Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.

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Abstract. The main focus of this review is the role of mammary stem cells in normal breast development and carcinogenesis. We have developed a new in vitro culture system that permits, for the first time, the propagation of mammary stem and progenitor cells in an undifferentiated state, which should facilitate the elucidation of pathways that regulate normal mammary stem-cell self-renewal and differentiation. Furthermore, we propose a model in which transformation of stem cells, or early progenitor cells, results in carcinogenesis. A key event in this process is the deregulation of normal self-renewal in these cells. Transformed mammary stem or progenitor cells undergo aberrant differentiation processes that result in generation of the phenotypic heterogeneity found in human and rodent breast cancers. This phenotypic diversity is driven by a small subset of mammary tumour stem cells. We will discuss the important implications of this mammary tumour stem-cell model.

Objective picardial adipose tissue is associated with coronary artery disease, however the causal relationship between perivascular adipose tissue and local atherogenesis is unclear. Methods and Results Apolipoprotein E deficient (ApoE−/−) mice underwent transplantation of visceral or subcutaneous adipose tissue immediately adjacent to the right common carotid artery. Carotid arteries with fat transplants were analyzed for atherosclerosis by surface oil-red-O staining and cross-sectional analysis. Vascular function of the carotid arteries was assessed using pressure myography. Visceral fat transplants were also performed to ApoE−/− mice with neutralization of P-selectin glycoprotein ligand-1 (Psgl-1). Atherosclerosis surface area and lesion thickness were greater in mice receiving the perivascular visceral fat compared to the subcutaneous fat. Mice with visceral fat transplants also displayed more complicated atherosclerotic lesions with evidence of atherothrombosis. Serum Mcp-1 was higher in mice receiving visceral fat transplants compared to subcutaneous transplants. Visceral fat transplantation also caused impaired endothelial-dependent relaxation of the carotid artery. Psgl-1 deficiency or neutralization of Psgl-1 with an anti-Psgl-1 antibody was protective against perivascular visceral adipose tissue-induced atherosclerosis and was associated with reduced Mcp-1 levels. Conclusions Perivascular visceral fat leads to endothelial dysfunction and accelerated atherosclerosis. This proatherogenic effect of perivascular adipose tissue is blocked by neutralization of Psgl-1.

The neuronal guidance molecule, Netrin-1, has been suggested to play a role in the adhesion and migration of the mammary gland epithelium. Human and mouse Cripto-1 induce proliferation, migration, invasion and colony formation by epithelial cells in 3D matrices. Here we investigate whether Netrin-1 affects these Cripto-1-dependent activities in mouse mammary epithelial cells. Overexpression of Cripto-1 in EpH4 and HC-11 cells (EpH4/Cripto-1 or HC-11/Cripto-1) was associated with low expression of Netrin-1 and increased expression of its receptor Neogenin compared to that of wild-type cells. No change was observed in the expression of the other Netrin-1 receptor, UNC5H1. Treating EpH4/Cripto-1 or HC-11/Cripto-1 mammary cells with exogenous soluble Netrin-1 resulted in increased expression of E-cadherin and UNC5H1, decreased expression of vimentin and decreased activation of Akt as determined by western blotting. Colony formation by Eph4/Cripto-1 cells in 3D gels was significantly reduced in proximity to a Netrin-1 source, and mammary glands of transgenic mice overexpressing human Cripto-1 showed altered ductal growth in proximity to implanted Netrin-1-releasing pellets. Terminal end buds in the treated transgenic mice mammary glands also showed increased expression of E-cadherin and UNC5H1 and decreased expression of active Akt determined by immunohistochemistry. Together, these results suggest that regulation of Netrin-1 expression is important in regulating Cripto-1-dependent invasion and migration of mammary epithelial cells.