It also supports use of the mousebased cell-separation system, which applies lineage markers CD24 and CD49f for prospective sorting and enrichment of the bMEC populations. bMECs were sorted into putative distinct populations according to the methodology used to distinguish mouse mammary AG-013736 epithelial cells adapted here to the specific characteristics of the bovine gland. Accordingly, mammary glands of 7- to 10-month-old heifers were dissociated into organoids, which were subsequently digested into a suspension of single cells, and Lin2 cells were then obtained using mouse antibodies to CD45, CD31and TER119. Propidium iodide -stained cells were gated by FACS before cell separation, and the relatively high amount of dead cells and cell debris resulting from tissue digestion was excluded. bMECs from the mammary glands of four heifers were individually sorted into the four populations: puStm, Basal, puPgt and Lum. mRNA was extracted from cells of each population and the PD325901 expression of genes, selected according to their involvement in marking and regulating breast/mammary gland cell hierarchy, was determined by RT-PCR. A main objective of separating bMECs is the prospective enrichment of stem cells. The expression of established markers, used to identify this rare population in various mammalian tissues, was therefore examined in the four bMEC populations . Nestin is an intermediate filament protein regarded as a neural stem cell marker. Recently, it was also inferred as a putative stem cell marker in the human breast . Among the bMEC populations, Nestin was highly expressed in the basal compared to the luminal compartment, but with no significant difference between the Basal and puStm cells. Nestin��s lowest expression level in the puPgt cells distinguished them from the Lum population. Attempts to localize Nestin expression in situ failed due to the lack of bovine cross reaction of the bovine antigen with available antibodies . Lgr5 is a G-protein-coupled receptor and a Wnt target gene, implicated as a stem cell marker in the intestinal and skin epithelium . Lgr5 has also been localized to a few, scattered epithelial cells in the basal layer of the adult mouse mammary gland . In contrast, expression analysis of Lgr5 in the bMECs revealed a significant two- to fourfold higher level of expression in the Lum cells compared to the other populations. This result might infer a unique role for Lgr5 in bovine mammary cell differentiation and highlights its possible use as a marker for the differentiated luminal bMEC population. The aldehyde-oxidizing enzyme ALDH1 was originally identified as a stem cell marker in the hematopoietic system and later also in the human breast . A recent study, performed in bovine mammary cells, associated high ALDH activity with luminal cells, contrary to lower activity in myoepithelial progenitors .