Journal of Bone and Mineral Metabolism

, Volume 25, Issue 1, pp 36–45

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

  • Mette Grøndahl Sørensen
  • Kim Henriksen
  • Sophie Schaller
  • Dennis Bang Henriksen
  • Finn Cilius Nielsen
  • Morten Hanefeld Dziegiel
  • Morten Asser Karsdal


Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14− population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.

Key words

human osteoclasts CDM morphology resorption osteoclastogenesis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Baron, R 2003

    General principles of bone biology

    eds. Primer on the Metabolic Bone Diseases and Disorders of Mineral MetabolismLippincott-RavenNew York19
    Google Scholar
  2. 2.
    Marks, SC, Hermey, DC 1996

    The structure and development of bone

    Bilezikian, JPRaisz, LGRodan, GA eds. Principles of Bone BiologyAcademic PressSan Diego314
    Google Scholar
  3. 3.
    Vaananen, HK 1993Mechanism of bone turnoverAnn Med25353359PubMedGoogle Scholar
  4. 4.
    Mundy, GR 1996

    Bone-resorbing cells

    eds. Primer on the Metabolic Bone Diseases and Disorders of Mineral MetabolismLippincott-RavenNew York1624
    Google Scholar
  5. 5.
    Suda, T, Udagawa, N, Takahashi, N 1996

    Cells of bone: osteoclast generation

    Bilezikian, JPRaisz, LGRodan, GA eds. Principles of Bone BiologyAcademic pressSan Diego87102
    Google Scholar
  6. 6.
    Baron, R, Neff, L, Tran, VP, Nefussi, JR, Vignery, A 1986Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclastsAm J Pathol122363378PubMedGoogle Scholar
  7. 7.
    Ibbotson, KJ, Roodman, GD, McManus, LM, Mundy, GR 1984Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cellsJ Cell Biol99471480PubMedCrossRefGoogle Scholar
  8. 8.
    Osdoby, P, Martini, MC, Caplan, AI 1982Isolated osteoclasts and their presumed progenitor cells, the monocyte, in cultureJ Exp Zool224331344PubMedCrossRefGoogle Scholar
  9. 9.
    Lacey, DL, Timms, E, Tan, HL, Kelley, MJ, Dunstan, CR, Burgess, T, Elliott, R, Colombero, A, Elliott, G, Scully, S, Hsu, H, Sullivan, J, Hawkins, N, Davy, E, Capparelli, C, Eli, A, Qian, YX, Kaufman, S, Sarosi, I, Shalhoub, V, Senaldi, G, Guo, J, Delaney, J, Boyle, WJ 1998Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activationCell93165176PubMedCrossRefGoogle Scholar
  10. 10.
    Roodman, GD 1999Cell biology of the osteoclastExp Hematol2712291241PubMedCrossRefGoogle Scholar
  11. 11.
    Teitelbaum, SL 2000Bone resorption by osteoclastsScience28915041508PubMedCrossRefGoogle Scholar
  12. 12.
    Lakkakorpi, PT, Vaananen, HK 1996Cytoskeletal changes in osteoclasts during the resorption cycleMicrosc Res Tech33171181PubMedCrossRefGoogle Scholar
  13. 13.
    Vaananen, HK, Horton, M 1995The osteoclast clear zone is a specialized cell-extracellular matrix adhesion structureJ Cell Sci10827292732PubMedGoogle Scholar
  14. 14.
    Lakkakorpi, P, Tuukkanen, J, Hentunen, T, Jarvelin, K, Vaananen, K 1989Organization of osteoclast microfilaments during the attachment to bone surface in vitroJ Bone Miner Res4817825PubMedCrossRefGoogle Scholar
  15. 15.
    Väänänen, K 1996

