Advertisement

Cell and Tissue Research

, Volume 353, Issue 1, pp 65–78 | Cite as

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

  • P. Hilkens
  • P. Gervois
  • Y. Fanton
  • J. Vanormelingen
  • W. Martens
  • T. Struys
  • C. Politis
  • I. Lambrichts
  • A. BronckaersEmail author
Regular Article

Abstract

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.

Keywords

Dental pulp stem cells Isolation method Mesenchymal trilineage differentiation Ultrastructural analysis 

Supplementary material

441_2013_1630_Fig8_ESM.jpg (61 kb)
Supplementary Fig. 1

Adipogenic differentiation of DPSC-EZ and DPSC-OG. Ultrastructurally, adipogenic differentiated cells are globularly shaped (f) with a cytoplasm filled with electron-dense vesicles (v in figure g and h), branched mitochondria (h, bMit) and dilated rough endoplasmatic reticulum (i, dER). Scale bars: a = 10 μm; b, c, d = 2 μm (JPEG 61 kb)

441_2013_1630_MOESM1_ESM.tif (8.8 mb)
High resolution image (TIFF 9009 kb)
441_2013_1630_Fig9_ESM.jpg (34 kb)
Supplementary Fig. 2

TEM analysis of 3D-chondrogenic differentiated DPSCs. Chondrogenic differentiated DPSC-EZ (a) and DPSC-OG (d) both display an elongated cell phenotype with a cytoplasm containing numerous ribosomes and electron-dense intracellular matrix vesicles (mv, figure e). Striated collagen fibers (cf) are abundant in the ECM (b). Cartilage fragments (ca) are present in the ECM of both cell types and are closely interacting with the surrounding DPSCs and ECM (c, f). Scale bars: a, d, e = 5 μm; b = 1 μm; c = 10 μm; f = 2 μm (JPEG 33 kb)

441_2013_1630_MOESM2_ESM.tif (4.1 mb)
High resolution image (TIFF 4213 kb)

