Health & Medical Cancer & Oncology

Hormones, Receptors in Hyperplastic Enlarged Lobular Units

Hormones, Receptors in Hyperplastic Enlarged Lobular Units

Abstract and Introduction

Abstract


Introduction: Breast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.
Methods: Mouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.
Results: Three populations of CD24 expressing cells were identified: CD24, CD24 and CD24. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24 cells, whilst CD24 cells have limited repopulation capacity.
Conclusion: Differential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24 population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.

Introduction


There is increasing evidence that normal tissue stem cells are the cells of origin of many cancers, and the identification of such cells is, therefore, key to understanding the aetiology of carcinogenesis. Stem cell identification strategies rely on the prospective isolation of candidate cell populations using cell surface markers, followed by in vivo functional assays in mouse models. The accurate definition and characterisation of a potential stem cell population, however, is dependent on its purity. The normal breast is a heterogeneous tissue, consisting of two epithelial layers, an inner luminal epithelial layer and an outer myoepithelial/basal layer, as well as non-epithelial cells. The enormous epithelial proliferation, differentiation and regression that occurs with each pregnancy provides indirect evidence for the existence of a mammary epithelial stem cell. Transplant data suggest three different mammary stem cell compartments may exist.and candidate cell types have been suggested (reviewed in ). Histological studies have suggested that stem cells within the mammary epithelium are likely to reside in a suprabasal location. However, the direct identification of mammary gland stem cells remains elusive. This is predominantly due to the paucity of appropriate markers for the separation of the luminal epithelial and myoepithelial/basal cell populations into their component subpopulations. Such separation is necessary in order to accurately assay populations of putative mammary stem cells by cleared fat pad transplantation.

CD24 has generated recent interest as a potential marker of human breast cancer stem cells. To explore the potential of this marker for isolating subpopulations of normal epithelial cells from the adult virgin mouse mammary gland, we used flow cytometry to investigate CD24 expression in mouse mammary cell preparations. Staining with CD24 revealed three distinct cell populations: CD24, CD24 and CD24. Analysis of cytoskeletal antigen staining and of gene expression patterns demonstrated that these populations represented luminal epithelial, myoepithelial and non-epithelial cells, respectively. Mammary fat pad repopulation assays revealed that the CD24 population is enriched for stem/progenitor activity.

CD24-based separation of mouse mammary luminal epithelial and myoepithelial cell populations represents a major advance towards the prospective isolation of mammary stem cells.



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