    Osteoclast function: biology and mechanisms

    Bilezikian, JPRaisz, LGRodan, GA eds. Principles of Bone BiologyAcademic PressSan Diego103113
    Google Scholar
  16. 16.
    Baron, R, Neff, L, Louvard, D, Courtoy, PJ 1985Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled borderJ Cell Biol10122102222PubMedCrossRefGoogle Scholar
  17. 17.
    Li, YP, Chen, W, Liang, Y, Li, E, Stashenko, P 1999Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidificationNat Genet23447451PubMedCrossRefGoogle Scholar
  18. 18.
    Taranta, A, Migliaccio, S, Recchia, I, Caniglia, M, Luciani, M, De Rossi, G, Dionisi-Vici, C, Pinto, RM, Francalanci, P, Boldrini, R, Lanino, E, Dini, G, Morreale, G, Ralston, SH, Villa, A, Vezzoni, P, Del Principe, D, Cassiani, F, Palumbo, G, Teti, A 2003Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosisAm J Pathol1625768PubMedGoogle Scholar
  19. 19.
    Sundquist, K, Lakkakorpi, P, Wallmark, B, Vaananen, K 1990Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorptionBiochem Biophys Res Commun168309313PubMedCrossRefGoogle Scholar
  20. 20.
    al Awqati, Q 1995Chloride channels of intracellular organellesCurr Opin Cell Biol7504508PubMedCrossRefGoogle Scholar
  21. 21.
    Schlesinger, PH, Blair, HC, Teitelbaum, SL, Edwards, JC 1997Characterization of the osteoclast ruffled border chloride channel and its role in bone resorptionJ Biol Chem2721863618643PubMedCrossRefGoogle Scholar
  22. 22.
    Toyomura, T, Oka, T, Yamaguchi, C, Wada, Y, Futai, M 2000Three subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential expression of the a3 isoform during osteoclast differentiationJ Biol Chem27587608765PubMedCrossRefGoogle Scholar
  23. 23.
    Manolson, MF, Yu, H, Chen, W, Yao, Y, Li, K, Lees, RL, Heersche, JN 2003The a3 isoform of the 100-kDa V-ATPase subunit is highly but differentially expressed in large (> or = 10 nuclei) and small (< or = nuclei) osteoclastsJ Biol Chem2784927149278PubMedCrossRefGoogle Scholar
  24. 24.
    Kornak, U, Schulz, A, Friedrich, W, Uhlhaas, S, Kremens, B, Voit, T, Hasan, C, Bode, U, Jentsch, TJ, Kubisch, C 2000Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosisHum Mol Genet920592063PubMedCrossRefGoogle Scholar
  25. 25.
    Kornak, U, Kasper, D, Bosl, MR, Kaiser, E, Schweizer, M, Schulz, A, Friedrich, W, Delling, G, Jentsch, TJ 2001Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and manCell104205215PubMedCrossRefGoogle Scholar
  26. 26.
    Vaananen, HK, Zhao, H, Mulari, M, Halleen, JM 2000The cell biology of osteoclast functionJ Cell Sci113377381PubMedGoogle Scholar
  27. 27.
    Gowen, M, Lazner, F, Dodds, R, Kapadia, R, Feild, J, Tavaria, M, Bertoncello, I, Drake, F, Zavarselk, S, Tellis, I, Hertzog, P, Debouck, C, Kola, I 1999Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralizationJ Bone Miner Res1416541663PubMedCrossRefGoogle Scholar
  28. 28.
    Littlewood-Evans, A, Kokubo, T, Ishibashi, O, Inaoka, T, Wlodarski, B, Gallagher, JA, Bilbe, G 1997Localization of cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistryBone (NY)208186Google Scholar
  29. 29.
    Nishi, Y, Atley, L, Eyre, DE, Edelson, JG, Superti-Furga, A, Yasuda, T, Desnick, RJ, Gelb, BD 1999Determination of bone markers in pycnodysostosis: effects of cathepsin K deficiency on bone matrix degradationJ Bone Miner Res1419021908PubMedCrossRefGoogle Scholar
  30. 30.
    Saftig, P, Hunziker, E, Wehmeyer, O, Jones, S, Boyde, A, Rommerskirch, W, Moritz, JD, Schu, P, von Figura, K 1998Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient miceProc Natl Acad Sci U S A951345313458PubMedCrossRefGoogle Scholar
  31. 31.
    Stroup, GB, Lark, MW, Veber, DF, Bhattacharyya, A, Blake, S, Dare, LC, Erhard, KF, Hoffman, SJ, James, IE, Marquis, RW, Ru, Y, Vasko-Moser, JA, Smith, BR, Tomaszek, T, Gowen, M 2001Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primateJ Bone Miner Res1617391746PubMedCrossRefGoogle Scholar
  32. 32.
    Everts, V, Aronson, DC, Beertsen, W 1985Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosisCalcif Tissue Int372531PubMedGoogle Scholar
  33. 33.
    Motyckova, G, Fisher, DE 2002Pycnodysostosis: role and regulation of cathepsin K in osteoclast function and human diseaseCurr Mol Med2407421PubMedCrossRefGoogle Scholar
  34. 34.
    Garnero, P, Borel, O, Byrjalsen, I, Ferreras, M, Drake, FH, McQueney, MS, Foged, NT, Delmas, PD, Delaisse, JM 1998The collagenolytic activity of cathepsin K is unique among mammalian proteinasesJ Biol Chem2733234732352PubMedCrossRefGoogle Scholar
  35. 35.
    Boyle, WJ, Simonet, WS, Lacey, DL 2003Osteoclast differentiation and activationNature (Lond)423337342CrossRefGoogle Scholar
  36. 36.
    Manolagas, SC 2000Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosisEndocr Rev21115137PubMedCrossRefGoogle Scholar
  37. 37.
    Nicholson, GC, Moseley, JM, Sexton, PM, Mendelsohn, FA, Martin, TJ 1986Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterizationJ Clin Invest78355360PubMedCrossRefGoogle Scholar
  38. 38.
    Horton, M 1990Vitronectin receptor: tissue specific expression or adaptation to culture? IntJ Exp Pathol71741759Google Scholar
  39. 39.
    Davies, J, Warwick, J, Totty, N, Philp, R, Helfrich, M, Horton, M 1989The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptorJ Cell Biol10918171826PubMedCrossRefGoogle Scholar
  40. 40.
    Okada, Y, Naka, K, Kawamura, K, Matsumoto, T, Nakanishi, I, Fujimoto, N, Sato, H, Seiki, M 1995Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorptionLab Invest72311322PubMedGoogle Scholar
  41. 41.
    Massey, HM, Flanagan, AM 1999Human osteoclasts derive from CD14-positive monocytesBr J Haematol106167170PubMedCrossRefGoogle Scholar
  42. 42.
    Nicholson, GC, Malakellis, M, Collier, FM, Cameron, PU, Holloway, WR, Gough, TJ, Gregorio-King, C, Kirkland, MA, Myers, DE 2000Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL)Clin Sci (Lond)99133140CrossRefGoogle Scholar
  43. 43.
    Li, C, Wong, W 2003

