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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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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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