Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels, which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks, the gels contain bilayered mammary epithelial structures, including luminal and myoepithelial cells, their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations.

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted, myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM), alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18, keratin 19, EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors.

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.

The developmental relationships between the different mammary epithelial cell lineages in the human mammary gland are not well defined. To characterize human breast epithelial cells (HBEC) with progenitor activity, we used flow cytometry and single cell sorting to analyze the distribution of cellular phenotypes in primary cultures of reduction mammoplasties and their associated ability to generate colonies in 2-dimensional (D) and 3-D (collagen gel) culture systems. This approach allowed two distinct types of HBEC progenitor populations to be distinguished on the basis of their differential expression of the MUC-1 glycoprotein, CALLA/CD10 and epithelial-specific antigen (ESA). The first type of progenitor, which is enriched in the MUC-1+/CAL-LA-/ESA+ subpopulation, generated colonies of tightly arranged cells in 2-D cultures and small alveolar-like colonies with a central lumen when cultured in a collagen matrix. The cells produced in the colonies and derived from these MUC-1+/CALLA-/ESA+ progenitors were found to express typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, which is present in the MUC-1-to +/-/CALLA +/- to +/ESA+ subpopulation, generated mixed colonies of both luminal and myoepithelial cells when seeded in 2-D and 3-D cultures. In 2-D cultures, the centrally located cells exhibited a luminal morphology and expressed ESA, but were heterogeneous in their expression of MUC-1. Radiating from the periphery of these ESA+ HBEC were highly refractile ESA- teardrop-shaped myoepithelial-like cells. When cultured in a collagen matrix, these bipotent progenitors generated large branched colonies composed of a heterogeneous population of cells, with some of the progeny cells expressing luminal epitopes (keratin 8/18, keratin 19 and MUC-1) and others expressing myoepithelial epitopes (keratin 14 and CD44v6). A third type of progenitor, which became apparent is passaged HBEC cultures and was enriched in the MUC-1-/CALLA+/ESA- subpopulation, was found to generate colonies of cells with an exclusively myoepithelial phenotype. These results provide definitive evidence for the existence of multilineage HBEC progenitors in normal adult human mammary tissue. The phenotypic profile of these cells suggest that these multilineage progenitors are a relatively undifferentiated cell since they express low levels of MUC-1 and that they have a luminal location within the mammary epithelium since they are ESA+. Furthermore, we suggest that the MUC-1+/CALLA-/ESA+ and the MUC-1- to +/-/CALLA +/- to +/ESA+ progenitors we have identified and characterized are candidate in vivo alveolar and ductal progenitors, respectively.