Separation by cell size enriches for mammary stem cell repopulation activity

Abstract

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.

Figure 1.

Cells >8 μm in size are enriched for mammary stem cell activity. (A): Top: Exponential dot plot depicting sizing beads and theoretical gates for different sized cell populations. Bottom: Linear dot plot depicting LIN⁻ cells, which were sorted by fluorescence-activated cell sorting on the basis of size. (B): Graph illustrates the number of secondary mammospheres formed per 5,000 cells, expressed as mammosphere efficiency of different cell sizes. The abilities of cells 9–12 μm in size and cells >12 μm in size were significantly increased as compared with LIN⁻ cells and cells 4–8 μm in size (*, p < .0001). Data represent mean ± SEM. Representative images of mammospheres from each group are shown. Scale bars = 100 μm. (C): Table indicates repopulation activity as a function of cell size. Whereas cells 4–8 μm in size lacked outgrowth potential, cells larger than 8 μm had increased outgrowth potential as compared with LIN⁻ and unsorted controls. Images depict representative outgrowths from 100 transplanted cells of each group. Scale bars = 5 mm. Abbreviations: FSC, forward scatter; LIN⁻, lineage-negative; SSC, side scatter.

Figure 2.

Cells >10 μm in size are enriched for outgrowth potential. (A): Dot plot depicting sizing beads and gating strategy for cells <10 μm and >10 μm in size (left). LIN⁻ cells <10 μm and >10 μm in size (middle), and LIN⁻CD24^HCD29^L and LIN⁻CD24⁺CD29^H cells (right) were sorted by fluorescence-activated cell sorting and transplanted into cleared fat pads of FvB mice. (B): Transplantation results demonstrated that when 100 cells were transplanted, cells <10 μm in size, LIN⁻CD24^HCD29^L cells, and LIN⁻ cells had limited repopulation ability. Repopulation of cells >10 μm in size was similar to that of mammary stem cells defined by LIN⁻CD24⁺CD29^H expression. Outgrowth potential is the number of outgrowths/number of recipient fat pads expressed as a percentage. Abbreviations: FSC, forward scatter; LIN⁻, lineage-negative; SSC, side scatter.