Our findings showed that the expression levels of stemness genes could be affected by CRC spheroid culture, and both spheroids displayed similar high expression levels of pluripotent stem cell genes (KLF4, OCT4 and C-MYC) when compared to their parental counterparts, while drastic higher expression level of SOX-2 and NANOG was found in Caco-2 and HT-29 spheres, respectively. Here, POU5F1 is linked to colorectal carcinoma.