Fig. 4.

In vitro differentiation of normal prostaspheres with extended culture. A, By d-10 of culture, the cells located in the prostasphere center began to differentiate, forming a double-layered structure of 100–150 μm in diameter. B, Immunofluorescent labeling of d-10 prostaspheres with p63 (green) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue) shows that the basal cells (aqua-green) are located in the outer layer, whereas the more centrally located cells are p63 negative. The green signal in the compacted prostasphere center was nonspecific and appeared when IgG was substituted for primary antibody (inset). C, Immunofluorescent labeling of d-10 prostaspheres with CK8 (green), NKX3.1 (red-purple), and DAPI nuclear stain (blue). The positively stained cells are located toward the prostasphere center and represent the differentiated cells. D–F, Prostaspheres cultured through d-30 began to grow laterally, forming ductal-like structures (D and E) with lumen formation (F). Scale bar, 50 μm (A–F). G, Steroid receptor and PSA expression levels in d-7 and d-30 prostaspheres (PS) and in parental PrEC grown in 2D culture as measured by real-time RT-PCR. Data were normalized to GAPDH in each sample and expressed relative to d-7 prostasphere levels set as 1. Day-30 prostasphere cells and the primary epithelial cell cultures from human prostate expressed AR, higher levels of all ER and PR, as well as PSA when compared with the undifferentiated d-7 prostasphere cells.