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The Use of Multiparameter Flow Cytometry and Cell Sorting to Characterize Native Human Bone Marrow Mesenchymal Stem Cells (MSC)

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Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1235))

Abstract

This chapter describes a method for identification, phenotypic analysis, and cell sorting of rare mesenchymal stem cells (MSCs) from human bone marrow (BM) aspirates. The native BM MSC population is identified based on the CD45−/lowCD271+ phenotype. The method consists of three related procedures: Procedure 1 involves a microbead-based pre-enrichment step. Two other procedures describe direct flow cytometric analysis of MSCs following the isolation of the mononuclear cell (MNC) fraction (Procedure 2) or more rapidly, following a simple ammonium chloride-based red cell lysis (Procedure 3). Recently described multi-lineage transcript expression in the CD45−/lowCD271+ cells suggests that the native BM MSC fraction could be further subdivided into functionally distinct subpopulations. The present protocols are hoped to help MSC biologists to enter this exciting field of research and to take it forward towards a better understanding of MSC biology in vivo.

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References

  1. Friedenstein AJ (1976) Precursor cells of mechanocytes. Int Rev Cytol 47:327–359. doi:10.1016/s0074-7696(08)60092-3

    Article  CAS  PubMed  Google Scholar 

  2. Friedenstein AJ (1995) Marrow stromal fibroblasts. Calcified Tissue Int 56:S17

    CAS  Google Scholar 

  3. Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147

    Article  CAS  PubMed  Google Scholar 

  4. Simmons PJ, Torok SB (1991) Identification of stromal cell precursors in human bone-marrow by a novel monoclonal-antibody, Stro-1. Blood 78:55–62

    CAS  PubMed  Google Scholar 

  5. Stewart K, Monk P, Walsh S, Jefferiss CM et al (2003) STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro. Cell Tissue Res 313:281–290

    Article  CAS  PubMed  Google Scholar 

  6. Quirici N, Soligo D, Bossolasco P et al (2002) Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 30:783–791

    Article  CAS  PubMed  Google Scholar 

  7. Jones EA, Kinsey SE, English A et al (2002) Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum 46:3349–3360

    Article  PubMed  Google Scholar 

  8. Harichandan A, Buehring H-J (2011) Prospective isolation of human MSC. Best Pract Res Clin Haematol 24:25–36. doi:10.1016/j.beha.2011.01.001

    Article  CAS  PubMed  Google Scholar 

  9. Jones EA, English A, Kinsey SE et al (2006) Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow. Cytometry B Clin Cytom 70:391–399

    Article  PubMed  Google Scholar 

  10. Tormin A, Li O, Brune JC et al (2011) CD146 expression on primary non-hematopoietic bone marrow stem cells correlates to in situ localization. Blood 117:5067–5077

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Qian H, Le Blanc K, Sigvardsson M (2012) Primary mesenchymal stem and progenitor cells from bone marrow lack expression of CD44. J Biol Chem. doi:10.1074/jbc.M112.339622

    Google Scholar 

  12. Jones E, English A, Churchman SM (2010) Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells. Arthritis Rheum 62:1944–1954

    Article  CAS  PubMed  Google Scholar 

  13. Churchman SM, Ponchel F, Boxall SA (2012) Transcriptional profile of native CD271+ multipotential stromal cells: Evidence for multiple fates, with prominent osteogenic and Wnt pathway signaling activity. Arthritis Rheum 64:2632–2643. doi:10.1002/art.34434

    Article  CAS  PubMed  Google Scholar 

  14. Battula VL, Treml S, Bareiss PM et al (2009) Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1. Haematologica 94:173–184. doi:10.3324/haematol.13740

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Cox G, Boxall SA, Giannoudis PV et al (2012) High abundance of CD271(+) multipotential stromal cells (MSCs) in intramedullary cavities of long bones. Bone 50:510–517

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Cuthbert R, Boxall SA, Tan HB et al (2012) Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use. Cytotherapy 14:431–440. doi:10.3109/14653249.2011.651533

    Article  CAS  PubMed  Google Scholar 

  17. Stolzing A, Jones E, McGonagle D et al (2008) Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 129:163–173. doi:10.1016/j.mad.2007.12.002

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Elena Jones .

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Boxall, S., Jones, E. (2015). The Use of Multiparameter Flow Cytometry and Cell Sorting to Characterize Native Human Bone Marrow Mesenchymal Stem Cells (MSC). In: Rich, I. (eds) Stem Cell Protocols. Methods in Molecular Biology, vol 1235. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1785-3_11

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  • DOI: https://doi.org/10.1007/978-1-4939-1785-3_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1784-6

  • Online ISBN: 978-1-4939-1785-3

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