Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells

Abstract

The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin − CD24 + CD29 h CD49f h ) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gland of mice expressing a controllable H2b-GFP transgene. In this system, the transgene expression can be repressed in a doxycycline-dependent fashion, allowing isolation of slowly dividing cells with retained nuclear GFP signal. Here, we show that H2b-GFP h cells reside within the predicted MaSC compartment and display greater mammary reconstitution unit frequency compared with H2b-GFP neg MaSCs. According to their transcriptome profile, H2b-GFP h MaSCs are enriched for pathways thought to play important roles in adult stem cells. We found Cd1d, a glycoprotein expressed on the surface of antigen-presenting cells, to be highly expressed by H2b-GFP h MaSCs, and isolation of Cd1d + MaSCs further improved the mammary reconstitution unit enrichment frequency to nearly a single-cell level. Additionally, we functionally characterized a set of MaSC-enriched genes, discovering factors controlling MaSC survival. Collectively, our data provide tools for isolating a more precisely defined population of MaSCs and point to potentially critical factors for MaSC maintenance.

Mammary gland development: cell fate specification, stem cells and the microenvironment

“…The reported likelihood of a single cell from this MRU-enriched population in a limiting dilution giving rise to a full glandular epithelium is ∼1/40-1/800, depending on the study (Badders et al, 2009;dos Santos et al, 2013;Plaks et al, 2013;Shackleton et al, 2006;Stingl et al, 2006;Zeng and Nusse, 2010). This can be compared with 1/1400 to 1/7600 in the unsorted Lin − epithelial population (Badders et al, 2009;Shackleton et al, 2006) or less than 1/3000 in the Lin .…”

“…The adult mammary gland contains cells that retain their parental DNA strand, suggesting that these cells divide infrequently and via asymmetric cell division, which are properties of stem and progenitor cells (Smith, 2005). These DNA label-retaining cells were indeed demonstrated to have fivefold higher gland-reconstituting capacity compared with nonlabeled and non-label-retaining epithelial cells in a recent study (dos Santos et al, 2013). Furthermore, the isolation and subsequent profiling of DNA-retaining CD29 high CD49f high CD24 + cells has been used to identify improved MRU markers.…”

“…The murine mammary epithelium is typically first isolated by selecting against markers of immune/hematopoietic (CD45), erythrocyte (Ter119) and endothelial (CD31) cells (commonly referred to as the lineage-marker negative, or Lin − population). These epithelial cells are then sorted for expression of moderate-to-high levels of CD24 (heat-stable antigen), high levels of CD29 (β1-integrin) and/or high levels CD49f (α6-integrin) (Badders et al, 2009;dos Santos et al, 2013;Plaks et al, 2013;Shackleton et al, 2006;Stingl et al, 2006;Zeng and Nusse, 2010). The expression pattern of these markers appears to vary between studies, making quantitative comparisons of the prevalence of different populations difficult (Fig.…”

“…Furthermore, the isolation and subsequent profiling of DNA-retaining CD29 high CD49f high CD24 + cells has been used to identify improved MRU markers. Through this work, an additional cell surface marker, CD1d, a glycoprotein typically found on antigen-presenting cells, was identified in the DNA labelretaining population (dos Santos et al, 2013) Another fruitful approach for isolating MRUs has been to identify elements of the Wnt signaling pathway that are active in mammary development, given the importance of Wnt signaling in stem cell behavior (Reya and Clevers, 2005) and the high activation of the Wnt/β-catenin pathway in DNA label-retaining cells (dos Santos et al, 2013). Indeed, a downstream target of Wnt, the G-protein- and myoepithelial cells (circled in red) from adult (8-12-week old) murine mammary glands show discordance between studies using similar methodologies.…”

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…No evidence was found for any increased incidence or growth of tumours in the SAFB1 +/− mice. Intriguingly, though, dos Santos et al [114] identified SLTM as a gene that is probably required for survival of mouse mammary stem cells.…”