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

  • Hyo-Jung Kim
  • Iel-Yong Sung
  • Yeong-Cheol Cho
  • Min-Su Kang
  • Gyu-Jin Rho
  • June-Ho Byun
  • Won-Uk Park
  • Myeong-Gyun Son
  • Bong-Wook Park
  • Hyeon-Jeong LeeEmail author
  • Young-Hoon KangEmail author
Original Article
  • 16 Downloads

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.

Keywords

Mesenchymal stem cells Dental follicle stem cells Spheroid Stemness Osteogenesis 

Notes

Acknowledgement

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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interest.

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.

References

  1. 1.
    Klingemanna H, Matzilevichb D, Marchand J. Mesenchymal stem cells sources and clinical applications. Transfus Med Hemother. 2008;35:272–7.Google Scholar
  2. 2.
    Ben-Ami E, Berrih-Aknin S, Miller A. Mesenchymal stem cells as an immunomodulatory therapeutic strategy for autoimmune diseases. Autoimmun Rev. 2011;10:410–5.CrossRefGoogle Scholar
  3. 3.
    Kwon A, Kim Y, Kim M, Kim J, Choi H, Jekarl DW, et al. Tissue-specific differentiation potency of mesenchymal stromal cells from perinatal tissues. Sci Rep. 2016;6:23544.CrossRefGoogle Scholar
  4. 4.
    Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, et al. Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant. 2010;19:667–79.CrossRefGoogle Scholar
  5. 5.
    Hsiao ST, Asgari A, Lokmic Z, Sinclair R, Dusting GJ, Lim SY, et al. Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue. Stem Cells Dev. 2012;21:2189–203.CrossRefGoogle Scholar
  6. 6.
    Kang YH, Lee HJ, Jang SJ, Byun JH, Lee JS, Lee HC, et al. Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles. Differentiation. 2015;90:48–58.CrossRefGoogle Scholar
  7. 7.
    Park BW, Kang EJ, Byun JH, Song MG, Kim HJ, Hah YS, et al. In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues. Differentiation. 2012;83:249–59.CrossRefGoogle Scholar
  8. 8.
    Eggenhofer E, Benseler V, Kroemer A, Popp FC, Geissler EK, Schlitt HJ, et al. Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion. Front Immunol. 2012;3:297.CrossRefGoogle Scholar
  9. 9.
    Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. Proc Natl Acad Sci U S A. 2006;103:2494–9.CrossRefGoogle Scholar
  10. 10.
    Li L, Chen X, Wang WE, Zeng C. How to improve the survival of transplanted mesenchymal stem cell in ischemic heart? Stem Cells Int. 2016;2016:9682757.Google Scholar
  11. 11.
    Zhang S, Liu P, Chen L, Wang Y, Wang Z, Zhang B. The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials. 2015;41:15–25.CrossRefGoogle Scholar
  12. 12.
    Cheng NC, Wang S, Young TH. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials. 2012;33:1748–58.CrossRefGoogle Scholar
  13. 13.
    Bartosh TJ, Ylöstalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K, et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A. 2010;107:13724–9.CrossRefGoogle Scholar
  14. 14.
    Potapova IA, Gaudette GR, Brink PR, Robinson RB, Rosen MR, Cohen IS, et al. Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. Stem Cells. 2007;25:1761–8.CrossRefGoogle Scholar
  15. 15.
    Guo L, Ge J, Zhou Y, Wang S, Zhao RC, Wu Y. Three-dimensional spheroid-cultured mesenchymal stem cells devoid of embolism attenuate brain stroke injury after intra-arterial injection. Stem Cells Dev. 2014;23:978–89.CrossRefGoogle Scholar
  16. 16.
    Wang CC, Chen CH, Hwang SM, Lin WW, Huang CH, Lee WY, et al. Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells. 2009;27:724–32.CrossRefGoogle Scholar
  17. 17.
    Park BW. Cryopreservation of dental tissue and subsequent isolation of mesenchymal stem cells. J Korean Assoc Oral Maxillofac Surg. 2015;41:1–2.CrossRefGoogle Scholar
  18. 18.
    Park BW, Jang SJ, Byun JH, Kang YH, Choi MJ, Park WU, et al. Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells. J Tissue Eng Regen Med. 2017;11:489–500.CrossRefGoogle Scholar
  19. 19.
    Kawashima N, Noda S, Yamamoto M, Okiji T. Properties of dental pulp-derived mesenchymal stem cells and the effects of culture conditions. J Endod. 2017;43:S31–4.CrossRefGoogle Scholar
  20. 20.
    Yamamoto M, Kawashima N, Takashino N, Koizumi Y, Takimoto K, Suzuki N, et al. Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells. Arch Oral Biol. 2014;59:310–7.CrossRefGoogle Scholar
  21. 21.
