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While accumulating evidence demonstrates the existence of prostate cancer stem cells (PCSCs), PCSCs have not been isolated and thoroughly characterized. We report here the enrichment and characterization of sphere-propagating cells with stem-like properties from DU145 PC cells in a defined serum-free medium (SFM). Approximately 1.25% of monolayer DU145 cells formed spheres in SFM and 26% of sphere cells formed secondary spheres. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins (34βE12 and CK18) and for cancer stem cell markers, including CD44, CD24, and integrin α2β1. Upon culturing spheres under differentiating media conditions in the presence of 10% serum, cells positive for CD44 and CD24 were substantially reduced. Furthermore, spheres could be generated from the sphere-derived adherent cell cultures and xenograft tumors, demonstrating the …