Stem/Progenitor Cells in Murine Mammary Gland: Isolation and Functional Characterization

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 879)

Abstract

The presence of adult functional mammary epithelial stem/progenitor cells in mammary gland and recent identification of enriched fraction of transplantable mammary stem cells (MaSCs) through specific cell surface markers have revolutionized the study to delineate the role of mammary stem/progenitor functions in both mammary gland development and mammary tumorigenesis. In this chapter, we have described detailed methods of isolation of mammary epithelial cells from murine mammary glands, enrichment of stem/progenitor fractions by fluorescence-activated cell sorting (FACS), in vitro mammosphere culture, and differentiation assays in two- and three-dimensional culture. We have also described the detailed protocol of in vivo cleared mammary fat pad transplantation assay for the assessment of the MaSC repopulating activity, which indicates their multilineage differentiation and self-renewal potential in vivo and is considered the “gold standard” assay of functional stem cells.

Key words

Mammary stem cells Mammary progenitor cells Mammary glands Cleared mammary fat pad Mammosphere Mice Fluorescence-activated cell sorting 

Notes

Acknowledgments

This work was supported in part by NIH grants R01CA079683, R01CA075253 (L-ZS), and Shelby Rae Tangg foundation (AB). We thank the Flow Cytometry Shared Resource Facility and department of Pathology of UT Health Science Center at San Antonio for their assistance in analysis and interpretation of the results.

References

  1. 1.
    Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE (2006) Generation of a functional mammary gland from a single stem cell. Nature 439:84–88PubMedCrossRefGoogle Scholar
  2. 2.
    Polyak K, Shipitsin M, Campbell-Marrotta L, Bloushtain-Qimron N, Park SY (2009) Breast tumor heterogeneity: causes and consequences. Breast Cancer Res 11(suppl 1):S18CrossRefGoogle Scholar
  3. 3.
    Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Brooks M, Reinhardt F, Su Y, Polyak K, Arendt LM, Kuperwasser C, Bierie B, Weinberg RA (2011) Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci USA 108(19):7950–7955PubMedCrossRefGoogle Scholar
  4. 4.
    Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HI, Eaves CJ (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997PubMedGoogle Scholar
  5. 5.
    Stingl J (2009) Detection and analysis of mammary gland stem cells. J Pathol 217:229–241PubMedCrossRefGoogle Scholar
  6. 6.
    Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270PubMedCrossRefGoogle Scholar
  7. 7.
    Medina D (2010) Of mice and women: a short history of mouse mammary cancer research with an emphasis on the paradigms inspired by the transplantation method. Cold Spring Harb Perspect Biol 2:a004523PubMedCrossRefGoogle Scholar
  8. 8.
    Medina D (1996) The mammary gland: a unique organ for the study of development and tumorigenesis. J Mammary Gland Biol Neoplasia 1:5–19PubMedCrossRefGoogle Scholar
  9. 9.
    Daniel CW, DeOme KB (1965) Growth of mouse mammary glands in vivo after monolayer culture. Science 149:634–636PubMedCrossRefGoogle Scholar
  10. 10.
    DeOme KB, Faulkin LJ Jr, Bern HA, Blair PB (1959) Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19:515–520PubMedGoogle Scholar
  11. 11.
    Young LJ, Medina D, DeOme KB, Daniel CW (1971) The influence of host and tissue age on life span and growth rate of serially transplanted mouse mammary gland. Exp Gerontol 6:49–56PubMedCrossRefGoogle Scholar
  12. 12.
    Stingl J, Eaves CJ, Kuusk U, Emerman JT (1998) Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation 63:201–213PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang M, Behbod F, Atkinson RL, Landis MD, Kittrell F, Edwards D, Medina D, Tsimelzon A, Hilsenbeck S, Green JE, Michalowska AM, Rosen JM (2008) Identification of tumor-initiating cells in a p53-null mouse model of breast cancer. Cancer Res 68:4674–4682PubMedCrossRefGoogle Scholar
  14. 14.
    Proia DA, Kuperwasser C (2006) Reconstruc­tion of human mammary tissues in a mouse model. Nat Protoc 1:206–214PubMedCrossRefGoogle Scholar
  15. 15.
    Illa-Bochaca I, Fernandez-Gonzalez R, Shelton DN, Welm BE, Ortiz-de-Solorzano C, Barcellos-Hoff MH (2010) Limiting-dilution transplantation assays in mammary stem cell studies. Methods Mol Biol 621:29–47PubMedCrossRefGoogle Scholar
  16. 16.
    Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam JS, Ailles LE, Wong M, Joshua B, Kaplan MJ, Wapnir I, Dirbas FM, Somlo G, Garberoglio C, Paz B, Shen J, Lau SK, Quake SR, Brown JM, Weissman IL, Clarke MF (2009) Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458:780–783PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Abhik Bandyopadhyay
    • 1
  • Qiaoxiang Dong
    • 1
  • Lu-Zhe Sun
    • 1
  1. 1.Department of Cellular and Structural BiologyUniversity of Texas Health Science CenterSan AntonioUSA

Personalised recommendations