PTN in the Mammary Gland Figure 6. PTN effect on ductal outgrowth and branching in developing mammary glands. (A) Whole mounts of representative mammary glands from mice treated with anti-PTN blocking antibody versus control. White arrows: distance of the outgrowth of a representative duct measured from the midpoint of the lymph node. Black arrowheads: branch points. Scale bar = 1.5 mm. (B to F) Controls (n = 9 mice, white), anti-PTN (n = 10 mice, grey). (B, C) Ductal length and number of branch points with the ductal length expressed in mm and the number of branch points per mm of duct. Data are means 6 SE; *P = 0.04; ** P = 0.006; n = 3 ducts per mouse. (D to F) Quantitation of Terminal Ends (TE) and Terminal End Buds (TEBs) of mammary ducts; (D) TEBs per duct, ns = not significant; (E) TEs per duct, * P = 0.03; (F) TEBs per total number of duct ends ( = TEBs + TEs), *P = 0.027. Data are means 6 SE; n = 6 ducts per mouse. doi:10.1371/journal.pone.0047876.g006 High CD29 and CD49f and low SCA-1 have previously been associated with the presence of mammary stem cells (MaSC) [61,62]. In addition, CD29 and CD49f belong to the integrin family and their activity is important for the attachment of cells to the extracellular matrix [67]. CD10, a known myoepithelial marker, was recently suggested as a progenitor cell marker with peptidase activity and has been shown to have a role in cleaving signaling molecules that induce differentiation [62,66]. The expression of CD29 and CD49f slightly increased while that of SCA-1 and CD10 decreased during the passaging and the 3D culturing of control MECs from passage #1 to #3 (Figure 8A). However, MECs treated with the PTN blocking antibody, showed no significant changes (CD29, CD49f) or only small changes (SCA-1, CD10) relative to MECs at passage #1 (Figure 8A). As mentioned above, treated MECs were able to form a higher number of spheres with correct apical organization and laminin deposition compared to control MECs (Figure 5). Overall our data suggests that endogenous PTN expression maintains the epithelial cells in an undifferentiated phenotype and that blocking its activity stimulates the expansion and differentiation of mammary progen- itor cells as indicated by the altered CD29, CD49f, SCA-1 and CD10 expression profile. A role for PTN as a stem and progenitor cell regulator has already been described in the brain, bone and bone marrow [29,31,68] our data also suggest such a role for MECs. PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47876