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A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability

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Abstract

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels, which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2–8 weeks, the gels contain bilayered mammary epithelial structures, including luminal and myoepithelial cells, their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations.

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Figure 1: Organized structures are generated in vivo from single-cell suspensions of primary human mammary cells.
Figure 2: CFC production in vivo as an indicator of human MRU repopulating activity.
Figure 3: MRUs are CD49f+EpCAMneg–low.

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Acknowledgements

C3H 10T1/2 mouse embryonic fibroblasts were a kind gift from G. Cunha, University of California, San Francisco. The authors acknowledge the excellent technical contributions of D. Wilkinson, G. Edin, the staff of the Flow Cytometry Facility of the Terry Fox Laboratory and the Centre for Translational and Applied Genomics. Mammoplasty tissue was obtained with the assistance of J. Sproul, P. Lennox, N. Van Laeken and R. Warren. This project was funded by grants from Genome British Columbia and Genome Canada, the Canadian Stem Cell Network and the Canadian Breast Cancer Foundation British Columbia and Yukon Division. P.E. was a recipient of a US Department of Defense Breast Cancer Research Program Studentship, a Terry Fox Foundation Research Studentship from the National Cancer Institute of Canada, a Canadian Imperial Bank of Commerce interdisciplinary award and a Canadian Stem Cell Network Studentship. J.S. held a Canadian Breast Cancer Foundation British Columbia and Yukon Division Fellowship and a Canadian National Science and Engineering Research Council Industrial Fellowship. A.R. held a Canadian Breast Cancer Foundation British Columbia and Yukon Division Fellowship and a Canadian Institutes of Health Research Fellowship. G.T. holds a Canadian Institutes of Health Research Pathology Training Fellowship. S.A. is supported by a Canada Research Chair in Molecular Oncology. The Centre for Translational and Applied Genomics laboratory is supported by a Canadian Institutes for Health Research Resource award.

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  1. John Stingl

    Present address: Present address: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.,

Authors and Affiliations

  1. Terry Fox Laboratory, British Columbia Cancer Agency, 675 West Tenth Avenue, Vancouver, V5Z 1L3, British Columbia, Canada

    Peter Eirew, John Stingl, Afshin Raouf & Connie J Eaves

  2. Molecular Oncology and Breast Cancer Program, British Columbia Cancer Agency, 675 West Tenth Avenue, Vancouver, V5Z 1L3, British Columbia, Canada

    Gulisa Turashvili & Samuel Aparicio

  3. Department of Pathology and Laboratory Medicine, University of British Columbia, 2211 Wesbrook Mall, Vancouver, V6T 2B5, British Columbia, Canada

    Samuel Aparicio

  4. Department of Cellular & Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, V6T 1Z3, British Columbia, Canada

    Joanne T Emerman

  5. Department of Medical Genetics, University of British Columbia, 2350 Health Sciences Mall, Vancouver, V6T 1Z3, British Columbia, Canada

    Connie J Eaves

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  1. Peter Eirew
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  2. John Stingl
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  3. Afshin Raouf
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  7. Connie J Eaves
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Contributions

P.E. designed and conducted most of the experiments and drafted the manuscript. J.S. initiated the work that led to the gel implant protocol, undertook preliminary experiments and contributed to the writing of the manuscript. A.R. critiqued the manuscript and participated in discussions of the experiments. G.T. and S.A. reviewed the histological preparations and contributed to the writing of the manuscript. J.T.E. helped organize the accrual of the mammoplasty material used. C.J.E. conceptualized the study and finalized the writing of the manuscript.

Corresponding author

Correspondence to Connie J Eaves.

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Supplementary Table 1 and Supplementary Fig. 1 (PDF 99 kb)

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Eirew, P., Stingl, J., Raouf, A. et al. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med 14, 1384–1389 (2008). https://doi.org/10.1038/nm.1791

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