Abstract
The mammary epithelium is composed of a variety of specialized cell types that function in a coordinated fashion to produce and eject milk through multiple cycles of pregnancy. The ability to identify and purify these subsets of cells in order to interrogate their growth and differentiation capacities, as well as to characterize the molecular mechanisms that regulate their behavior, is essential in identifying the processes associated with breast cancer initiation and progression. This methods chapter outlines the step-by-step methods for dissociating human breast reduction specimens to a single cell suspension of viable cells. As well, strategies for purifying four distinct subsets of epithelial cells by using fluorescence-activated cell sorting and protocols for interrogating the growth and differentiation properties of these purified cells at clonal densities in adherent culture are also described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wellings SR, Jensen HM, Marcum RG (1975) An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J Natl Cancer Inst 55(2):231–273
Shehata M, Teschendorff A, Sharp G, Novcic N, Russell A, Avril S, Prater M, Eirew P, Caldas C, Watson CJ, Stingl J (2012) Phenotypic and functional characterization of the luminal cell hierarchy of the mammary gland. Breast Cancer Res 14(5):R134. doi:10.1186/bcr3334
Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ, Fox SB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, Lindeman GJ (2009) Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med 15(8):907–913. doi:10.1038/nm.2000
Eirew P, Stingl J, Raouf A, Turashvili G, Aparicio S, Emerman JT, Eaves CJ (2008) A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med 14(12):1384–1389. doi:10.1038/nm.1791
Eirew P, Stingl J, Eaves CJ (2010) Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay. Nat Protoc 5(12):1945–1956. doi:10.1038/nprot.2010.148
Stingl J, Eaves CJ, Zandieh I, Emerman JT (2001) Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res Treat 67(2):93–109
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(4):201–213. doi:10.1111/j.1432-0436.1998.00201.x
O'Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA (1991) Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting. Differentiation 46(3):209–221
Petersen OW, Hoyer PE, van Deurs B (1987) Frequency and distribution of estrogen receptor-positive cells in normal, nonlactating human breast tissue. Cancer Res 47(21):5748–5751
Villadsen R, Fridriksdottir AJ, Ronnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, Bissell MJ, Petersen OW (2007) Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol 177(1):87–101. doi:10.1083/jcb.200611114
Oakes SR, Naylor MJ, Asselin-Labat ML, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA, Pfeffer PL, Lindeman GJ, Visvader JE, Ormandy CJ (2008) The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes Dev 22(5):581–586. doi:10.1101/gad.1614608
Keller PJ, Arendt LM, Skibinski A, Logvinenko T, Klebba I, Dong S, Smith AE, Prat A, Perou CM, Gilmore H, Schnitt S, Naber SP, Garlick JA, Kuperwasser C (2012) Defining the cellular precursors to human breast cancer. Proc Natl Acad Sci U S A 109(8):2772–2777. doi:10.1073/pnas.1017626108
Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS (2003) Stem cells in normal breast development and breast cancer. Cell Prolif 36(Suppl 1):59–72
Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567. doi:10.1016/j.stem.2007.08.014
Eirew P, Kannan N, Knapp DJ, Vaillant F, Emerman JT, Lindeman GJ, Visvader JE, Eaves CJ (2012) Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells. Stem Cells 30(2):344–348. doi:10.1002/stem.1001
Visvader JE, Stingl J (2014) Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev 28(11):1143–1158. doi:10.1101/gad.242511.114
Acknowledgements
The authors thank Alasdair Russell for scientific discussions. They also thank Prof. Carlos Caldas and Linda Jones for facilitating and coordinating access to human tissue. They would like to acknowledge the support of The University of Cambridge, Breast Cancer Campaign, Cancer Research UK (core grant C14303/A17197), Hutchison Whampoa Limited, and NIHR Cambridge Biomedical Research Centre.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Shehata, M., Stingl, J. (2017). Purification of Distinct Subsets of Epithelial Cells from Normal Human Breast Tissue. In: Martin, F., Stein, T., Howlin, J. (eds) Mammary Gland Development. Methods in Molecular Biology, vol 1501. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6475-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6475-8_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6473-4
Online ISBN: 978-1-4939-6475-8
eBook Packages: Springer Protocols