Fig. 1.

Isolation and growth of prostaspheres from primary cultures of normal human PrEC. A, Normal human PrEC grown in 2D culture were used as a source for stem/progenitor cells. A small fraction (0.2–1.0%) of PrEC was differentially selected and formed prostaspheres in a 3D matrigel culture system. At the early stage of prostasphere formation, single prostate progenitor cells divided and formed two- (B), four- (C), and eight-cell (D) structures. E, At d-3 of culture, early sphere formation was observed. F, Typical d-4 prostasphere 25–40 μm in diameter. G, Prostaspheres continued to grow, reaching approximately 60–90 μm in diameter by d-7. H, Day-7 prostasphere after 12-h bromodeoxyuridine incorporation (pink) revealed proliferation rates of 30–50% in the progenitor cells. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). I–Q, To document clonal origin of prostaspheres from single cells, equal amounts of Hoechst-labeled and -unlabeled PrEC cells were mixed and placed in 3D culture. I and J, At culture d-2, a dividing progenitor cell at two-cell stage (I) transferred Hoechst stain to the daughter cell as seen using the blue fluorescent channel (J). K–L, Culture d-4 prostasphere with a brightfield (K) or blue fluorescent channel (L) shows all progenitor cells in the spheroid positive for Hoechst blue staining. M and N, Another prostasphere in the mixed culture at d-4 (M) was entirely negative under the blue fluorescent channel (N). O–Q, Day-4 prostaspheres from mixed 3D cultures were transferred to 2D culture for overnight attachment and outgrowth. Blue fluorescent channel viewing shows Hoechst-labeled nuclei (O), whereas red fluorescent channel viewing shows propidium iodide nuclear counterstain (P). When merged (Q), all cells in a single prostasphere are shown to be Hoechst positive (pink). Scale bar, 50 μm.