Advertisement

Cytotechnology

, 58:163 | Cite as

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

  • A. Dumas
  • M. A. Le Drévo
  • M. F. Moreau
  • C. Guillet
  • M. F. Baslé
  • D. Chappard
Original Research

Abstract

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b cells increased the number of AlkP+ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.

Keywords

Osteoprogenitor Immunodepletion CD11b Alkaline phosphatase mRNA profiles 

Abbreviations

AlkP

Alkaline phosphatase

MSC

Mesenchymal stem cells

FACS

Fluorescent activating cell sorting

CAM

Cell adhesion molecule

Notes

Acknowledgments

This work was made possible by grants from Contrat de Plan Etat—Région “Pays de la Loire”, INSERM and the Bioregos Program. We thank Laurence Preisser and Marie-Hélène Guilleux from SCCAN Service Commun de Cytométrie et d’Analyses Nucléotidiques, IFR132 for their assistance with RT-PCR quantification.

References

  1. Atmani H, Chappard D, Baslé MF (2003) Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol. J Cell Biochem 89:364–372. doi: 10.1002/jcb.10507 CrossRefGoogle Scholar
  2. Aubin JE (1998) Bone stem cells. J Cell Biochem Suppl 30–31:73–82. doi: 10.1002/(SICI)1097-4644(1998)72:30/31+<73::AID-JCB11>3.0.CO;2-L CrossRefGoogle Scholar
  3. Aubin JE (1999) Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell–cell interactions in osteoblast differentiation. J Cell Biochem 72:396–410. doi: 10.1002/(SICI)1097-4644(19990301)72:3<396::AID-JCB9>3.0.CO;2-6 CrossRefGoogle Scholar
  4. Baddoo M, Hill K, Wilkinson R et al (2003) Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem 89:1235–1249. doi: 10.1002/jcb.10594 CrossRefGoogle Scholar
  5. Bianco P, Gehron Robey P (2000) Marrow stromal stem cells. J Clin Invest 105:1663–1668. doi: 10.1172/JCI10413 CrossRefGoogle Scholar
  6. Bianco P, Riminucci M, Gronthos S et al (2001) Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180–192. doi: 10.1634/stemcells.19-3-180 CrossRefGoogle Scholar
  7. Canalis E, Delany AM (2002) Mechanisms of glucocorticoid action in bone. Ann N Y Acad Sci 966:73–81CrossRefGoogle Scholar
  8. Dennis JE, Esterly K, Awadallah A et al (2007) Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration. Stem Cells 25:2575–2582. doi: 10.1634/stemcells.2007-0204 CrossRefGoogle Scholar
  9. Friedenstein AJ, Chailakhyan RK, Gerasimov UV (1987) Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20:263–272Google Scholar
  10. Gindraux F, Selmani Z, Obert L et al (2007) Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule. Cell Tissue Res 327:471–483. doi: 10.1007/s00441-006-0292-3 CrossRefGoogle Scholar
  11. Hebert E (2000) Endogenous lectins as cell surface transducers. Biosci Rep 20:213–237. doi: 10.1023/A:1026484722248 CrossRefGoogle Scholar
  12. Hernigou P, Poignard A, Beaujean F et al (2005) Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 87:1430–1437. doi: 10.2106/JBJS.D.02215 CrossRefGoogle Scholar
  13. Kadiyala S, Young RG, Thiede MA et al (1997) Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 6:125–134. doi: 10.1016/S0963-6897(96)00279-5 CrossRefGoogle Scholar
  14. Kassem M (2004) Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells 6:369–374. doi: 10.1089/clo.2004.6.369 CrossRefGoogle Scholar
  15. Komori T (2006) Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 99:1233–1239. doi: 10.1002/jcb.20958 CrossRefGoogle Scholar
  16. Kruyt MC, Dhert WJ, Oner C et al (2004) Optimization of bone-tissue engineering in goats. J Biomed Mater Res 69B:113–120. doi: 10.1002/jbm.b.10073 CrossRefGoogle Scholar
  17. Marom R, Shur I, Solomon R et al (2005) Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells. J Cell Physiol 202:41–48. doi: 10.1002/jcp.20109 CrossRefGoogle Scholar
