Separation by cell size enriches for mammary stem cell repopulation activity

Stem Cells Transl Med. 2013 Mar;2(3):199-203. doi: 10.5966/sctm.2012-0121. Epub 2013 Feb 13.


Mammary gland reconstitution experiments, as well as lineage tracing experiments, have provided evidence for the existence of adult mammary stem cells (MaSCs). In addition, cell sorting techniques for specific cell surface markers (CD24(+)CD29(H)CD49f(H)Sca1(-)) have been used to prospectively isolate MaSC-enriched populations. Although these markers enrich for cell subpopulations that harbor MaSCs, they do not identify regenerative stem cells uniquely. Here, we report that MaSCs can be further defined by the property of cell size. Fluorescence-activated cell sorting was used to analyze sizing beads and further separate populations of cells with varying degrees of forward scatter (FSC). Cells with a low FSC that were approximately <10 μm in size lacked outgrowth potential and failed to reconstitute the mammary gland when transplanted into the cleared fat pads of syngeneic mice. In contrast, cells >10 μm in size with a higher FSC had increased outgrowth potential as compared with lineage-negative (LIN(-)) control cells. Limiting dilution transplantation assays indicated that the repopulating ability of LIN(-)CD24(+)CD29(H) cells that were >10 μm in size was significantly increased as compared with cells marked by CD24 and CD29 alone. These results suggest that MaSCs can be further isolated by sorting based on size/FSC. These findings have critical implications for understanding mammary gland stem cell biology, an important requisite step for understanding the etiology of breast cancer.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • CD24 Antigen / metabolism
  • Cell Lineage
  • Cell Proliferation
  • Cell Separation* / methods
  • Cell Size*
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Epithelial Cells / transplantation
  • Female
  • Flow Cytometry
  • Integrin beta1 / metabolism
  • Mammary Glands, Animal / cytology*
  • Mammary Glands, Animal / metabolism
  • Mammary Glands, Animal / transplantation
  • Mice
  • Mice, Transgenic
  • Spheroids, Cellular
  • Stem Cell Transplantation
  • Stem Cells / cytology*
  • Stem Cells / metabolism
  • Time Factors


  • Biomarkers
  • CD24 Antigen
  • Cd24a protein, mouse
  • Integrin beta1