demonstrated that iCSCs persist certain somatic cell memory even after up-regulation of pluripotent marker genes through repro- gramming. Our findings revealed that up-regulation of gene sets for the Core and Myc modules is sufficient to confer the properties of self-renewal and pluripotency, and sequential down-regulation of gene sets for the PRC module is required to install the proper iPSC signature on somatic cells. These findings demonstrate that iCSCs and iPSCs share a reprogramming pathway from somatic nuclei into pluripotent and self-renewable nuclei, and then diverge to iCSCs or iPSCs. Materials and Methods Ethics statement Experiments with mice were performed according to the institutional guideline of Kyoto University, Japan. Our animal experiments (W-3-6) are reviewed and permitted by the animal research committee of Kyoto University, Japan. Cell culture Human fetal lung fibroblasts (TIG1) provided by the JCRB Cell Bank were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, USA) containing 10% FBS, and were infected with Oct4, Sox2, Klf4 and c-Myc retroviruses. At day 4 after infection, the cells were reseeded into a 10 cm culture dish on feeder cells. At day 5 after infection, culture medium was changed to iPSC medium (DMEM/Nutrient Mixture F-12 Ham (DMEM/ F12) (Sigma-Aldrich) supplemented with 20% of knockout serum replacement (Invitrogen, USA), 10 ng/ml bFGF (Peprotech, USA), L-glutamine, and non-essential amino acids and 2- mercaptoethanol). Colonies, which could self-renew and expand, were picked up and reseeded onto feeder cells around day 30. To isolate human iPSCs and induced epithelial stem cells (iESCs), each colony was picked up and reseeded into Matrigel-coated dishes with mouse embryonic fibroblast (MEF)-conditioned iPSC medium. For embryoid body (EB) formation, small iESC aggregates were formed by overnight hanging drop culture in MEF-conditioned iPSC medium. Aggregates were grown in bacterial culture dishes for 5–7 days, and then cultured in gelatin-coated dish with DMEM containing 10% FBS. For tumor-derived cell culture, tumors were cut into small pieces, dissociated with collagenase, and plated on gelatin-coated dishes with 10% FBS containing DMEM. Established iCSC lines were maintained in gelatin-coated dish with DMEM/F12 (Wako, Japan) supplemented with 15% FBS, 1000 U/ml LIF (Chemicon, USA), L-glutamine, penicillin-streptomycin, sodium bicarbonate, sodium pyruvate, and 2-mercaptoethanol through passage using 0.25% trypsin/EDTA. RT-PCR For RT-PCR analyses, total RNA of cultured cells was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized from 1 mg total RNA with Superscript III (Invitrogen) using random hexamers following the manufacturer’s instructions. All PCR experiments were performed with the annealing temperature at 57uC. Primer sequences used in this study are summarized in Table S1. Immunocytochemistry Cultured cells were fixed with 4% PFA (paraformaldehyde)/ PBS (phosphate-buffered saline) for 10 minutes at room temper- ature, washed with PBST (0.1% Triton X-100 in PBS), then pre- treated with blocking solution (3% BSA and 2% skim milk Reprogramming into Cancer Stem Cell (DIFCO, USA) in PBST) at 4uC overnight. The cells were incubated with primary antibodies; anti-OCT4 (1:50; Santa Cruz Biotechnology, USA), anti-SOX2 (1:500; Abcam, UK) and anti- NANOG (1:200; ReproCELL, Japan), anti-CDH1 (1:200; Ta- KaRa, Japan), anti-SSEA4 (1:500; Hybridoma Bank, USA), and anti-TRA-1-60 (1:500; Millipore, USA) antibodies at 4uC over- night. They were then incubated with secondary antibodies; anti- rabbit IgG or anti-mouse IgG conjugated with Alexa 488 (1:500; Molecular Probes, USA) in blocking buffer for 1 hour at room temperature. The cells were counterstained with DAPI (4,6- diamidino-2-phenylindole) and mounted with a SlowFade light antifade kit (Invitrogen). Tumor formation A cell suspension of 5.06105 iESCs or iCSCs in 200 ml DMEM was subcutaneously injected into the inguinal region or trans- planted into the kidney capsules of immunodeficient SCID mice (CLEA, Japan). Tumors were surgically dissected out 5–7 weeks after implantation, fixed with 4% paraformaldehyde in PBS and embedded in paraffin. Sections 5 mm in thickness were stained with hematoxylin and eosin. Microarray and data processing For microarray analyses, 1 mg total RNA was labeled according to standard Affymetrix protocols and hybridized to the Affymetrix human genome U133 Plus 2.0 Array (samples of human origin). Raw data were normalized by the MAS 5.0 method using the bioconductor package on R program (http://www.r-project.org/). Heat maps of the gene expression profile for all genes in each cell line were visualized by MeV program (http://www.tm4.org/mev/ ). Hierarchical clusters were calculated by Pearson’s correlation coefficient (r) and visualized by the pvclust package on R program. For scatter plot analyses, raw data were normalized by Robust Multichip Average (RMA). Scatter plots were visualized using the bioconductor package on R program. Results and Discussion Isolation of iESCs and iCSCs Human iPSCs were picked up as colonies about 30 days after retroviral transduction of Oct4, Sox2, Klf4 and c-Myc into the somatic fibroblasts TIG1, a cell line isolated from human fetal lung (Fig. 1A). The efficiency of iPSC generation was less than 1%, and the other populations of cells were differentially reprogrammed. Some of the differentially reprogrammed somatic cells showed the property of self-renewal formed colonies consisting of cells (iESCs) with epithelial cell morphology in MEF-conditioned iPSC medium (Fig. 1A). iESCs were maintained with epithelial cell morphology for around 10 passages, since were prone to differentiate (Fig. S1A). Therefore, to analyze the differentiation potential of iESCs, EB formation was induced by suspension culture (Fig. 1B). Successfully formed iESC EBs showing pluripotency as analyzed by RT-PCR (Fig. 2A) were attached on the bottom to culture dishes to isolate self-renewal stem cells. Consequently, the first (1st) iCSC lines isolated by picking cell clumps of expanded EBs in three independent experiments were stably maintained for more than 20 passages, or re-plated from frozen-stored cells (Fig. 1B). To verify the identity of the 1st iCSCs, the second (2nd) iCSCs were isolated from tumors generated by serial transplantation with injection of the 1st iCSCs into the inguinal regions of immuno- deficient SCID mice in two independent experiments (Fig. 1C). The 2nd iCSCs resembling the 1st iCSCs were stably maintained with features of epithelial cell morphology and robust cell growth for more than 20 passages (Fig. 1C). Next, to explore the PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e48699