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A Method to Isolate, Purify, and Characterize Human Periodontal Ligament Stem Cells

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Oral Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 666))

Abstract

Human periodontal ligament stem cells (PDLSCs) are a unique population of mesenchymal stem cells (MSCs) which demonstrate the capacity to generate cementum- and periodontal ligament-like structures in vivo. As such, PDLSCs represent a promising cell-based therapy in reconstructive dentistry for the treatment of periodontal disease. The present chapter describes two methods for isolating PDLSCs from human PDL tissue including traditional plastic adherence and immunomagnetic selection based on the expression of MSC-associated surface markers STRO-1 antigen, CD146 (MUC-18), CD29 (integrin β-1), CD44, and CD106 (VCAM-1). Although no single antibody demonstrates specificity for MSCs, isolation based on the expression of individual markers results in homogeneous preparations of PDLSCs. Methods to further characterize the immunophenotype and multipotent capacity of PDLSCs to differentiate into adipocytes, osteoblast- and cementoblast-like cells in vitro, and cementum- and periodontal ligament-like tissues in vivo are also described.

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References

  1. Melcher, A. H. (1985) Cells of periodontium: their role in the healing of wounds. Ann. R. Coll. Surg. Engl. 67, 130–131.

    PubMed  CAS  Google Scholar 

  2. Pitaru, S., McCulloch, C. A., and Narayanan, S. A. (1994) Cellular origins and differentiation control mechanisms during periodontal development and wound healing. J. Periodont. Res. 29, 81–94.

    Article  PubMed  CAS  Google Scholar 

  3. Gould, T. R., Melcher, A. H., and Brunette, D. M. (1980) Migration and division of progenitor cell populations in periodontal ligament after wounding. J. Periodont. Res. 15, 20–42.

    Article  PubMed  CAS  Google Scholar 

  4. Seo, B. M., Miura, M., Gronthos, S., Bartold, P. M., Batouli, S., Brahim, J., Young, M., Robey, P. G., Wang, C. Y., and Shi, S. (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 364, 149–155.

    Article  PubMed  CAS  Google Scholar 

  5. Gronthos, S., Mrozik, K., Shi, S., and Bartold, P. M. (2006) Ovine periodontal ligament stem cells: isolation, characterization, and differentiation potential. Calcif. Tissue Int. 79, 310–317.

    Article  PubMed  CAS  Google Scholar 

  6. Shi, S., Bartold, P. M., Miura, M., Seo, B. M., Robey, P. G., and Gronthos, S. (2005) The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod. Craniofac. Res. 8, 191–199.

    Article  PubMed  CAS  Google Scholar 

  7. Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., and Shi, S. (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 97, 13625–13630.

    Article  PubMed  CAS  Google Scholar 

  8. Trubiani, O., Di Primio, R., Traini, T., Pizzicannella, J., Scarano, A., Piattelli, A., and Caputi, S. (2005) Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament. Int. J. Immunopathol. Pharmacol. 18, 213–221.

    PubMed  CAS  Google Scholar 

  9. Chen, S. C., Marino, V., Gronthos, S., and Bartold, P. M. (2006) Location of putative stem cells in human periodontal ligament. J. Periodont. Res. 41, 547–553.

    Article  PubMed  CAS  Google Scholar 

  10. Nagatomo, K., Komaki, M., Sekiya, I., Sakaguchi, Y., Noguchi, K., Oda, S., Muneta, T., and Ishikawa, I. (2006) Stem cell properties of human periodontal ligament cells. J. Periodont. Res. 41, 303–310.

    Article  PubMed  CAS  Google Scholar 

  11. Jo, Y. Y., Lee, H. J., Kook, S. Y., Choung, H. W., Park, J. Y., Chung, J. H., Choung, Y. H., Kim, E. S., Yang, H. C., and Choung, P. H. (2007) Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng. 13, 767–773.

    Article  PubMed  CAS  Google Scholar 

  12. Ivanovski, S., Haase, H. R., and Bartold, P. M. (2001) Isolation and characterization of fibroblasts derived from regenerating human periodontal defects. Arch. Oral Biol. 46, 679–688.

    Article  PubMed  CAS  Google Scholar 

  13. Luan, X., Ito, Y., Dangaria, S., and Diekwisch, T. G. (2006) Dental follicle progenitor cell heterogeneity in the developing mouse periodontium. Stem Cells Dev. 15, 595–608.

    Article  PubMed  CAS  Google Scholar 

  14. Techawattanawisal, W., Nakahama, K., Komaki, M., Abe, M., Takagi, Y., and Morita, I. (2007) Isolation of multipotent stem cells from adult rat periodontal ligament by neurosphere-forming culture system. Biochem. Biophys. Res. Commun. 357, 917–923.

