Oct4/GFP and Nestin/GFP cells initiate tumor growth. (A-F) Studies show differences in tumor initiation of different cell populations isolated from H1299, A549, and U1752 cell lines. (A) By FACS, 106 GFP (+) cells were isolated from H1299 cells stably expressing Oct4/GFP, Nestin/GFP, or SM22/GFP expression vectors and injected subcutaneously into nude mice. (B) 105 GFP (+) cells isolated form Vector/GFP and Oct4/GFP cells were injected into nude mice. GFP (−) cells were also isolated from Oct4/GFP (+) cells that became GFP (−) after being in cell culture for 6 weeks. (C-D) By FACS, 105 GFP (+) were isolated from A549 and U1752 cells stably expressing Vector/GFP, Oct4/GFP, Nestin/GFP, SM22/GFP expression vectors and injected subcutaneously into nude mice. GFP (−) cells were isolated from Oct4/GFP cells. (E) 105 GFP (+) and GFP (−) cells isolated from U1752 Oct4/GFP cells were injected into nude mice with Matrigel. These studies show that Oct4/GFP and Nestin/GFP cells are more tumorigenic than Vector/GFP, GFP (−), and SM22 cells, which did not form tumors at these cell concentrations. In addition, Nestin/GFP cells grow faster than Oct4/GFP cells. (F) 104 GFP (+) cells isolated from Oct4/GFP and Nestin/GFP tumors re-established tumor after repeated injections into nude mice.