Spheres from 293T cells express embryonic transcription factors implicated in self-renewal. A) RT-PCR demonstrated higher expression of embryonic stem cell specific genes including Oct4, Nanog, Sox2 and Klf4 in 3D spheres of 293T cells but no expression was observed in 2D monolayer cells. B) Genes implicated in promoting survival of cancer stem cells such as β-catenin, survivin and Rex-1 were upregulated in 3D spheres vs. 2D monolayer cells. C) Serial passage for more than 4 weeks without additional PKH-26 label (Red) reveals serial dilution to a single long term label-retaining cell per sphere suggestive of asymmetric self-renewal. D) Immunocytochemistry reveals the majority of 293T cells as keratin 14 positive.