    DNA-chip analyser (dChip)

    Parmigiani, GGarret, ESIrizarry, RZeger, SL eds. The Analysis of Gene Expression Data: Methods and SoftwareSpringerNew York120141
    CrossRefGoogle Scholar
  44. 44.
    Engsig, MT, Chen, QJ, Vu, TH, Pedersen, AC, Therkidsen, B, Lund, LR, Henriksen, K, Lenhard, T, Foged, NT, Werb, Z, Delaisse, JM 2000Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bonesJ Cell Biol151879889PubMedCrossRefGoogle Scholar
  45. 45.
    Henriksen, K, Gram, J, Schaller, S, Dahl, BH, Dziegiel, MH, Bollerslev, J, Karsdal, MA 2004Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type IIAm J Pathol16415371545PubMedGoogle Scholar
  46. 46.
    Schaller, S, Henriksen, K, Sveigaard, C, Heegaard, A, Hélix, N, Stahlhut, M, Ovejero, MC, Johansen, JV, Solberg, H, Andersen, TL, Hougaard, D, Shiøt, CB, Sørensen, BH, Lichtenberg, J, Christophersen, P, Foged, NT, Delaissé, J, Engsig, MT, Karsdal, MA 2004The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats without changing bone formationJ Bone Miner Res1911441153PubMedCrossRefGoogle Scholar
  47. 47.
    Henriksen, K, Karsdal, M, Delaisse, JM, Engsig, MT 2003RANKL and vascular endothelial growth factor (VEGF) induce osteoclast chemotaxis through an ERK1/2-dependent mechanismJ Biol Chem2784874548753PubMedCrossRefGoogle Scholar
  48. 48.
    Karsdal, MA, Hjorth, P, Henriksen, K, Kirkegaard, T, Nielsen, KL, Lou, H, Delaisse, JM, Foged, NT 2003Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expressionJ Biol Chem2784497544987PubMedCrossRefGoogle Scholar
  49. 49.
    Quinn, JM, Neale, S, Fujikawa, Y, McGee, JO, Athanasou, NA 1998Human osteoclast formation from blood monocytes, peritoneal macrophages, and bone marrow cellsCalcif Tissue Int62527531PubMedCrossRefGoogle Scholar
  50. 50.
    Shalhoub, V, Elliott, G, Chiu, L, Manoukian, R, Kelley, M, Hawkins, N, Davy, E, Shimamoto, G, Beck, J, Kaufman, SA, Van, G, Scully, S, Qi, M, Grisanti, M, Dunstan, C, Boyle, WJ, Lacey, DL 2000Characterization of osteoclast precursors in human bloodBr J Haematol111501512PubMedCrossRefGoogle Scholar
  51. 51.
    Blair, HC, Teitelbaum, SL, Ghiselli, R, Gluck, S 1989Osteoclastic bone resorption by a polarized vacuolar proton pumpScience245855857PubMedCrossRefGoogle Scholar
  52. 52.
    Silver, IA, Murrills, RJ, Etherington, DJ 1988Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclastsExp Cell Res175266276PubMedCrossRefGoogle Scholar
  53. 53.
    Yoshimori, T, Yamamoto, A, Moriyama, Y, Futai, M, Tashiro, Y 1991Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cellsJ Biol Chem2661770717712PubMedGoogle Scholar
  54. 54.
    Karsdal, MA, Henriksen, K, Sorensen, MG, Gram, J, Schaller, S, Dziegiel, MH, Heegaard, AM, Christophersen, P, Martin, TJ, Christiansen, C, Bollerslev, J 2005Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorptionAm J Pathol166467476PubMedGoogle Scholar
  55. 55.
    Bowman, EJ, Siebers, A, Altendorf, K 1988Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cellsProc Natl Acad Sci U S A8579727976PubMedCrossRefGoogle Scholar
  56. 56.
    Falgueyret, JP, Oballa, RM, Okamoto, O, Wesolowski, G, Aubin, Y, Rydzewski, RM, Prasit, P, Riendeau, D, Rodan, SB, Percival, MD 2001Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and LJ Med Chem4494104PubMedCrossRefGoogle Scholar
