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Characterization of Normal Human Breast Epithelial Cell Subpopulations Isolated by Fluorescence-Activated Cell Sorting and Their Clonogenic Growth In Vitro

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Methods in Mammary Gland Biology and Breast Cancer Research

Abstract

Luminal-cell-restricted, myoepithelial-cell-restricted, and mixed progenitors of the human mammary epithelium can be isolated by fluorescence-activated cell sorting of cells harvested from 3-day primary cultures of normal mammary tissue. Useful markers that allow these populations to be distinguished are MUC1, CALLA, α6 integrin, and epithelial-specific antigen. The progenitor content of the sorted cell subpopulations is detected in a 9-to 12-day colony assay in which the cells are cultured in a serum-free medium with NIH 3T3 feeders to optimize the growth of luminal and myoepithelial progenitors plated at low densities. Efficiencies of colony formation of 1–20% and colony sizes of > 100 cells are routinely observed in such cultures. The different types of colonies can be readily identified by their gross morphology and their expression of a variety of luminal- and myoepithelial-cellspecific proteins.

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Abbreviations

(FACS):

fluorescence-activated cell sorting

(HBEC):

human breast epithelial cells

(F12/DME/H):

Ham’s F12/Dulbecco’s modified Eagle’s medium/Hepes

(P):

penicillin

(S):

streptomycin

(BSA):

bovine serum albumin

(INS):

insulin

(HC):

hydrocortisone

(CT):

cholera toxin

(TRIS):

tris hydroxymethyl amino methane hydrochloride

(FBS):

fetal bovine serum

(FGFR):

fibroblast growth factor receptor

(HFN):

Hank’s balanced salt solution supplemented with sodium azide and FBS

(PI):

propidium iodide

(MUC1):

mammary gland mucin

(CALLA):

common acute lymphoblastic leukemia antigen

(FITC):

fluorescein isothiocyanate

(PE):

R-phycoerythrin

(ESA):

epithelial-specific antigen

(Ep-CAM):

epithelial cell adhesion molecule

(a6):

α6 integrin

(IgG):

immunoglobulin

(EGF):

epidermal growth factor

(SF medium):

serum-free medium

(HMF):

human mammary fibroblasts

(rbc):

red blood cell

(APAAP):

alkaline phosphatase anti-alkaline phosphatase

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Author information

Authors and Affiliations

  1. Department of Anatomy, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

    John Stingl & Joanne T. Emerman

  2. Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada

    Connie J. Eaves

  3. Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada

    Connie J. Eaves

Authors
  1. John Stingl
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  2. Connie J. Eaves
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  3. Joanne T. Emerman
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Editor information

Editors and Affiliations

  1. Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, 14263, USA

    Margot M. Ip

  2. Roswell Park Cancer Research Institute, Buffalo, New York, USA

    Bonnie B. Asch

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Stingl, J., Eaves, C.J., Emerman, J.T. (2000). Characterization of Normal Human Breast Epithelial Cell Subpopulations Isolated by Fluorescence-Activated Cell Sorting and Their Clonogenic Growth In Vitro . In: Ip, M.M., Asch, B.B. (eds) Methods in Mammary Gland Biology and Breast Cancer Research. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-4295-7_17

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  • DOI: https://doi.org/10.1007/978-1-4615-4295-7_17

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4613-6927-1

  • Online ISBN: 978-1-4615-4295-7

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