Lrp5 in Mammary Stem Cells Figure 2. Lrp5 is Expressed by Basal Mammary Epithelial Cells. A) BALB/c virgin MECs were stained with CD24, CD49f, and Lrp5 prior to FACS analysis. MECs were gated for positive (blue) and negative (red) Lrp5 expression and the subsequent CD24/CD49f profile of the populations were overlaid. B) Basal MECs were gated based on the magnitude of CD24/CD49f expression level, as CD24low/CD49flow (1, red) or CD24high/CD49fhigh (2, black). The relative Lrp5 expression levels of each population were then overlaid as histograms. C) Basal, Luminal, Lrp5+, Lrp5-, and whole population (WP) MEC fractions (gates show in A) were isolated by FACS and subsequently stained for K5 and K8 to confirm their purity and cellular phenotype. doi:10.1371/journal.pone.0006594.g002 Lrp6 expression is greater in basal cells expressing high levels of CD49f and CD24 (Fig. 3B). Furthermore, staining of isolated Lrp6 positive cells for K5 and K8 showed that these cells are predominantly basal (Fig. 3C). We conclude that most basal cells express both Lrp5 and Lrp6. High Lrp5 Expression Identifies a Stem Cell Enriched Population Previously, CD24+CD49fhigh antibody staining has been shown to identify a population of MECs highly enriched in stem cell activity, termed mammary repopulating units (MRU) [33,34]. The stem cell frequency of the MRU fraction was estimated to be 1/ 60–1/90 cells based on ductal outgrowth upon transplantation into cleared fat pads [34,35]. To evaluate the functional stem cell activity of Lrp5-expressing cells, we separated high- and low Lrp5- expressing cells. Flow cytometric analysis using differential C49f/ CD24 staining pattern showed that the Lrp5-high cells were enriched in the MRU fraction (Fig. 4A). Approximately 50% of Lrp5-high cells were distributed to a previously undefined region of the CD24/CD49f FACS profile. Further analysis revealed this fraction primarily contained von Willebrand factor (VWB) positive endothelial cells (Fig. 4B). Although CD31 positive cells were excluded by previous gating, sorting and staining of Lrp5 negative and positive populations, confirmed that approximately 40% of the Lrp5-positive cell fraction was VWB positive (Fig. 4C). Transplantation of Lrp5 high cells in limiting dilutions into cleared fat pads demonstrated that Lrp5 high cells possess significantly augmented ductal stem cell activity compared to the whole population (Fig. 5A). The estimated stem cell frequency was found to increase from 1/7,760 cells in the whole population to 1/ 485 in the Lrp5-high fraction (a 16-fold enrichment in mammary stem cells; Fig. 5B). In addition, Lrp5 negative cells were depleted of ductal stem cell activity (1/107,272 cells) compared to the whole population, and the overall recovery of stem cell function in the Lrp5-high fraction was 80%. Based on this one marker (and assuming a 50% dilution with VWB-positive endothelial cells), the overall stem cell enrichment is only 3-fold lower than in the MRU fractionation protocol (p = 0.049, Fig. 5B). Using limiting cell dilutions (10k C57Bl6 MECs) and scoring both the robust outgrowths and partial outgrowths, we confirmed that Lrp5-/cells have less stem cell activity. Interestingly, outgrowths from mixtures of control and Lrp52/2 cells contained labeled (control) and unlabeled Lrp52/2 cells. In other words, outgrowths were not clonal and Lrp52/2 cells can contribute to outgrowths in the presence of wild type cells (Figure S3). Although stem cell populations are clearly depleted in Lrp52/2 glands, this has no obvious effect on function of glands during pregnancy; indeed there are no differences in lactation of multiparous Lrp52/2 breeders, or in their mammary ductal trees (Figure S4), despite at least 8 months of estrus cycling and 3 pregnancies. We conclude that regenerative basal stem cells are not required to contribute to lobuloalveolar development. Lrp5 Expression is Required for Maintenance of the Basal Cell Layer Since Lrp5 positive cells are localized to the basal epithelial layer and ductal stem cell activity is depleted in Lrp5 mammary glands, we quantified basal cells in populations of PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6594