done in mice as early as the 1960s. Research studies found that serial transplantations (described below) of epithelial cells into empty fat pads of other mice resulted in the regeneration of all the epithelial cell lineages for several successive generations. The presence of stem cells in these studies was indicated by the fact that the transplanted cells satisfied the two important criteria for being stem cells: the ability to differentiate into multiple lineages and the ability to self-renew. The sections below detail the methods of identifying mammary stem cells, the discovery of stem and progenitor cells, and the types of stem cells that have been proposed to give rise to the epithelial cell lineages in the breast.
Methods of Identifying Mammary Stem Cells
Two major techniques have been used to identify mammary stem cells. A single stem cell that can generate complete bi-layered glands with both luminal and basal cells is known as a Mammary Repopulating Unit (MRU), and the number of stem cells identified by any method used is quantified in terms of MRUs.
Transplantation Assays
Transplantation assays to identify the presence of stem or progenitor cells involve first isolating tissue fragments or specific cell populations from the epithelial layer of the breast. While earlier techniques used more heterogeneous populations, newer cell isolation reagents have the ability to isolate and test individual cells or more homogenous populations. These cells are then transplanted to the cleared fat pads of other mice that contain the supporting tissue but no glandular or ductal cells. If the transplanted cells had stem cell capacity, they would generate either one more of the three epithelial cell types depending upon whether they were multipotent or unipotent in nature. Further self-renewal capacity, which is an important characteristic of stem cells, can be tested by obtaining cells from the first transplantation and transferring them again to another recipient animal and observing whether they have the capacity to regenerate all epithelial cell types in the luminal and basal layer. In this method, the accuracy of identifying stem cells depends on the ability to isolate and purify cells that are being transplanted.
Lineage Tracing
Lineage tracing or tracking is a more recent technique that was created with the ability to genetically mark specific cells in the breast with molecular signatures known as reporters. Once marked, these parent cells would be able to transmit the reporter to all the daughter cells that arise through cell proliferation and differentiation. Thus the lineage of individual cells can be tracked through this method, making it easy to identify multipotent stem cells in the breast. Since both the cell type and the timing at which the reporter is activated can be controlled, lineage tracing can identify stem cells with more accuracy. Some of the drawbacks of this technique include technical difficulty in labeling specific cell populations and the off-target expression of the reporter molecule.
Mammary Stem Cells and the Epithelial Differentiation Hierarchy
The search for mammary stem and progenitor cells is based on the understanding of a differentiation hierarchy that is present in the mammary epithelial cells. This differentiation hierarchy gives rise to three adult cell types: the myoepithelial cell, the ductal cell, and the alveolar cell. Currently there are two modes of differentiation that have been proposed to explain the origin of these cells based upon the techniques used. In both cases, the primary cell of origin is a stem cell with self-renewing capacity and multipotency, termed mammary stem cell. In the first model, the stem cell gives rise to a single progenitor that is long lived but not self-renewing, which in turn gives rise to both luminal (ductal, alveolar) and myoepithelial progenitor cells, which are more restricted in their differentiation capacity. These are termed bipotent progenitors. In the second model, proposed based on lineage tracing studies, cell maintenance in the adult breast is proposed to occur from unipotent progenitor cells (which in turn arise from the single progenitor). These cells are capable of producing cells of only a single lineage (luminal, alveolar, or myoepithelial). In both cases, the restricted progenitor cells give rise to the adult cells. The existence of bipotent progenitor cells was initially thought to occur only during development, with a switch to unipotent cells occurring at birth. However, recent studies have shown that bipotent progenitors continue to exist in the adult and may contribute to the long-term maintenance of the breast. This is an active area of study that is currently determining the roles of each of these modes of differentiation in the developing and adult breast.
Identification of Adult Mammary Stem Cells (MaSCs) and Luminal Progenitor Cells
The identification a specific cell that could regenerate the entire epithelial breast lineage with intact morphological features such as ducts and glands occurred in 2006. Researchers were able to isolate a single mouse breast cell based on its specific surface proteins, visualize it under a microscope, and demonstrate its capacity to regenerate a complete functional mammary gland when transplanted into a cleared fat pad of a recipient mouse. Clonal outgrowths obtained from the recipient mouse were further transplanted into another recipient and were once again shown to regenerate a complete functional gland, which meant that the outgrowth must been generated from the self-renewing mammary stem cell. These cells were found to be localized to the basal cell population in the gland where the myoepithelial cells are present. MaSCs are difficult to identify with precision because they represent a small percentage of the basal cells, and markers to identify them are still being researched. Advances in cell dissection, isolation, and sorting techniques will also enable more accurate separation of MaSCs from other cell types in the basal cell population. Identification of lineage-restricted luminal and myoepithelial progenitors has had significantly more success, with a specific population of progenitors having been identified based both on cell surface markers and the presence or absence of receptors for the hormones estrogen and progesterone. After the identification of MaSCs in the mouse in 2006, distinct luminal progenitor cell populations were also identified. Following this, MaSCs and luminal progenitors have also been identified in humans by various research groups.
More recently, a study published in 2013 has identified a small population of cells in the human breast that exhibit significant multilineage differentiation capacity. When these cells were isolated from a larger population of mammary epithelial cells and cultured in the lab, they exhibited a capacity to differentiate into both luminal and myoepithelial cells. They were also shown to be capable of forming alveolar structures in culture. When these human cells were transplanted on to the cleared fat pads of immunodeficient mice, they also formed glandular and ductal outgrowths, showing a distinct capacity to repopulate the breast with all three cell types present. These cells were termed endogenous plastic somatic (ePS) cells. Further study demonstrated that when ePS cells were isolated from this epithelial population each of these cells were capable of differentiating into all three germ layers, indicating the presence of pluripotency characteristics. However, these cells were shown to be distinct from embryonic stem cells, induced pluripotent stem cells, or multipotent mesenchymal stem cells in that they cannot proliferate indefinitely.
Potential Role of MaSCs in Breast Cancer
One of the more direct implications of the presence of stem cells in the breast is their potential role in breast cancer. Breast cancer is one of the most common cancers among women, and significant basic and clinical research efforts have focused on understanding the biology of breast cancer to develop effective therapeutic strategies. The self-renewing nature of stem cells presents an ideal source for the origin of highly proliferative malignant tumors. With increasing knowledge of the role that stem cells may play in cancer (cancer stem cells) and the presence of stem and progenitor cells in the breast, there has been several research efforts focused on determining the presence or role of potential breast cancer stem cells. Breast cancer stem cells were first identified in 2003 based on the presence or absence of specific molecules on the cell surface. A combination of the expression of the molecule CD44 and the lack