References

  1. About I, Bottero MJ, De Denato P, Camps J, Franquin JC, Mitsiadis TA (2000) Human dentin production in vitro. Exp Cell Res 258:33–41PubMedCrossRefGoogle Scholar
  2. Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S (2008) Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26:1787–1795PubMedCrossRefGoogle Scholar
  3. Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, Geurtsen W (2011) Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth. Calcif Tissue Int 88:130–141PubMedCrossRefGoogle Scholar
  4. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H (2000) Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 66:129–138PubMedCrossRefGoogle Scholar
  5. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317PubMedCrossRefGoogle Scholar
  6. Ellerstrom C, Hyllner J, Strehl R (2010) Single cell enzymatic dissociation of human embryonic stem cells: a straightforward, robust, and standardized culture method. Methods Mol Biol 584:121–134PubMedCrossRefGoogle Scholar
  7. Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235–242PubMedCrossRefGoogle Scholar
  8. Feng J, Mantesso A, De Bari C, Nishiyama A, Sharpe PT (2011) Dual origin of mesenchymal stem cells contributing to organ growth and repair. Proc Natl Acad Sci USA 108:6503–6508PubMedCrossRefGoogle Scholar
  9. Friedenstein AJ (1961) Osteogenetic activity of transplanted transitional epithelium. Acta Anat (Basel) 45:31–59CrossRefGoogle Scholar
  10. Gagari E, Rand MK, Tayari L, Vastardis H, Sharma P, Hauschka PV, Damoulis PD (2006) Expression of stem cell factor and its receptor, c-kit, in human oral mesenchymal cells. Eur J Oral Sci 114:409–415PubMedCrossRefGoogle Scholar
  11. Graziano A, D’aquino R, Laino G, Proto A, Giuliano MT, Pirozzi G, De Rosa A, Di Napoli D, Papaccio G (2008) Human CD34+ stem cells produce bone nodules in vivo. Cell Prolif 41:1–11PubMedCrossRefGoogle Scholar
  12. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97:13625–13630PubMedCrossRefGoogle Scholar
  13. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, Denbesten P, Robey PG, Shi S (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81:531–535PubMedCrossRefGoogle Scholar
  14. Huang GT, Sonoyama W, Chen J, Park SH (2006) In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res 324:225–236PubMedCrossRefGoogle Scholar
  15. Huang GT, Yamaza T, Shea LD, Djouad F, Kuhn NZ, Tuan RS, Shi S (2010) Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 16:605–615PubMedCrossRefGoogle Scholar
  16. Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M (2008) A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells 26:2408–2418PubMedCrossRefGoogle Scholar
  17. Jing W, Xiao J, Xiong Z, Yang X, Huang Y, Zhou M, Chen S, Lin Y, Tian W (2011) Explant culture: an efficient method to isolate adipose-derived stromal cells for tissue engineering. Artif Organs 35:105–112PubMedGoogle Scholar
  18. Karamzadeh R, Eslaminejad MB, Aflatoonian R (2012) Isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods. J Vis Exp 69:pii 4372Google Scholar
  19. Karaoz E, Dogan BN, Aksoy A, Gacar G, Akyuz S, Ayhan S, Genc ZS, Yuruker S, Duruksu G, Demircan PC, Sariboyaci AE (2010) Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol 133:95–112PubMedCrossRefGoogle Scholar
  20. Karaoz E, Demircan PC, Saglam O, Aksoy A, Kaymaz F, Duruksu G (2011) Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells. Histochem Cell Biol 136:455–473PubMedCrossRefGoogle Scholar
  21. Kiraly M, Porcsalmy B, Pataki A, Kadar K, Jelitai M, Molnar B, Hermann P, Gera I, Grimm WD, Ganss B, Zsembery A, Varga G (2009) Simultaneous PKC and CAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons. Neurochem Int 55:323–332PubMedCrossRefGoogle Scholar
  22. Laino G, D’aquino R, Graziano A, Lanza V, Carinci F, Naro F, Pirozzi G, Papaccio G (2005) A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (lab). J Bone Miner Res 20:1394–1402PubMedCrossRefGoogle Scholar
  23. Laino G, Graziano A, D’aquino R, Pirozzi G, Lanza V, Valiante S, De Rosa A, Naro F, Vivarelli E, Papaccio G (2006) An approachable human adult stem cell source for hard-tissue engineering. J Cell Physiol 206:693–701PubMedCrossRefGoogle Scholar
  24. Malone AM, Anderson CT, Tummala P, Kwon RY, Johnston TR, Stearns T, Jacobs CR (2007) Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism. Proc Natl Acad Sci USA 104:13325–13330PubMedCrossRefGoogle Scholar
  25. Martens W, Wolfs E, Struys T, Politis C, Bronckaers A, Lambrichts I (2012) Expression pattern of basal markers in human dental pulp stem cells and tissue. Cells Tissues Organs 196:490–500PubMedCrossRefGoogle Scholar
  26. Mikami Y, Ishii Y, Watanabe N, Shirakawa T, Suzuki S, Irie S, Isokawa K, Honda MJ (2011) Cd271/p75(NTR) inhibits the differentiation of mesenchymal stem cells into osteogenic, adipogenic, chondrogenic, and myogenic lineages. Stem Cells Dev 20:901–913PubMedCrossRefGoogle Scholar
  27. Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, Helwig B, Beerenstrauch M, Abou-Easa K, Hildreth T, Troyer D, Medicetty S (2003) Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells 21:50–60PubMedCrossRefGoogle Scholar
  28. Nakashima M, Iohara K, Sugiyama M (2009) Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration. Cytokine Growth Factor Rev 20:435–440PubMedCrossRefGoogle Scholar
  29. Patel M, Smith AJ, Sloan AJ, Smith G, Cooper PR (2009) Phenotype and behaviour of dental pulp cells during expansion culture. Arch Oral Biol 54:898–908PubMedCrossRefGoogle Scholar
  30. Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, Becchetti E, Marchionni C, Alviano F, Fossati V, Staffolani N, Franchina M, Grossi A, Bagnara GP (2005) Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 80:836–842PubMedCrossRefGoogle Scholar
  31. Shi S, Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 18:696–704PubMedCrossRefGoogle Scholar
  32. Spath L, Rotilio V, Alessandrini M, Gambara G, De Angelis L, Mancini M, Mitsiadis TA, Vivarelli E, Naro F, Filippini A, Papaccio G (2010) Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials. J Cell Mol Med 14:1635–1644PubMedCrossRefGoogle Scholar
  33. Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ (1995) Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP). J Biol Chem 270:9420–9428PubMedCrossRefGoogle Scholar
  34. Struys T, Moreels M, Martens W, Donders R, Wolfs E, Lambrichts I (2010) Ultrastructural and immunocytochemical analysis of multilineage differentiated human dental pulp- and umbilical cord-derived mesenchymal stem cells. Cells Tissues Organs 193:366–378PubMedCrossRefGoogle Scholar
  35. Struys T, Moreels M, Martens W, Donders R, Wolfs E, Lambrichts I (2011) Ultrastructural and immunocytochemical analysis of multilineage differentiated human dental pulp- and umbilical cord-derived mesenchymal stem cells. Cells Tissues Organs 193:366–378PubMedCrossRefGoogle Scholar
  36. Struys T, Ketkar-Atre A, Gervois P, Leten C, Hilkens P, Martens W, Bronckaers A, Dresselaers T, Politis C, Lambrichts I, Himmelreich U (2012) Magnetic resonance imaging of human dental pulp stem cells in vitro and in vivo. Cell Transplant (in press)Google Scholar
  37. Tsukamoto Y, Fukutani S, Shin-Ike T, Kubota T, Sato S, Suzuki Y, Mori M (1992) Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch Oral Biol 37:1045–1055PubMedCrossRefGoogle Scholar
  38. Waddington RJ, Youde SJ, Lee CP, Sloan AJ (2009) Isolation of distinct progenitor stem cell populations from dental pulp. Cells Tissues Organs 189:268–274PubMedCrossRefGoogle Scholar
  39. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, Fu YS, Lai MC, Chen CC (2004) Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 22:1330–1337PubMedCrossRefGoogle Scholar
  40. Zhang W, Walboomers XF, Shi S, Fan M, Jansen JA (2006) Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng 12:2813–2823PubMedCrossRefGoogle Scholar
  41. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • P. Hilkens
    • 1
  • P. Gervois
    • 1
  • Y. Fanton
    • 1
  • J. Vanormelingen
    • 1
  • W. Martens
    • 1
  • T. Struys
    • 1
  • C. Politis
    • 1
    • 2
  • I. Lambrichts
    • 1
  • A. Bronckaers
    • 1
    Email author
  1. 1.Department of Functional Morphology, Laboratory of Histology, Biomedical Research Institute (BIOMED)Hasselt UniversityDiepenbeekBelgium
  2. 2.Department of Oral and Maxillofacial Surgery, KU LeuvenUniversity Hospitals LeuvenLeuvenBelgium

Personalised recommendations