ORIGINAL ARTICLE

 

Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp

 

 

Leliane Macedo de Souza^I; Juliana Duarte Bittar^II; Izabel Cristina Rodrigues da Silva^III; Orlando Ayrton de Toledo^IV; Marcelo de Macedo Bírígido^V; Marcio José Poças-Fonseca^VI

^IDDS, MS, Department of Dentistry, School of Health Sciences, University of Brasilia, Brasília, DF, Brazil
^IIDDS, Department of Dentistry, School of Health Sciences, University of Brasilia, Brasília, DF, Brazil
^IIIBSc, MS, PhD, Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, UnB, Brasília, DF, Brazil
^IVDDS, MS, DS, PhD, Professor, Department of Dentistry, School of Health Sciences, University of Brasilia, Brasília, DF, Brazil
^VBSc, MS, DS, PhD, Associate Professor, Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, UnB, Brasília, DF, Brazil.
^VIBSc, DDS, MS, DS, Associate Professor, Department of Genetics and Morphology, Institute of Biological Sciences, University of Brasilia, UnB, Brasília, DF, Brazil

Correspondence to

 

 

---

ABSTRACT

AIM: This study was developed to compare the morphological, proliferative and immunophenotypic profiles of pulp cells from permanent and primary teeth, obtained by two isolation methods.
METHODS: Normal human impacted third molars and exfoliated primary teeth were collected and cut around the cementoenamel junction. Pulp cells cultures were established by two approaches: enzyme digestion (3 mg/mL type I colagenase and 4 mg/mL dispase), or culture of the tissue explants in cell culture dishes. Morphological and proliferative analyses, as well as immunophenotype characterization with monoclonal antibodies against CD117, CD34 and CD45 surface receptors were performed.
RESULTS: For the permanent teeth, on the 4th day of culture, the cell number was significantly higher for the outgrowth method. By the end of the studied period (14th day), the enzymatic method was more efficient in promoting culture growth. On the other hand, for primary teeth, enzymatic digestion always promoted a higher cell proliferation. The immunophenotypic profiles were CD117+/ CD34-/ CD45- and CD117+/ CD34+/ CD45- for cells from permanent and primary teeth, respectively.
CONCLUSIONS: The findings of this study indicate that both isolation methods can be efficiently used. The cell population displayed an immunophenotype compatible to the one of stem cells, with remarkable positive expression of CD117.

Keywords: stem cells, cell culture techniques, dental pulp, primary dentition, permanent dentition.

---

 

 

Full text available only in PDF format.

 

 

Acknowledgements

This work was supported by FINEP/MCT and FUB - Brazil

 

References

1. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod. 2007; 33: 377-90.         [ Links ]

2. Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, et al. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J Endod. 2008; 34: 962-69.         [ Links ]

3. Nakashima M. Tissue engineering in endodontics. Aust Endod J. 2005; 31: 111-3.         [ Links ]

4. Nakashima M, Akamine A. The application of tissue engineering to regeneration of pulp and dentin in endodontics. J Endod. 2005; 31: 711-8.         [ Links ]

5. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA. 2000; 97: 13625-30.         [ Links ]

6. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, ET AL. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002; 81: 531-5.         [ Links ]

7. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003; 100: 5807-12.         [ Links ]

8. Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes MSM, Pereira LV, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs. 2006; 184: 105-16.         [ Links ]

9. Chen SC, Marino V, Gronthos S, Bartold PM. Location of putative stem cells in human periodontal ligament. J Periodontal Res. 2006; 41: 547-53.         [ Links ]

10. Kamata N, Fujimoto R, Tomonari M, Taki M, Nagayama M, Yasumoto S. Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase. J Oral Pathol Med. 2004; 33: 417-23.         [ Links ]

11. Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. J Dent Res. 2004; 83: 27-34.         [ Links ]

12. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004; 364: 149-55.         [ Links ]

13. Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos S, et al. Comparison of stem cells mediated osteogenesis and dentinogenesis. J Dent Res. 2003; 82: 976-81.         [ Links ]

14. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H. Odontoblast differentiation of human dental pulp cells in explants cultures. Calcif Tissue Int. 2000; 66: 129-38.         [ Links ]

15. Huang GT, Shagramanova K, Chan SW. Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro. J Endod. 2006; 32: 1066-73.         [ Links ]

16. Laino G, Carinci F, Graziano A, D’aquino R, Lanza V, De Rosa A, et al. In vitro bone production using stem cells derived from human dental pulp. J Craniofac Surg. 2006; 17: 511-5.         [ Links ]

17. Huang GT, Sonoyama W, Chen J, Park SH. In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res. 2006; 324: 225-36.         [ Links ]

18. Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, et al. Multipotent mesenquimal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation. 2005; 80: 836-42.         [ Links ]

19. Stanislawski L, Carreau JP, Pouchelet M, Chen ZH, Goldberg M. In vitro culture of human dental pulp cells: some aspects of cells emerging early from the explants. Clin Oral Investig. 1997; 1: 131-40.         [ Links ]

20. Laino G, D’aquino R, Graziano A, Lanza V, Carinci F, Naro F, et al. A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue. J Bone Miner Res. 2005; 20: 1394-1402.         [ Links ]

21. Suchanek J, Soukup T, Visek B, Ivancakova R, Kucerova L, Mokry J. Dental pulp stem cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2009; 153: 31-5.         [ Links ]

22. Ikeda E, Hirose M, Kotobuki N, Shimaoka H, Tadokoro M, Maeda M, et al. Osteogenic differentiation of human dental papilla mesenchymal cells. Biochem Biophys Res Commun. 2006; 342: 1257-62.         [ Links ]

23. Freshney IR, Stacey GN, Auerbach JM. Culture of human stem cells: culture of specialized cells. New York: Wiley; 2007.         [ Links ]

24. Sonoyama W, Yamaza T, Gronthos S, Shi S. Multipotent stem cells in dental pulp. In: Freshney IR, Stacey GN, Aurebach JM. Culture of human stem cells: culture of specialized cells. New York: Wiley; 2007. p.187- 206.         [ Links ]

25. Castro-Malaspina H, Gay RE, Resnick G, Kapoor N, Meyers P, Chiarieri D, et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood. 1980; 56: 289-301.         [ Links ]

26. Papaccio G, Graziano A, D’aquino R, Graziano MF, Pirozzi G, Menditti D, et al. Long-term cryopreservation of dental pulp stem cells (SBPDPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol. 2006; 208: 319-25.         [ Links ]

27. Gagari E, Rand MK, Tayari L, Vastardis H, Sharma P, Hauschka PV, et al. Expression of stem cell factor and its receptor, c-kit, in human oral mesenchymal cells. Eur J Oral Sci. 2006; 114: 409-15.         [ Links ]

28. Gangenahalli GU, Singh VK, Verma YK, Gupta P, Sharma RK, Chandra R, et al. Three-dimensional structure prediction of the interaction of CD34 with the SH3 domain of CRK-L. Stem Cells Dev. 2005; 14: 470-7.         [ Links ]

 

 

Correspondence to:
Marcio José Poças-Fonseca
Universidade de Brasília - Instituto de Ciências
Biológicas - Departamento de Genética e
Morfologia - Campus Universitário Darcy
Ribeiro - 70910-900 Brasília - DF, Brazil.
Phone: + 55 (61) 3107-3090
Fax: +55 (61) 3107-2923
E-mail: mpossas@unb.br

Received for publication: March 02, 2010
Accepted: July 19, 2010