352 Remodeling of Prostatic Tissue by Progenitor Cell Figure 5. Three-dimensional (3D) culture of NHPrE1 and BHPrE1 cells. A: NHPrE1 and BHPrE1 cells form globular structures similar in size and shape after 9 days in a 3D-on-top assay in 2% Matrigel as shown by bright-ﬁeld microscopy. Confocal imaging (F-actin polymerization shown by phalloidin staining) shows a degree of polarization but no obvious glandular formation in these structures. B: NHPrE1 or BHPrE1 cells produce very different structures when completely embedded in Matrigel after 9 days. NHPrE1 can give rise to elegantly branched structures, whereas BHPrE1 cells form globular structures of similar size and shape to those seen in the 3D-on-top assay. Abbreviations: BHPrE1, a sponta- neously immortalized benign human prostate epithelial cell line from BHPrE0; NHPrE1: a spontaneously immortalized ‘‘normal’’ human prostate epithelial cell line, that was established from parent NHPrE0 cells. of additional progenitor and differentiation markers are listed in supporting information. Branching Morphogenesis in 3D Culture by NHPrE1 and BHPrE1 Cells 3D culture is a valuable in vitro model for studying tissue branching, tubulogenesis, and morphogenesis. Tumorigenic cells often fail to appropriately organize and polarize in 3D culture [30]. Therefore, to determine whether NHPrE1 and BHPrE1 cells could organize appropriately in 3D culture con- ditions, cells were either embedded in 2% Matrigel (3D-on- top) or completely within Matrigel, according to previously established methods [31]. When embedded in 2% Matrigel plus HPrE-conditional medium (3D-on-top assay), polarized acinar structures were formed by both cell lines, as conﬁrmed with F-actin immunolocalization (Fig. 5A). When embedded completely in Matrigel, NHPrE1 cells sometimes initiated