    Moritani Y, Usui M, Sano K, Nakazawa K, Hanatani T, Nakatomi M, et al. Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells. J Periodontal Res. 2018;53:870–82.CrossRefGoogle Scholar
  22. 22.
    Xiao L, Kumazawa Y, Okamura H. Cell death, cavitation and spontaneous multi-differentiation of dental pulp stem cells-derived spheroids in vitro: a journey to survival and organogenesis. Biol Cell. 2014;106:405–19.CrossRefGoogle Scholar
  23. 23.
    Park E, Patel AN. Changes in the expression pattern of mesenchymal and pluripotent markers in human adipose-derived stem cells. Cell Biol Int. 2010;34:979–84.CrossRefGoogle Scholar
  24. 24.
    Li Y, Wu Q, Wang Y, Li L, Bu H, Bao J. Senescence of mesenchymal stem cells (Review). Int J Mol Med. 2017;39:775–82.CrossRefGoogle Scholar
  25. 25.
    Marx V. Cell culture: a better brew. Nature. 2013;496:253–8.CrossRefGoogle Scholar
  26. 26.
    Gustafsson MV, Zheng X, Pereira T, Gardin K, Jin S, Lundkvist J, et al. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell. 2005;9:617–28.CrossRefGoogle Scholar
  27. 27.
    Zhang D, Kilian KA. The effect of mesenchymal stem cells shape on the maintenance of multipotency. Biomaterials. 2013;34:3962–9.CrossRefGoogle Scholar
  28. 28.
    Rosso F, Giordano A, Barbarisi M, Barbarisi A. From cell-ECM interaction to tissue engineering. J Cell Physiol. 2004;199:174–80.CrossRefGoogle Scholar
  29. 29.
    Mathews S, Mathew SA, Gupta PK, Bhonde R, Totey S. Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells. J Tissue Eng Regen Med. 2014;8:143–52.CrossRefGoogle Scholar
  30. 30.
    Rustad KC, Wong VW, Sorkin M, Glotzbach JP, Major MR, Rajadas J, et al. Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biometric hydrogel scaffold. Biomaterials. 2012;33:80–90.CrossRefGoogle Scholar
  31. 31.
    Kapur SK, Wang X, Shang H, Yun S, Li X, Feng G, et al. Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids. Biofabication. 2012;4:025004.CrossRefGoogle Scholar
  32. 32.
    Dang SM, Kyba M, Perlingeiro R, Daley GQ, Zandstra PW. Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng. 2002;78:442–53.CrossRefGoogle Scholar
  33. 33.
    Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng. 2003;83:173–80.CrossRefGoogle Scholar
  34. 34.
    Koike M, Sakaki S, Amano Y, Kurosawa H. Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies. J Biosci Bioeng. 2007;104:294–9.CrossRefGoogle Scholar
  35. 35.
    Spelke DP, Ortmann D, Khademhosseini A, Ferreira L, Karp JM. Methods for embryoid body formation: the microwell approach. Methods Mol Biol. 2011;690:151–62.CrossRefGoogle Scholar
  36. 36.
    Yeh HY, Liu BH, Hsu SH. The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes. Biomaterials. 2012;33:8943–54.CrossRefGoogle Scholar
  37. 37.
    Frith JE, Thomson B, Genever PG. Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods. 2010;16:735–49.CrossRefGoogle Scholar
  38. 38.
    Sakai Y, Yoshida S, Yoshiura Y, Mori R, Tamura T, Yahiro K, et al. Effect of microwell chip structure on cell microsphere production of various animal cells. J Biosci Bioeng. 2010;110:223–9.CrossRefGoogle Scholar
  39. 39.
    Zimmermann JA, McDevitt TC. Pre-conditioning mesenchymal stromal cell spheroids for immunomodulatory paracrine factor secretion. Cytotherpay. 2014;16:331–45.CrossRefGoogle Scholar
  40. 40.
    Sun Y, Wang Y, Zhou L, Zou Y, Huang G, Gao G, et al. Spheroid-cultured human umbilical cord-derived mesenchymal stem cells attenuate hepatic ischemia-reperfusion injury in rats. Sci Rep. 2018;8:2518.CrossRefGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society 2019

Authors and Affiliations

  1. 1.Department of Oral and Maxillofacial Surgery, College of MedicineUniversity of UlsanDong-gu, UlsanRepublic of Korea
  2. 2.Department of Oral and Maxillofacial SurgeryChangwon Gyeongsang National University HospitalSeongsan-gu, ChangwonRepublic of Korea
  3. 3.Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life ScienceGyeongsang National UniversityJinju-siRepublic of Korea
  4. 4.Department of Dentistry, Institute of Health ScienceGyeongsang National University School of MedicineJinjuRepublic of Korea
  5. 5.Department of Dental TechnologyJinju Health CollegeJinjuRepublic of Korea
  6. 6.Department of DentistryHanil HospitalJinjuRepublic of Korea

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