title: 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.
authors.affiliation: Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. dossanto@cshl.edu
authors.forename: Camila O
authors.initials: CO
authors.lastname: dos Santos
authors.forename: Clare
authors.initials: C
authors.lastname: Rebbeck
authors.forename: Elena
authors.initials: E
authors.lastname: Rozhkova
authors.forename: Amy
authors.initials: A
authors.lastname: Valentine
authors.forename: Abigail
authors.initials: A
authors.lastname: Samuels
authors.forename: Lolahon R
authors.initials: LR
authors.lastname: Kadiri
authors.forename: Pavel
authors.initials: P
authors.lastname: Osten
authors.forename: Elena Y
authors.initials: EY
authors.lastname: Harris
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Uren
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Smith
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Hannon
mesh_terms: D000818:Animals; D055610:Antigens, CD1d / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D005260:Female; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology*; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*
chemical_list: D055610:Antigens, CD1d; D015415:Biomarkers; C526879:Cd1d1 protein, mouse; D006657:Histones; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins