| The murine mammary system is a complex milieu of epithelial cell types that function together to support lactogenesis. The breast tissue is spatially and temporally regulated, with most growth occurring postnatally. The mature mammary gland displays two characteristic architectural features: ducts and terminal end buds. There are different cell types found in these two features that perform independent functions in the mature organ. In general, the mature gland is a bilayer system with a single layer of fibroblast-like myoepithelial cells encompassing an inner luminal epithelial layer. Upon pregnancy, the induction of alveolar secretory cells is initiated, which eventually leads to milk production. Milk is then channeled through the entire system via contraction of the myoepithelium, and eventually dispersed through the nipple. The mouse mammary gland is capable of multiple stages of development: growth, lactation and involution. These stages are regulated by cytokines and hormones depending on the developmental stage of the mouse and the physiological context (i.e. estrus). Numerous studies have shown the mammary epithelium is capable of regeneration upon transplantation. Recent reports have isolated fractions of cells enriched for duct forming ability and self-renewal. In addition, a distinct progenitor population was also identified that lacked the ability to form ducts in vivo but retained bipotentiality in vitro. This thesis builds on previous marker studies of mammary epithelial cells, using existing phenotypes to further enrich the mammary stem cell population. My data shows Thy-1, or CD90, is differentially expressed in mammary cells. In addition, Thy-1+CD24medCD49f hi cells are enriched for in vivo engraftment of ductal epithelium. These cells are approximately 10-fold enriched for duct-forming ability. Importantly, the Thy-1+CD24medCD49 hi phenotype enriched for cells that are capable of self-renewal and retain the full differentiation potential of the parent stem cells. This data provided a new phenotype for the stem cells (MaSC). The Thy-1 - CD24medCD49hi cells are not only diminished for engraftment but have significantly decreased self-renewal, suggesting they are a potential multipotent progenitor population. I have named them mammary multipotent progenitors, or MMPPs. A novel protein staining methodology revealed the Thy-1+CD24medCD49hi and Thy-1- CD24medCD49hi cells have keratin profile similar to myoepithelial cells, whereas the further differentiated progenitors initiated a luminal epithelial transcriptional program. In addition, my work shows the first description of murine Epithelial Specific Antigen (or EpCAM) using anantibody produced by the Developmental Studies Hybridoma Bank (NIH, Iowa) in the mammary gland. This antibody successfully discriminates luminal cells from myoepithelial cells, and the staining patterns mirrors ESA in the human mammary system. To further investigate the transcriptional regulation of the mammary populations, I performed microarray analysis of the Thy-1+CD24medCD49hi (MaSC), Thy-1- CD24medCD49hi(MMPP), MaCFC progenitors, differentiated myoepithelial (MYO) and differentiated luminal epithelial (EPI) cells. This work is the first description of transcriptome analysis of differentiated luminal epithelial cells directly isolated from fresh mouse tissue. The results show Thy-1+CD24medCD49 hi cells express gene programs consistent with their predicted basal location in vivo. In addition, I identified a set of genes that may be used to discriminate stem cells from myoepithelial cells. Discovering gene sets that were specific to hierarchical mammary populations prompted the hypothesis that DNA damage responses and cytokine signaling may also affect those populations in different ways. I went on to investigated how ROS and TGFbeta affect the cellular hierarchy. My studies show that stem cells with low intracellular ROS have a growth advantage in vivo. In addition, I found that exogenous TGFbeta ligand had little effect on MaSC and MMPP colony formation in vitro, but did induce a motility phenotype in early progenitor MaCFC cells. In conclusion, my thesis work led to a number of interesting and novel insights into mammary biology. By using the tools described in this thesis, it is now easier to identify which types of cells within the breast are susceptible for transformation. Knowing the cell(s) of origin for breast cancers will be the key to future therapeutic strategies. |
