Fig. 2. NANOG overexpressing cells can be serially passaged in the absence of feeders. (A) Wild-type and NANOG overexpressing cells were seeded on laminin-coated plates at a density of 2x10⁴ cells per cm² and passaged in the absence of conditioned media. Photos were taken of the third passage after withdrawal of conditioned media. Whereas wild-type cells stopped proliferating and showed differentiated morphology, NANOG overexpressing cells continued to proliferate for more than 15 passages and formed colonies with ES cell morphology. (B) OCT4 expression was examined on the 15th passage and was still positive in most of the cells. (C) TRA-1-60 staining performed 20 passages after CM withdrawal showed that the majority of the cells (more than 90%) remained undifferentiated. (I) MEF cells only, (II) wild-type cells grown on feeders, (III) clone grown on feeders, (IV) clone grown for 20 passages without feeders or CM. (D) After 15 passages in the absence of CM, the cells still formed teratomas composed of multiple cell types following injection into immunodeficient mice.