Oct4/GFP (+) cells give rise to cells expressing nestin and NeuN. 105 GFP (+) cells were isolated from Oct4/GFP cells from A549 and H1299 cells and were injected subcutaneously into nude mice. IHC was performed on the tumors for nestin and NeuN from (A-C) A549 Oct4/GFP and (D-F) H1299 Oct4/GFP cells. (A,D) Represent negative controls. (B,E) Cancer cells expressing nestin (black arrow) were located along the periphery of the tumor acini (white arrow). (C,F) Cancer cells expressing NeuN (black arrows) were located toward the center of the tumor acini. (G-J) By FACS, GFP (+) cells were isolated from H1299 Oct4/GFP cells and single cells grown on glass cover slips for 2 weeks. Immunoflourscent imaging was performed on colonies for expression of GFP, Nestin, or NeuN. Shown are representative images of single colonies. (G) Immunflourescent image for GFP showing only a portion of cells in the colony express GFP. Arrows show the border of the colony. (H) Dual immunoflourescent for NeuN (red with white arrows) and GFP (green). (I) Immunoflourescent imaging for nestin (red) and (J) its corresponding phase contrast image. White arrows show cells expressing nestin.