    Article  PubMed  CAS  Google Scholar 

  15. Seo, B. M., Miura, M., Sonoyama, W., Coppe, C., Stanyon, R., and Shi, S. (2005) Recovery of stem cells from cryopreserved periodontal ligament. J. Dent. Res. 84, 907–912.

    Article  PubMed  Google Scholar 

  16. Pihlstrom, B. L., Michalowicz, B. S., and Johnson, N. W. (2005) Periodontal diseases. Lancet. 366, 1809–1820.

    Article  PubMed  Google Scholar 

  17. Polimeni, G., Xiropaidis, A. V., and Wikesjo, U. M. (2006) Biology and principles of periodontal wound healing/regeneration. Periodontol 2000. 41, 30–47.

    Article  PubMed  Google Scholar 

  18. Lin, N. H., Gronthos, S., and Bartold, P. M. (2008) Stem cells and periodontal regeneration. Aust. Dent. J. 53, 108–121.

    Article  PubMed  Google Scholar 

  19. Zannettino, A. C., Paton, S., Arthur, A., Khor, F., Itescu, S., Gimble, J. M., and Gronthos, S. (2008) Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J. Cell. Physiol. 214, 413–421.

    Article  PubMed  CAS  Google Scholar 

  20. Shi, S., and Gronthos, S. (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J. Bone Miner. Res. 18, 696–704.

    Article  PubMed  Google Scholar 

  21. Filshie, R. J., Zannettino, A. C., Makrynikola, V., Gronthos, S., Henniker, A. J., Bendall, L. J., Gottlieb, D. J., Simmons, P. J., and Bradstock, K. F. (1998) MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies. Leukemia. 12, 414–421.

    Article  PubMed  CAS  Google Scholar 

  22. Fedarko, N. S., Fohr, B., Robey, P. G., Young, M. F., and Fisher, L. W. (2000) Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack. J. Biol. Chem. 275, 16666–16672.

    Article  PubMed  CAS  Google Scholar 

  23. Ingram, R. T., Clarke, B. L., Fisher, L. W., and Fitzpatrick, L. A. (1993) Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity. J. Bone Miner. Res. 8, 1019–1029.

    Article  PubMed  CAS  Google Scholar 

  24. Kuznetsov, S. A., Krebsbach, P. H., Satomura, K., Kerr, J., Riminucci, M., Benayahu, D., and Robey, P. G. (1997) Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J. Bone Miner. Res. 12, 1335–1347.

    Article  PubMed  CAS  Google Scholar 

  25. Muraglia, A., Cancedda, R., and Quarto, R. (2000) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J. Cell Sci. 113, 1161–1166.

    PubMed  CAS  Google Scholar 

  26. Gronthos, S., Brahim, J., Li, W., Fisher, L. W., Cherman, N., Boyde, A., DenBesten, P., Robey, P. G., and Shi, S. (2002) Stem cell properties of human dental pulp stem cells. J. Dent. Res. 81, 531–535.

    Article  PubMed  CAS  Google Scholar 

  27. Gronthos, S., Zannettino, A. C., Hay, S. J., Shi, S., Graves, S. E., Kortesidis, A., and Simmons, P. J. (2003) Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J. Cell Sci. 116, 1827–1835.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Mesenchymal Stem Cell Group, Division of Hematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, Australia

    Krzysztof Mrozik & Stan Gronthos

  2. University of Southern California, Los Angeles, CA, USA

    Songtao Shi

  3. Dental School, Colgate Australian Clinical Dental Research Centre, University of Adelaide, Adelaide, Australia

    P. Mark Bartold

Authors
  1. Krzysztof Mrozik
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  2. Stan Gronthos
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  3. Songtao Shi
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  4. P. Mark Bartold
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Editor information

Editors and Affiliations

  1. Faculty of Dentistry, Department of Oral Sciences, University of Otago, Great King Street 310, Dunedin, 9016, New Zealand

    Gregory J. Seymour

  2. Faculty of Dentistry, Department of Oral Sciences, University of Otago, Great King Street 310, Dunedin, 9016, New Zealand

    Mary P. Cullinan

  3. Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Great King Street 310, Dunedin, 9016, New Zealand

    Nicholas C.K. Heng

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Mrozik, K., Gronthos, S., Shi, S., Bartold, P.M. (2010). A Method to Isolate, Purify, and Characterize Human Periodontal Ligament Stem Cells. In: Seymour, G., Cullinan, M., Heng, N. (eds) Oral Biology. Methods in Molecular Biology, vol 666. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-820-1_17

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  • DOI: https://doi.org/10.1007/978-1-60761-820-1_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-819-5

  • Online ISBN: 978-1-60761-820-1

  • eBook Packages: Springer Protocols

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