Isolation of lung cancer cells expressing Oct4 or nestin. Lung cancer cell lines were stably expressed with Oct4 or nestin promoters that drive GFP expression (Oct4/GFP and Nestin/GFP). (A) H1299 cell showing GFP expression after stable transfection with Oct4/GFP reporter. By FACS, GFP (+) cells were sorted from the Oct4/GFP cells and placed into cell culture. After 6 weeks cells were sorted for the GFP (+) and GFP (−) populations. (A) FACS analysis shows >99% purity of GFP (+) and GFP (−) cell populations. (B) Quantitative RT-PCR shows significantly higher expression of Oct4 and Sox2 in GFP (+) cells compared to GFP (−) cells (n = 3). (C) Immunoflourescent imaging showing Oct4/GFP (+) cells stain for Oct4. (D) The GFP (+) cells were sorted from H1299 cells stably expressing Nestin/GFP reporter and placed into cell culture. After approximately 8 weeks, the GFP (+) and GFP (−) cells were isolated by FACS. (E) Quantitative RT-PCR shows higher expression of nestin in GFP (+) cells compared to GFP (−) cells. (F) Dual immunoflourescent image shows NeuN (red) is not expressed in Nestin/GFP (+) (green) cells. (G-I) GFP (+) cells were isolated from Oct4/GFP and Nestin/GFP cells. Quantitative RT-PCR demonstrates differential expression of stem regulations and BMP type I receptors between Oct4/GFP and Nestin/GFP cells (n = 2).