Isolation and Characterization of Mouse Side Population Cells

  • Aysegul V. Ergen
  • Mira Jeong
  • Kuanyin K. Lin
  • Grant A. Challen
  • Margaret A. Goodell
Part of the Methods in Molecular Biology book series (MIMB, volume 946)


The side population (SP) is a subpopulation of mouse bone marrow cells highly enriched for hematopoietic stem cell activity. The SP is identified using flow cytometry as a minor population that efficiently effluxes the DNA-binding dye Hoechst 33342 relative to the rest of the bone marrow. Phenotypic and functionally analysis has established SP cells as highly phenotypically homogeneous and functional active. In this chapter we describe a detailed protocol for the purification of murine bone marrow SP cells based on Hoechst dye efflux in combination with the presence of HSC surface markers.

Key words

SP Hoechst 33342 Dye efflux Hematopoietic stem cell Purification Side population 



The authors are supported by grants from the NIH, the Ellison Foundation, and the American Heart Association. M.J. was supported by University of Science and Technology though UST Post-Doc Research Program. G.A.C. is a scholar of the American Society of Hematology.


  1. 1.
    Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183(4):1797–1806PubMedCrossRefGoogle Scholar
  2. 2.
    Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G et al (1997) Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3(12):1337–1345PubMedCrossRefGoogle Scholar
  3. 3.
    Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U et al (2004) A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 101(39):14228–14233PubMedCrossRefGoogle Scholar
  4. 4.
    Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24(1):3–12PubMedCrossRefGoogle Scholar
  5. 5.
    Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 65(14):6207–6219PubMedCrossRefGoogle Scholar
  6. 6.
    Dean M (2009) ABC transporters, drug resistance, and cancer stem cells. J Mammary Gland Biol Neoplasia 14(1):3–9PubMedCrossRefGoogle Scholar
  7. 7.
    Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ et al (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7(9):1028–1034PubMedCrossRefGoogle Scholar
  8. 8.
    Moserle L, Indraccolo S, Ghisi M, Frasson C, Fortunato E, Canevari S et al (2008) The side population of ovarian cancer cells is a primary target of IFN-alpha antitumor effects. Cancer Res 68(14):5658–5668PubMedCrossRefGoogle Scholar
  9. 9.
    Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP (2002) Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 99(19):12339–12344PubMedCrossRefGoogle Scholar
  10. 10.
    Scharenberg CW, Harkey MA, Torok-Storb B (2002) The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 99(2):507–512PubMedCrossRefGoogle Scholar
  11. 11.
    Venezia TA, Merchant AA, Ramos CA, Whitehouse NL, Young AS, Shaw CA et al (2004) Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2(10):e301PubMedCrossRefGoogle Scholar
  12. 12.
    Morrison SJ, Weissman IL (1994) The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1(8):661–673PubMedCrossRefGoogle Scholar
  13. 13.
    Osawa M, Hanada K, Hamada H, Nakauchi H (1996) Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273(5272):242–245PubMedCrossRefGoogle Scholar
  14. 14.
    Christensen JL, Weissman IL (2001) Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA 98(25):14541–14546PubMedCrossRefGoogle Scholar
  15. 15.
    Hirao A, Arai F, Suda T (2004) Regulation of cell cycle in hematopoietic stem cells by the niche. Cell Cycle 3(12):1481–1483PubMedCrossRefGoogle Scholar
  16. 16.
    Chen CZ, Li M, de Graaf D, Monti S, Gottgens B, Sanchez MJ et al (2002) Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells. Proc Natl Acad Sci USA 99(24):15468–15473PubMedCrossRefGoogle Scholar
  17. 17.
    Balazs AB, Fabian AJ, Esmon CT, Mulligan RC (2006) Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 107(6):2317–2321PubMedCrossRefGoogle Scholar
  18. 18.
    Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121(7):1109–1121PubMedCrossRefGoogle Scholar
  19. 19.
    Challen GA, Boles NC, Chambers SM, Goodell MA (2010) Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1. Cell Stem Cell 6(3):265–278PubMedCrossRefGoogle Scholar
  20. 20.
    Kent DG, Copley MR, Benz C, Wohrer S, Dykstra BJ, Ma E et al (2009) Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood 113(25):6342–6350PubMedCrossRefGoogle Scholar
  21. 21.
    Morita Y, Ema H, Nakauchi H (2010) Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J Exp Med 207(6):1173–1182PubMedCrossRefGoogle Scholar
  22. 22.
    Beerman I, Bhattacharya D, Zandi S, Sigvardsson M, Weissman IL, Bryder D et al (2010) Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc Natl Acad Sci USA 107(12):5465–5470PubMedCrossRefGoogle Scholar
  23. 23.
    Simpson C, Pearce DJ, Bonnet D, Davies D (2006) Out of the blue: a comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources. J Immunol Methods 310(1–2):171–181PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Aysegul V. Ergen
    • 1
  • Mira Jeong
    • 1
    • 2
  • Kuanyin K. Lin
    • 1
  • Grant A. Challen
    • 1
  • Margaret A. Goodell
    • 3
  1. 1.The Center for Cell and Gene Therapy, Baylor College of MedicineHoustonUSA
  2. 2.University of Science and TechnologyDaejeonSouth Korea
  3. 3.Stem Cells and Regenerative Medicine Center, Baylor College of MedicineHoustonUSA

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