In addition to the expression of typical prostate CSC biomarkers (CD44, CD117, CD133, and ALDH1A1) and crucial stemness- and pluripotency-regulating transcription factors (Sox2, Oct4, Nanog, and KLF4), the augmented expression of essential stemness- and pluripotency-associated factors in PC (Bmi-1, ABCG2, EZH2, TG2, Trop2, and KLK3) in CD117+ cells isolated from PC spheroids demonstrated the CSC-enrichment efficiency of our 3D PC cell culture model. This evidence concerns the gene NANOG and pachyonychia congenita.