

  8. Lew, W. Z., Feng, S. W., Lin, C. T., & Huang, H. M. (2019). Use of 0.4-Tesla static magnetic field to promote reparative dentine formation of dental pulp stem cells through activation of p38 MAPK signalling pathway. International Endodontic Journal, 52, 28–43.

    PubMed  Article  Google Scholar 

  9. Bu, N. U., Lee, H. S., Lee, B. N., Hwang, Y. C., Kim, S. Y., Chang, S. W., Choi, K. K., Kim, D. S., & Jang, J. H. (2020). In vitro characterization of dental pulp stem cells cultured in two microsphere-forming culture plates. Journal of Clinical Medicine, 9, 242.

    CAS  PubMed Central  Article  Google Scholar 

  10. Shuai, Y., Liao, L., Su, X., Yu, Y., Shao, B., Jing, H., Zhang, X., Deng, Z., & Jin, Y. (2016). Melatonin treatment improves mesenchymal stem cells therapy by preserving stemness during long-term in vitro expansion. Theranostics, 6, 1899–1917.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Zhang, S., Buttler-Buecher, P., Denecke, B., Arana-Chavez, V. E., & Apel, C. (2018). A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system. Archives of Oral Biology, 91, 1–8.

    CAS  PubMed  Article  Google Scholar 

  12. Yin, Q., Xu, N., Xu, D., Dong, M., Shi, X., Wang, Y., Hao, Z., Zhu, S., Zhao, D., Jin, H., & Liu, W. (2020). Comparison of senescence-related changes between three- and two-dimensional cultured adipose-derived mesenchymal stem cells. Stem Cell Research & Therapy, 11, 226.

    CAS  Article  Google Scholar 

  13. Abdel Meguid, E., Ke, Y., Ji, J., & El-Hashash, A. H. K. (2018). Stem cells applications in bone and tooth repair and regeneration: New insights, tools, and hopes. Journal of Cellular Physiology, 233, 1825–1835.

    CAS  PubMed  Article  Google Scholar 

  14. Kaku, M., Akiba, Y., Akiyama, K., Akita, D., & Nishimura, M. (2015). Cell-based bone regeneration for alveolar ridge augmentation–cell source, endogenous cell recruitment and immunomodulatory function. Journal of Prosthodontic Research, 59, 96–112.

    PubMed  Article  Google Scholar 

  15. Kitami, M., Kaku, M., Rocabado, J. M., Ida, T., Akiba, N., & Uoshima, K. (2016). Prolonged survival of transplanted osteoblastic cells does not directly accelerate the healing of calvarial bone defects. Journal of Cellular Physiology, 231, 1974–1982.

    CAS  PubMed  Article  Google Scholar 

  16. Han, H. W., Asano, S., & Hsu, S. H. (2019). Cellular spheroids of mesenchymal stem cells and their perspectives in future healthcare. Applied Sciences, 9, 627.

    CAS  Article  Google Scholar 

  17. Ryu, N. E., Lee, S. H., & Park, H. (2019). Spheroid culture system methods and applications for mesenchymal stem cells. Cells, 8, 1620.

    CAS  PubMed Central  Article  Google Scholar 

  18. Petrenko, Y., Syková, E., & Kubinová, Š (2017). The therapeutic potential of three-dimensional multipotent mesenchymal stromal cell spheroids. Stem Cell Research & Therapy, 8, 94.

    Article  CAS  Google Scholar 

  19. Baker, B. M., & Chen, C. S. (2012). Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. Journal of Cell Science, 125, 3015–3024.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Yamamoto, M., Kawashima, N., Takashino, N., Koizumi, Y., Takimoto, K., Suzuki, N., Saito, M., & Suda, H. (2014). Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells. Archives of Oral Biology, 59, 310–317.

    CAS  PubMed  Article  Google Scholar 

  21. Lee, S. H., Inaba, A., Mohindroo, N., Ganesh, D., Martin, C. E., Chugal, N., Kim, R. H., Kang, M. K., Park, N. H., & Shin, K. H. (2017). Three-dimensional sphere-forming cells are unique multipotent cell population in dental pulp cells. Journal of Endodontics, 43, 1302–1308.

    PubMed  Article  Google Scholar 

  22. Zhang, S., Liu, P., Chen, L., Wang, Y., Wang, Z., & Zhang, B. (2015). The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials, 41, 15–25.

    PubMed  Article  CAS  Google Scholar 

  23. He, D., Wang, R. X., Mao, J. P., Xiao, B., Chen, D. F., & Tian, W. (2017). Three-dimensional spheroid culture promotes the stemness maintenance of cranial stem cells by activating PI3K/AKT and suppressing NF-κB pathways. Biochemical and Biophysical Research Communications, 488, 528–533.

    CAS  PubMed  Article  Google Scholar 

  24. Imamura, A., Kajiya, H., Fujisaki, S., Maeshiba, M., Yanagi, T., Kojima, H., & Ohno, J. (2020). Three-dimensional spheroids of mesenchymal stem/stromal cells promote osteogenesis by activating stemness and Wnt/β-catenin. Biochemical and Biophysical Research Communications, 523, 458–464.

    CAS  PubMed  Article  Google Scholar 

  25. Lewis, N. S., Lewis, E. E., Mullin, M., Wheadon, H., Dalby, M. J., & Berry, C. C. (2017). Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence. Journal of Tissue Engineering, 8, 2041731417704428.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Adine, C., Ng, K. K., Rungarunlert, S., Souza, G. R., & Ferreira, J. N. (2018). Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands. Biomaterials, 180, 52–66.

    CAS  PubMed  Article  Google Scholar 

  27. Ferreira, J. N., Hasan, R., Urkasemsin, G., Ng, K. K., Adine, C., Muthumariappan, S., & Souza, G. R. (2019). A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids. Journal of Tissue Engineering and Regenerative Medicine, 13, 495–508.

    CAS  PubMed  Article  Google Scholar 

  28. Cui, X., Hartanto, Y., & Zhang, H. (2017). Advances in multicellular spheroids formation. Journal of The Royal Society Interface, 14, 20160877.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  29. Kim, M., Yun, H. W., Park, D. Y., Choi, B. H., & Min, B. H. (2018). Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells. Tissue Engineering and Regenerative Medicine, 15, 427–436.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Yan, L., & Wu, X. (2020). Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity. Cell Biology and Toxicology, 36, 165–178.

    CAS  PubMed  Article  Google Scholar 

  31. Tatsuhiro, F., Seiko, T., Yusuke, T., Reiko, T. T., & Kazuhito, S. (2018). Dental pulp stem cell-derived, scaffold-free constructs for bone regeneration. International Journal of Molecular Sciences, 19, 1846.

    PubMed Central  Article  CAS  Google Scholar 

  32. Tsai, A. C., Liu, Y., Yuan, X., & Ma, T. (2015). Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate. Tissue Engineering Part A, 21, 1705–1719.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Gurumurthy, B., Bierdeman, P. C., & Janorkar, A. V. (2017). Spheroid model for functional osteogenic evaluation of human adipose derived stem cells. Journal of Biomedical Materials Research Part A, 105, 1230–1236.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Rumiński, S., Kalaszczyńska, I., Długosz, A., & Lewandowska-Szumieł, M. (2019). Osteogenic differentiation of human adipose-derived stem cells in 3D conditions - comparison of spheroids and polystyrene scaffolds. European Cells & Materials, 37, 382–401.

    Article  Google Scholar 

  35. Yannarelli, G., Pacienza, N., Cuniberti, L., Medin, J., Davies, J., & Keating, A. (2013). The potential role of epigenetics on multipotent cell differentiation capacity of mesenchymal stromal cells. Stem Cells, 31, 215–220.

    CAS  PubMed  Article  Google Scholar 

  36. Cheng, N. C., Chen, S. Y., Li, J. R., & Young, T. H. (2013). Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Translational Medicine, 2, 584–594.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Zhou, Y., Chen, H., Li, H., & Wu, Y. (2017). 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension. Journal of Cellular and Molecular Medicine, 21, 1073–1084.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Faruqu, F. N., Zhou, S., Sami, N., Gheidari, F., Lu, H., & Al-Jamal, K. T. (2020). Three-dimensional culture of dental pulp pluripotent-like stem cells (DPPSCs) enhances Nanog expression and provides a serum-free condition for exosome isolation. FASEB BioAdvances, 2, 419–433.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Safwani, W. K., Makpol, S., Sathapan, S., & Chua, K. (2014). Impact of adipogenic differentiation on stemness and osteogenic gene expression in extensive culture of human adipose-derived stem cells. Archives of Medical Science, 10, 597–606.

    PubMed  PubMed Central  Article  Google Scholar 

  40. Deynoux, M., Sunter, N., Ducrocq, E., Dakik, H., Guibon, R., Burlaud-Gaillard, J., Brisson, L., Rouleux-Bonnin, F., le Nail, L. R., Hérault, O., Domenech, J., Roingeard, P., Fromont, G., & Mazurier, F. (2020). A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids. PLoS One, 15, e0225485.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Hotamisligil, G. S., & Davis, R. J. (2016). Cell signaling and stress responses. Cold Spring Harbor Perspectives in Biology, 8, a006072.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. Gharibi, B., Ghuman, M. S., & Hughes, F. J. (2012). Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRβ-induced MSC self-renewal. Journal of Cellular and Molecular Medicine, 16, 2789–2801.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Kim, M. H., Takeuchi, K., & Kino-Oka, M. (2019). Role of cell-secreted extracellular matrix formation in aggregate formation and stability of human induced pluripotent stem cells in suspension culture. Journal of Bioscience and Bioengineering, 127, 372–380.

    CAS  PubMed  Article  Google Scholar 

  44. Sart, S., Tomasi, R. F., Barizien, A., Amselem, G., Cumano, A., & Baroud, C. N. (2020). Mapping the structure and biological functions within mesenchymal bodies using microfluidics. Science Advances, 6, eaaw7853.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Zelzer, E., Mamluk, R., Ferrara, N., Johnson, R. S., Schipani, E., & Olsen, B. R. (2004). VEGFA is necessary for chondrocyte survival during bone development. Development, 131, 2161–2171.

    CAS  PubMed  Article  Google Scholar 

  46. Huang, C., Xue, M., Chen, H., Jiao, J., Herschman, H. R., O’Keefe, R. J., & Zhang, X. (2014). The spatiotemporal role of COX-2 in osteogenic and chondrogenic differentiation of periosteum-derived mesenchymal progenitors in fracture repair. PLoS One, 9, e100079.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Smyrek, I., Mathew, B., Fischer, S. C., Lissek, S. M., Becker, S., & Stelzer, E. H. K. (2019). E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity. Biology Open, 8, bio037051.

    CAS  PubMed  Google Scholar 

  48. Passanha, F. R., Geuens, T., Konig, S., van Blitterswijk, C. A., & LaPointe, V. L. (2020). Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11. Biomaterials, 254, 120127.

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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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Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Sheng-Wei Feng.

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Ethical Approval and Consent to Participate

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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Not applicable.

Conflict of Interest

The authors declare that they have no competing interests.

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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

Keywords

  • Mesenchymal stem cells
  • 3DM-spheroid culture
  • Cell differentiation
  • Gene expression
  • MAPK
  • NF-kB
  • Signaling