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

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Tissue Engineering and Regenerative Medicine Aims and scope

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 mono-layer 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.

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Acknowledgement

This work was supported by the grant from National Research Foundation (2017R1D1A1B03035677), Republic of Korea.

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Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, College of Medicine, University of Ulsan, 877 Bangeojinsunhwando-ro, Dong-gu, Ulsan, 44033, Republic of Korea

    Hyo-Jung Kim, Iel-Yong Sung & Yeong-Cheol Cho

  2. Department of Oral and Maxillofacial Surgery, Changwon Gyeongsang National University Hospital, 11 Samjeongja-ro, Seongsan-gu, Changwon, 51472, Republic of Korea

    Min-Su Kang & Young-Hoon Kang

  3. Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, 15 Jinju-daero 816 beon-gil, Jinju-si, Gyeongsangnam-do, 52727, Republic of Korea

    Gyu-Jin Rho & Hyeon-Jeong Lee

  4. Department of Dentistry, Institute of Health Science, Gyeongsang National University School of Medicine, Jinju, 816-15, Republic of Korea

    June-Ho Byun & Young-Hoon Kang

  5. Department of Dental Technology, Jinju Health College, Jinju, Republic of Korea

    Won-Uk Park

  6. Department of Dentistry, Hanil Hospital, Jinju, Republic of Korea

    Myeong-Gyun Son & Bong-Wook Park

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  1. Hyo-Jung Kim
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Correspondence to Hyeon-Jeong Lee or Young-Hoon Kang.

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Ethical statement

This study protocol was approved by the institutional review board of Changwon Gyeongsang National University Hospital (IRB No. GNUH IRB-2018-11-002-001) and informed consent was confirmed by IRB.

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Kim, HJ., Sung, IY., Cho, YC. et al. Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties. Tissue Eng Regen Med 16, 513–523 (2019). https://doi.org/10.1007/s13770-019-00203-0

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  • DOI: https://doi.org/10.1007/s13770-019-00203-0

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