In contrast, cells at the bottom of the hierarchy display limited growth rate, as they are post-mitotic or near that period. bMECs of the sorted populations were seeded at a low density that enabled statistical estimation of their proliferation rate by day 7 of culture . Propagation rate was calculated as the average number of cells added per hour to the culture after seeding. Interestingly, the puStm and Basal populations exhibited significantly higher growth than the puPgt and Lum populations , with the highest value being maintained by the puStm cells. Apparently, this population preserved the highest number of proliferating cells over this culture period. The Lum population displayed negative growth, consistent with the considerable presence of mature cells in this population. MaSCs differentiate in vivo into the full repertoire of epithelial cells that comprise the functional mammary gland . To test whether this ability can be recapitulated in vitro, cells sorted into the four populations: puStm, Basal, puPgt and Lum, were cultured separately for 7 days and sorted again according to CD24 and CD49f Nutlin-3 Mdm2 inhibitor expression . Preliminary studies established constitutive CD49f expression throughout culture, while CD24 expression was KRX-0401 markedly reduced . Thus, the second sorting was based only on distinction between CD24- and CD49fexpressing and non-expressing populations . Subpopulations P1 through P4 were cultured separately and the resulting colonies were morphologically analyzed and stained for the lineage markers CK14 and CK18 . Two types of colonies were observed: organized colonies which were typically round and compact . These colonies consisted of densely clustered polygonal cells expressing CK14 at the rim and CK18 at the center, thus resembling a duct-like alignment; non-organized colonies consisting of less dense, elongated cells comprising a non-descript outline and promiscuous expression of CK14 and/or CK18. Irrespective of CD24 and/or CD49f expression levels in the individual populations collected at the second sorting, most colonies that developed from cells of the original puStm and Basal populations maintained an organized phenotype, while most of those that originated from the puPgt and Lum populations were unorganized. Taken together, these results indicated that essential properties of the puStm and Basal populations are preserved in culture, as cells of these populations assemble into an organized duct-like alignment of two distinct layers. In addition, the findings imply that CD24 and CD49f expression levels may not be useful for distinguishing cultured bMEC populations. Under non-adherent culture conditions, mammospheres develop from MaSCs that have escaped anoikis.