The future of mammary stem cell biology: the power of in vivo transplants

The recent review by Smith and Medina [1] of in vivo transplantation models and their role in investigating mammary stem cell (MaSC) biology provides comprehensive coverage of the history and complexity of the ‘gold standard ’ MaSC assay in mice. This includes a description of the pioneering studies that showed that mammary epithelial outgrowths can be generated in cleared mammary fat pads transplanted with explants or admixtures of mammary cells [2]. However, this approach clearly does not lend itself to prospective analysis of isolated subpopulations in order to identify which cells possess in vivo regenerative activity. More recently, success in obtaining complex mammary gland structures from transplanted suspensions of single cells has now made this possible [3-7]. Moreover, the regenerated structures have been shown to contain daughter cells with the same in viv

Ovarian steroid hormones: what's hot in the stem cell pool?

The vital role of ovarian hormones in the development of the normal breast foreshadowed their importance in mammary stem cell regulation. Two recent papers reveal that 17β-estradiol and progesterone control the size and repopulating ability of the mammary stem cell compartment. This likely occurs via paracrine signaling from steroid receptor-positive luminal cells to steroid receptor-negative stem cells. These findings illuminate roles for the female sex steroids in mobilizing the stem cell pool in the normal breast, and also provide a crucial link between the known hormonal risks of breast cancer and the potential stem cell origin of this disease

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further

Age- and Pregnancy-Associated DNA Methylation Changes in Mammary Epithelial Cells

Summary Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24+CD61+CD29hi, CD24+CD61+CD29lo, and CD24+CD61−CD29lo cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFβ signaling as key regulators of CD24+CD61+CD29lo cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells

Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways

INTRODUCTION: Molecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks. METHODS: RNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways. RESULTS: The four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets. CONCLUSIONS: The conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelial-mesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models

Gata-3 and mammary cell fate

Genomic regulatory networks specify how cellular gene expression responds to external temporal and spatial stimuli, ensuring that correct cell fate decisions are made and the appropriate cell phenotypes are adopted. In mammary epithelial cells, the hierarchy of stem and progenitor cells and the genetically specified program of transcriptional activity are beginning to be elucidated and integrated. A novel role for Gata-3 in specifying and maintaining mammary cell fate has recently been identified. These reports offer an understanding of how mammary cells assume and maintain a variety of cell behaviours and functions, and how a mammary cell may potentially subvert these constraints during carcinogenesis

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24(+) CD49f(hi) Mammary Stem Cell-Enriched Compartment

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland

Optimizing the noise versus bias trade-off for Illumina whole genome expression BeadChips

Five strategies for pre-processing intensities from Illumina expression BeadChips are assessed from the point of view of precision and bias. The strategies include a popular variance stabilizing transformation and model-based background corrections that either use or ignore the control probes. Four calibration data sets are used to evaluate precision, bias and false discovery rate (FDR). The original algorithms are shown to have operating characteristics that are not easily comparable. Some tend to minimize noise while others minimize bias. Each original algorithm is shown to have an innate intensity offset, by which unlogged intensities are bounded away from zero, and the size of this offset determines its position on the noise–bias spectrum. By adding extra offsets, a continuum of related algorithms with different noise–bias trade-offs is generated, allowing direct comparison of the performance of the strategies on equivalent terms. Adding a positive offset is shown to decrease the FDR of each original algorithm. The potential of each strategy to generate an algorithm with an optimal noise–bias trade-off is explored by finding the offset that minimizes its FDR. The use of control probes as part of the background correction and normalization strategy is shown to achieve the lowest FDR for a given bias

Identification of murine mammary stem cells: implications for studies of mammary development and carcinogenesis

The epithelial components of the mammary gland are thought to arise from a stem cell capable of both self-renewal and multi-lineage differentiation. Furthermore, there is increasing evidence that mammary carcinomas originate in these cells or their immediate progeny. The recent identification of murine mammary stem cells should facilitate their molecular characterization and help to elucidate their role in mammary carcinogenesis. In addition, an understanding of the biology of these cells including the pathways that regulate their self-renewal and differentiation may suggest new approaches for the prevention and treatment of breast cancer

Estrogen regulation of mammary gland development and breast cancer: amphiregulin takes center stage

Estrogen-mediated proliferation is fundamental to normal mammary gland development. Recent studies have demonstrated that amphiregulin is a critical paracrine regulator of estrogen action during ductal morphogenesis. These studies implicate a critical role for amphiregulin in mammary stem cell differentiation as well as breast cancer initiation and progression