Tissue Eng Regen Med.  2019 Oct;16(5):513-523. 10.1007/s13770-019-00203-0.

Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties

• Kim HJ
• Sung IY
• Cho YC
• Kang MS
• Rho GJ
• Byun JH
• Park WU
• Son MG
• Park BW
• Lee HJ
• Kang YH

• KMID: 2464071
• DOI: http://doi.org/10.1007/s13770-019-00203-0

Abstract

BACKGROUND
Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional monolayer cultured MSCs.
METHODS
In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties.
RESULTS
In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis.
CONCLUSION
To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.

Keyword

Mesenchymal stem cells; Dental follicle stem cells; Spheroid; Stemness; Osteogenesis

MeSH Terms

Adipogenesis
Aging
Apoptosis
Bone and Bones
Cell Cycle
Cell Cycle Checkpoints
Dental Sac
Extracellular Matrix
Humans*
In Vitro Techniques
Mesenchymal Stromal Cells
Methods
Molar, Third
Osteogenesis
Regeneration
Stem Cells*