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Isolation of Mouse Mammary Epithelial Subpopulations: A Comparison of Leading Methods

  • Matthew J. SmalleyEmail author
  • Howard Kendrick
  • Julie M. Sheridan
  • Joseph L. Regan
  • Michael D. Prater
  • Geoffrey J. Lindeman
  • Christine J. Watson
  • Jane E. Visvader
  • John Stingl
Article

Abstract

Isolation of mammary epithelial subpopulations, including stem and progenitor cells, has become a standard technique in recent years. However, a number of methods and approaches for this have developed and the relative benefits of the different approaches, and the reason for their development, have not always been clear. Here, three of the leading laboratories working on the separation of mammary cell subpopulations have summarised their methods, highlighted their differences and similarities and also discussed the reasoning behind the approaches they have taken. This article will assist workers establishing mammary cell separation protocols in their laboratories to make informed choices about the methods they should use.

Keywords

Mammary Stem Progenitor Epithelial Sorting Isolation 

Abbreviations

CFCs

Colony Forming Cells

DAPI

4',6-diamidino-2-phenylindole

DMEM

Dulbecco’s Modified Eagle’s Medium

DPBS

Dulbecco’s Phosphate Buffered Saline

EDTA

ethylenediaminetetraacetic acid

EGTA

ethylene glycol tetraacetic acid

F12

Ham’s F12 Medium

FCS

Foetal Calf Serum

FMO

Fluorescence Minus One

FSC-H

Forward Scatter – Height

FSC-W

Forward Scatter – Width

HBSS

Hank’s Balanced Salt Solution

HEPES

hydroxyethyl piperazineethanesulfonic acid

MaSCs

Mammary Stem Cells

MRUs

Mammary Repopulating Units

SSC-H

Side Scatter – Height

SSC-W

Side Scatter – Width

Notes

Acknowledgments

MJS and HK are supported by Cardiff University and would like to thank Kelly Soady for her assistance in protocol development. JLR is supported by Breakthrough Breast Cancer. JEV, GJL and JMS are supported by the National Health and Medical Research Council and Victorian Breast Cancer Research Consortium. JS would like to acknowledge the support of The University of Cambridge, The Breast Cancer Campaign, Hutchison Whampoa Limited and Cancer Research UK. CJW would to acknowledge the support of the Breast Cancer Campaign.

References

  1. 1.
    Alvi AJ, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco MM, et al. Functional and molecular characterisation of mammary side population cells. Breast Canc Res. 2003;5(1):R1–8.CrossRefGoogle Scholar
  2. 2.
    Asselin-Labat ML, Shackleton M, Stingl J, Vaillant F, Forrest NC, Eaves CJ, et al. Steroid hormone receptor status of mouse mammary stem cells. J Natl Cancer Inst. 2006;98(14):1011–4.PubMedCrossRefGoogle Scholar
  3. 3.
    Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, et al. Generation of a functional mammary gland from a single stem cell. Nature. 2006;439(7072):84–8.PubMedCrossRefGoogle Scholar
  4. 4.
    Sleeman KE, Kendrick H, Ashworth A, Isacke CM, Smalley MJ. CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Canc Res. 2006;8(1):R7.CrossRefGoogle Scholar
  5. 5.
    Sleeman KE, Kendrick H, Robertson D, Isacke CM, Ashworth A, Smalley MJ. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J Cell Biol. 2007;176(1):19–26.PubMedCrossRefGoogle Scholar
  6. 6.
    Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, et al. Purification and unique properties of mammary epithelial stem cells. Nature. 2006;439(7079):993–7.PubMedGoogle Scholar
  7. 7.
    Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol. 2002;245(1):42–56.PubMedCrossRefGoogle Scholar
  8. 8.
    Dundas SR, Ormerod MG, Gusterson BA, O’Hare MJ. Characterization of luminal and basal cells flow-sorted from the adult rat mammary parenchyma. J Cell Sci. 1991;100(Pt 3):459–71.PubMedGoogle Scholar
  9. 9.
    O’Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA. Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting. Differentiation. 1991;46(3):209–21.PubMedCrossRefGoogle Scholar
  10. 10.
    Smalley MJ. Isolation, culture and analysis of mouse mammary epithelial cells. In: Ward A, Tosh D, editors. Methods Mol Biol: Springer; 2010. p. 139–70.Google Scholar
  11. 11.
    Prater M, Shehata M, Watson CJ, Stingl J. Enzymatic dissociation, flow cytometric analysis and culture of normal mouse mammary tissue. Methods Mol Biol: Springer; In press.Google Scholar
  12. 12.
    Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G, Rock J, et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature. 2011;479(7372):189–93.PubMedCrossRefGoogle Scholar
  13. 13.
    Molyneux G, Geyer FC, Magnay FA, McCarthy A, Kendrick H, Natrajan R, et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell. 2010;7(3):403–17.PubMedCrossRefGoogle Scholar
  14. 14.
    Boulanger CA, Wagner KU, Smith GH. Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-beta1 expression. Oncogene. 2005;24(4):552–60.PubMedCrossRefGoogle Scholar
  15. 15.
    Moraes RC, Zhang X, Harrington N, Fung JY, Wu MF, Hilsenbeck SG, et al. Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia. Development. 2007;134(6):1231–42.PubMedCrossRefGoogle Scholar
  16. 16.
    Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell. 2009;138(6):1083–95.PubMedCrossRefGoogle Scholar
  17. 17.
    Regan JL, Kendrick H, Magnay FA, Vafaizadeh V, Groner B, Smalley MJ. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer. Oncogene. 2011. doi: 10.1038/onc.2011.289.
  18. 18.
    Britt KL, Kendrick H, Regan JL, Molyneux G, Magnay FA, Ashworth A, et al. Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells. Breast Canc Res. 2009;11(2):R20.CrossRefGoogle Scholar
  19. 19.
    Regan JL, Kendrick H, Magnay FA, Vafaizadeh V, Groner B, Smalley MJ. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer. Oncogene. 2012;31(7):869–83.PubMedCrossRefGoogle Scholar
  20. 20.
    Shehata M, Teschendorff A, Sharp G, Novcic N, Russell IA, Avril S, et al. Phenotypic and functional characterization of the luminal cell hierarchy of the mammary gland. Submitted.Google Scholar
  21. 21.
    Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, et al. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol. 2007;9(2):201–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Vaillant F, Lindeman GJ, Visvader JE. Jekyll or Hyde: does Matrigel provide a more or less physiological environment in mammary repopulating assays? Breast Canc Res. 2011;13(3):108.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Matthew J. Smalley
    • 1
    Email author
  • Howard Kendrick
    • 1
  • Julie M. Sheridan
    • 2
    • 3
  • Joseph L. Regan
    • 4
  • Michael D. Prater
    • 5
  • Geoffrey J. Lindeman
    • 2
    • 7
  • Christine J. Watson
    • 6
  • Jane E. Visvader
    • 2
    • 3
  • John Stingl
    • 5
  1. 1.European Cancer Stem Cell Research Institute, Cardiff School of BiosciencesCardiff UniversityCardiffUK
  2. 2.Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia
  3. 3.Department of Medical BiologyUniversity of MelbourneParkvilleAustralia
  4. 4.Breakthrough Breast Cancer Research CentreThe Institute of Cancer ResearchLondonUK
  5. 5.Cambridge Research InstituteCancer Research UKCambridgeUK
  6. 6.Department of PathologyUniversity of CambridgeCambridgeUK
  7. 7.Department of Medicine, Royal Melbourne HospitalUniversity of MelbourneParkvilleAustralia

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