Separation by Cell Size Enriches for Mammary Stem Cell Repopulation Activity

• Published in: Stem cells translational medicine Vol. 2; no. 3; pp. 199 - 203
• Main Authors: Machado, Heather L., Kittrell, Frances S., Edwards, David, White, Amanda N., Atkinson, Rachel L., Rosen, Jeffrey M., Medina, Daniel, Lewis, Michael T.
• Format: Journal Article
• Language: English
• Published: United States AlphaMed Press 01.03.2013; Oxford University Press
• Subjects: Adult stem cells; Animals; Antibiotics; Biomarkers - metabolism; Breast cancer; CD24 Antigen - metabolism; Cell Lineage; Cell Proliferation; Cell Separation - methods; Cell Size; Cell surface; Cell surface markers; Data analysis; Epidermal growth factor; Epithelial Cells - cytology; Epithelial Cells - metabolism; Epithelial Cells - transplantation; Etiology; FACS; Female; Flow Cytometry; Integrin beta1 - metabolism; Laboratories; Mammary gland; Mammary Glands, Animal - cytology; Mammary Glands, Animal - metabolism; Mammary Glands, Animal - transplantation; Mice; Mice, Transgenic; Microscopy; Spheroids, Cellular; Stem Cell Transplantation; Stem cells; Stem Cells - cytology; Stem Cells - metabolism; Surface markers; Time Factors; Tissue-Specific Progenitor and Stem Cells; Transplantation
• ISSN: 2157-6564; 2157-6580
• DOI: 10.5966/sctm.2012-0121

Cell sorting techniques for specific cell surface markers (CD24+CD29HCD49fHSca1−) have been used to prospectively isolate mammary stem cell (MaSC)‐enriched populations, but these markers do not identify regenerative stem cells uniquely. This study found that MaSCs can be further enriched by size fractionation. These findings have critical implications for understanding mammary gland stem cell biology, an important requisite step for understanding the etiology of breast cancer. 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+CD29HCD49fHSca1−) 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+CD29H 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.