  57. 57.
    Hall, TJ, Higgins, W, Tardif, C, Chambers, TJ 1991A comparison of the effects of inhibitors of carbonic anhydrase on osteoclastic bone resorption and purified carbonic anhydrase isozyme IICalcif Tissue Int49328332PubMedGoogle Scholar
  58. 58.
    Kellinsalmi, M, Monkkonen, H, Monkkonen, J, Leskela, HV, Parikka, V, Hamalainen, M, Lehenkari, P 2005In vitro comparison of clodronate, pamidronate and zoledronic acid effects on rat osteoclasts and human stem cell-derived osteoblastsBasic Clin Pharmacol Toxicol97382391PubMedCrossRefGoogle Scholar
  59. 59.
    Garnero, P, Ferreras, M, Karsdal, MA, Nicamhlaoibh, R, Risteli, J, Borel, O, Qvist, P, Delmas, PD, Foged, NT, Delaisse, JM 2003The type I collagen fragments ICTP and CTX-I reveal distinct enzymatic pathways of bone collagen degradationJ Bone Miner Res18859867PubMedCrossRefGoogle Scholar
  60. 60.
    Henriksen, K, Sorensen, MG, Nielsen, RH, Gram, J, Schaller, S, Dziegiel, MH, Everts, V, Bollerslev, J, Karsdal, MA 2006Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidificationJ Bone Miner Res215866PubMedCrossRefGoogle Scholar
  61. 61.
    Udagawa, N, Takahashi, N, Akatsu, T, Tanaka, H, Sasaki, T, Nishihara, T, Koga, T, Martin, TJ, Suda, T 1990Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cellsProc Natl Acad Sci U S A8772607264PubMedCrossRefGoogle Scholar
  62. 62.
    Kotake, S, Udagawa, N, Hakoda, M, Mogi, M, Yano, K, Tsuda, E, Takahashi, K, Furuya, T, Ishiyama, S, Kim, KJ, Saito, S, Nishikawa, T, Takahashi, N, Togari, A, Tomatsu, T, Suda, T, Kamatani, N 2001Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patientsArthritis Rheum4410031012PubMedCrossRefGoogle Scholar
  63. 63.
    Weitzmann, MN, Cenci, S, Rifas, L, Haug, J, Dipersio, J, Pacifici, R 2001T cell activation induces human osteoclast formation via receptor activator of nuclear factor kappaB ligand-dependent and -independent mechanismsJ Bone Miner Res16328337PubMedCrossRefGoogle Scholar
  64. 64.
    Horwood, NJ, Kartsogiannis, V, Quinn, JM, Romas, E, Martin, TJ, Gillespie, MT 1999Activated T lymphocytes support osteoclast formation in vitroBiochem Biophys Res Commun265144150PubMedCrossRefGoogle Scholar
  65. 65.
    Teitelbaum, SL, Ross, FP 2003Genetic regulation of osteoclast development and functionNat Rev Genet4638649PubMedCrossRefGoogle Scholar
  66. 66.
    Meredith, JE,Jr, Winitz, S, Lewis, JM, Hess, S, Ren, XD, Renshaw, MW, Schwartz, MA 1996The regulation of growth and intracellular signaling by integrinsEndocr Rev17207220PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Tokyo 2007

Authors and Affiliations

  • Mette Grøndahl Sørensen
    • 1
  • Kim Henriksen
    • 1
  • Sophie Schaller
    • 1
  • Dennis Bang Henriksen
    • 2
  • Finn Cilius Nielsen
    • 3
  • Morten Hanefeld Dziegiel
    • 4
  • Morten Asser Karsdal
    • 1
  1. 1.Nordic Bioscience A/SHerlevDenmark
  2. 2.Sanos Bioscience A/SRødovreDenmark
  3. 3.Department of Clinical BiochemistryUniversity Hospital of CopenhagenCopenhagenDenmark
  4. 4.HS BlodbankUniversity Hospital of CopenhagenCopenhagenDenmark

Personalised recommendations