  18. Muschler GF, Nakamoto C, Griffith LG (2004) Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg Am 86-A:1541–1558Google Scholar
  19. Ohgushi H, Caplan AI (1999) Stem cell technology and bioceramics: from cell to gene engineering. J Biomed Mater Res 48:913–927. doi: 10.1002/(SICI)1097-4636(1999)48:6<913::AID-JBM22>3.0.CO;2-0 CrossRefGoogle Scholar
  20. Owen M, Friedenstein AJ (1988) Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 136:42–60Google Scholar
  21. Owen TA, Aronow M, Shalhoub V et al (1990) Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 143:420–430. doi: 10.1002/jcp.1041430304 CrossRefGoogle Scholar
  22. Peister A, Mellad JA, Larson BL et al (2004) Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103:1662–1668. doi: 10.1182/blood-2003-09-3070 CrossRefGoogle Scholar
  23. Pereira RM, Delany AM, Canalis E (2001) Cortisol inhibits the differentiation and apoptosis of osteoblasts in culture. Bone 28:484–490. doi: 10.1016/S8756-3282(01)00422-7 CrossRefGoogle Scholar
  24. Phinney DG, Kopen G, Isaacson RL et al (1999) Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 72:570–585. doi: 10.1002/(SICI)1097-4644(19990315)72:4<570::AID-JCB12>3.0.CO;2-W CrossRefGoogle Scholar
  25. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74. doi: 10.1126/science.276.5309.71 CrossRefGoogle Scholar
  26. Rider DA, Nalathamby T, Nurcombe V et al (2007) Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells. J Mol Histol 38:449–458. doi: 10.1007/s10735-007-9128-z CrossRefGoogle Scholar
  27. Ringe J, Kaps C, Burmester GR et al (2002) Stem cells for regenerative medicine: advances in the engineering of tissues and organs. Naturwissenschaften 89:338–351. doi: 10.1007/s00114-002-0344-9 CrossRefGoogle Scholar
  28. Seshi B, Kumar S, Sellers D (2000) Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages. Blood Cells Mol Dis 26:234–246. doi: 10.1006/bcmd.2000.0301 CrossRefGoogle Scholar
  29. Shiota M, Heike T, Haruyama M et al (2007) Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties. Exp Cell Res 313:1008–1023. doi: 10.1016/j.yexcr.2006.12.017 CrossRefGoogle Scholar
  30. Shur I, Zilberman M, Benayahu D et al (2005) Adhesion molecule expression by osteogenic cells cultured on various biodegradable scaffolds. J Biomed Mater Res A 75:870–876. doi: 10.1002/jbm.a.30507 Google Scholar
  31. Stewart M, Thiel M, Hogg N (1995) Leukocyte integrins. Curr Opin Cell Biol 7:690–696. doi: 10.1016/0955-0674(95)80111-1 CrossRefGoogle Scholar
  32. Tropel P, Noel D, Platet N et al (2004) Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res 295:395–406. doi: 10.1016/j.yexcr.2003.12.030 CrossRefGoogle Scholar
  33. Turksen K, Aubin JE (1991) Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J Cell Biol 114:373–384. doi: 10.1083/jcb.114.2.373 CrossRefGoogle Scholar
  34. Van Vlasselaer P, Falla N, Snoeck H et al (1994) Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin. Blood 84:753–763Google Scholar
  35. Werntz JR, Lane JM, Burstein AH et al (1996) Qualitative and quantitative analysis of orthotopic bone regeneration by marrow. J Orthop Res 14:85–93. doi: 10.1002/jor.1100140115 CrossRefGoogle Scholar
  36. Yoshikawa T, Ohgushi H, Nakajima H et al (2000) In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution. Transplantation 69:128–134. doi: 10.1097/00007890-200001150-00022 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • A. Dumas
    • 1
  • M. A. Le Drévo
    • 1
  • M. F. Moreau
    • 1
  • C. Guillet
    • 2
  • M. F. Baslé
    • 1
  • D. Chappard
    • 1
  1. 1.INSERM, U922 “Remodelage osseux et biomatériaux”, LHEA—Faculté de MédecineAngers CedexFrance
  2. 2.Service Commun de cytométrie et d’analyse nucléotidique (SCCAN)Angers Cedex 9France

Personalised recommendations