Stem cells and cell hierarchy in the bovine mammary gland

Abstract Text:

Elucidating cell hierarchy and lineage commitment in the mammary gland is fundamental for understanding its development, and for establishing methodologies aimed at increased production via stem-cell manipulation. Here, we demonstrate the existence of bovine mammary stem cells and describe enrichment and transplantation methodologies, and attempts made to manipulate this population. Lin^- bovine mammary epithelial cells (bMECs) from Holstein heifers were sorted according to CD24 and CD49f expression into four populations. The CD24^medCD49f^pos-enriched population maintained high expression of basal markers. In culture, it generated luminal and basal clones and had high floating-sphere formation and growth rate. Upon transplantation into cleared mouse mammary fat pad, it gave rise to multilayered outgrowths with self-renewing properties. This population was positioned at the top of the cell hierarchy and referred to as the stem cell population. A more committed, bipotent basal population generated both luminal and basal clones in vitro, but was almost completely restricted to generating unilayered basal outgrowths. Together with the luminally restricted progenitor population, it may serve as a reservoir for the highly differentiated luminal cells. Two markers, E-cadherin and miR-200c, whose expression levels correlate with differentiation, assisted in more comprehensive delineation of the bovine mammary cell hierarchy. Xanthosine administration did not affect the proportion of stem cells in bovine implants transplanted into the cleared mouse fat pad. However, it had a latent negative effect on cell proliferation and may, therefore, interfere with mammary gland development, but also limit tumor growth.  To analyze the development of bovine mammary morphology in the mouse mammary stroma, bMECs were transplanted into the cleared mammary fat pad of immunodeficient mice. Multilayered hollow spheres developed within fibrotic areas. They shared morphological and immunohistochemical characteristics with the heifer gland, but did not extend via ductal morphology. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro, paracrine inhibition of bovine epithelial mammosphere development by adipocytes was recorded, and antagonized by FGF administration. This indicates an active equilibrium between inhibitory and promotive effects exerted by the adipose and fibrotic regions of the stroma, respectively. Together, these findings imply that unique bovine mammary cell properties are integrated within a conserved mammary cell hierarchy paradigm delineated in their mouse and human counterparts.

Keywords: bovine, cell hierarchy, mammary gland, stem cells