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Purification and unique properties of mammary epithelial stem cells

Abstract

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer1. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential2,3,4. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.

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Figure 1: The MRU assay.
Figure 2: Phenotypic characterization of MRUs.
Figure 3: Functional and molecular characterization of subsets of mammary cells.
Figure 4: Characterization of dye efflux and cycling properties of MRUs and Ma-CFCs.

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References

  1. Al Hajj, M. & Clarke, M. F. Self-renewal and solid tumour stem cells. Oncogene 23, 7274–7282 (2004)

    Article  CAS  PubMed  Google Scholar 

  2. Kordon, E. C. & Smith, G. H. An entire functional mammary gland may comprise the progeny from a single cell. Development 125, 1921–1930 (1998)

    CAS  PubMed  Google Scholar 

  3. Dontu, G. et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17, 1253–1270 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Stingl, J., Raouf, A., Emerman, J. T. & Eaves, C. J. Epithelial progenitors in the normal human mammary gland. J. Mamm. Gland Biol. Neoplasia 10, 49–59 (2005)

    Article  Google Scholar 

  5. Sakakura, T. in The Mammary Gland: Development, Regulation, and Function (eds Neville, M. C. & Daniel, C. W.) 37–66 (Plenum, New York, 1987)

    Book  Google Scholar 

  6. Smalley, M. J., Titley, J. & O'Hare, M. J. Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting. In Vitro Cell. Dev. Biol. Anim. 34, 711–721 (1998)

    Article  CAS  PubMed  Google Scholar 

  7. Stingl, J., Eaves, C. J., Zandieh, I. & Emerman, J. T. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 67, 93–109 (2001)

    Article  CAS  PubMed  Google Scholar 

  8. Smith, G. H. Experimental mammary epithelial morphogenesis in an in vivo model: evidence for distinct cellular progenitors of the ductal and lobular phenotype. Breast Cancer Res. Treat. 39, 21–31 (1996)

    Article  CAS  PubMed  Google Scholar 

  9. Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297, 2256–2259 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Hadjantonakis, A. K., Macmaster, S. & Nagy, A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2, 11 (2002)

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wlem, B. E. et al. Sca-1pos cells in the mouse mammary gland represent an enriched progenitor cell population. Dev. Biol. 245, 42–56 (2002)

    Article  Google Scholar 

  12. Tani, H., Morris, R. J. & Kaur, P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc. Natl Acad. Sci. USA 97, 10960–10965 (2000)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Li, A., Simmons, P. J. & Kaur, P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc. Natl Acad. Sci. USA 95, 3902–3907 (1998)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jones, C. et al. Expression profiling of purified normal human luminal and myoepithelial breast cells: identification of novel prognostic markers for breast cancer. Cancer Res. 64, 3037–3045 (2004)

    Article  CAS  PubMed  Google Scholar 

  15. Carter, W. G., Kaur, P., Gil, S. G., Gahr, P. J. & Wayner, E. A. Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J. Cell Biol. 111, 3141–3154 (1990)

    Article  CAS  PubMed  Google Scholar 

  16. Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  17. Li, Y. et al. Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells. Proc. Natl Acad. Sci. USA 100, 15853–15858 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. & Mulligan, R. C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797–1806 (1996)

    Article  CAS  PubMed  Google Scholar 

  19. Zhou, S. et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nature Med. 7, 1028–1034 (2001)

    Article  CAS  PubMed  Google Scholar 

  20. Spangrude, G. J. & Johnson, G. R. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc. Natl Acad. Sci. USA 87, 7433–7437 (1990)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Uchida, N. et al. ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood 103, 4487–4495 (2004)

    Article  CAS  PubMed  Google Scholar 

  22. Bhattacharya, S. et al. Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest. Ophthalmol. Vis. Sci. 44, 2764–2773 (2003)

    Article  PubMed  Google Scholar 

  23. Hierlihy, A. M., Seale, P., Lobe, C. G., Rudnicki, M. A. & Megeney, L. A. The post-natal heart contains a myocardial stem cell population. FEBS Lett. 530, 239–243 (2002)

    Article  CAS  PubMed  Google Scholar 

  24. Alvi, A. J. et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res. 5, R1–R8 (2003)

    Article  PubMed  Google Scholar 

  25. Smith, G. H. Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands. Development 132, 681–687 (2005)

    Article  CAS  PubMed  Google Scholar 

  26. Glimm, H., Oh, I. & Eaves, C. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0 . Blood 96, 4185–4193 (2000)

    CAS  PubMed  Google Scholar 

  27. Stingl, J., Emerman, J. T. & Eaves, C. J. in Methods in Molecular Biology: Basic Cell Culture Protocols (eds Helgason, C. D. & Miller, C. L.) 249–263 (Humana, New Jersey, 2005)

    Google Scholar 

  28. Young, L. J. T. in Methods in Mammary Gland Biology and Breast Cancer Research (eds Ip, M. M. & Asch, B. B.) 67–74 (Kluwer/Plenum, New York, 2000)

    Book  Google Scholar 

  29. Szilvassy, S. J., Nicolini, F. E., Eaves, C. J. & Miller, C. L. in Methods in Molecular Medicine: Hematopoietic Stem Cell Protocols (eds Jordon, C. T. & Klug, C. A.) 167–187 (Humana, New Jersey, 2002)

    Google Scholar 

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Acknowledgements

This work was supported by grants from the Canadian Stem Cell Network and Genome BC/Canada. J.S. held Postdoctoral Fellowships from the Canadian Breast Cancer Foundation (BC/Yukon Chapter) and the Natural Sciences and Engineering Research Council of Canada; P.E. held a Stem Cell Network Studentship; I.R. and D.C. held British Columbia Cancer Foundation Summer Studentships; and M.S. held a Scholarship from the National Health and Medical Research Council of Australia. The authors thank the Ontario Genomics Innovation Centre for performing the microarrays, the Terry Fox Laboratory FACS Facility for assistance in cell sorting, G. Edin and K. M. Saw for technical assistance, and A. Eaves, S. Aparicio, C. Fisher and D. Kent for scientific discussion. Author Contributions J.S., P.E., I.R. and D.C. performed most of the experiments described in the manuscript. M.S. and F.V. performed some of the single cell transplants. H.I.L. analysed the microarray data and J.S., P.E. and C.J.E. designed the studies and wrote the manuscript.

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Correspondence to Connie J. Eaves.

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Competing interests

The microarray data can be accessed online at StemBase (http://www.scgp.ca:8080/StemBase/) and at the Gene Expression Omnibus (series accession number GSE3711) at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3711. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

This figure illustrates how a quantitative in vivo transplantation assay was used to demonstrate the two hallmarks of mouse mammary epithelial stem cells: their ability as single cells to generate a complete new gland containing both ductal and alveolar components as well as progenitors detectable as in vitro colony-forming cells, and their ability to self-renew as assessed by the transplantation of cells from primary outgrowths into secondary mice

Supplementary Figure 2

This figure shows the rapidity with which the expression of Sca-1 is upregulated in mouse mammary epithelial cells cultured in serum-free media.

Supplementary Tables 1-4

These report the frequencies and distribution of MRUs in unfractionated mouse mammary epithelial cells and various subsets of these, including CD24highCD49flow (Ma-CFC-enriched), CD24medCD49fhigh (MRU-enriched), CD24lowCD49flow (MYO), SP and non-SP subpopulations, as well as cells sorted based on their differential Rhodamine 123 efflux activity.

Supplementary Table 5

This lists genes found to be expressed at twofold or higher levels and at statistically significant higher levels in the MRU fraction as compared to the CFC and MYO fractions. The statistical tests used include DEDS and LIMMA.

Supplementary Table 6

This lists genes found to be expressed at twofold or lower levels and at statistically significant lower levels in the MRU fraction as compared to the CFC and MYO fractions. The statistical tests used include DEDS and LIMMA.

Supplementary Table 7

This table lists the GEO sample accession numbers for series GSE3711.

Supplementary Methods

Supplementary Methods describing the experiments in which mixtures of GFP+ and CFP+ cells were transplanted, the immunocytochemistry protocols used, and the microarray analyses and quantitative real-time PCR analyses performed.

Supplementary Notes

References for Supplementary Methods.

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Stingl, J., Eirew, P., Ricketson, I. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006). https://doi.org/10.1038/nature04496

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