Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine

• Bovine mammary subpopulations are separated according to in vitro morphology.
• The phenotype of the two different subpopulations is assessed.
• In vivo ability to regenerate bovine mammary structures is assessed.
• Adult mammary stem cells are retained during in vitro culture.
• Adult mammary stem cells can be found only in the myoepithelial fraction.

Adult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors.We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells.When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted.We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology.