Reprogramming into Cancer Stem Cell pluripotency of iESCs, 1st iCSCs, and 2nd CSCs, the cells were transplanted into kidney capsules or inguinal regions of SCID mice. Formation of teratomas containing ecotoderm, mesoderm, and endoderm derivatives was detected with hematoxylin and eosin staining at high frequency in all cell types (Fig. 2B), indicating that the three cell types were stem cells acquiring pluripotency, despite having epithelial cell morphology. Gene expression profile of iESCs and iCSCs To examine the gene expression in iESCs and iCSCs, global gene expression profiles detected by gene expression microarray analyses were compared. Among TIG1, iESCs, 1st iCSCs, and iPSCs, iESCs and iCSCs closely resemble each other. Interest- ingly, iESCs and iCSCs were more similar to somatic cells, and TIG1 rather than human pluripotent cells (iPSCs and ES cells), even with the consistent high expression of the exogenous Oct4, Sox2, Klf4, and c-Myc (Fig. S2 and Fig. 3A and 3B). Consistent with this, data on heat map analyses demonstrated that iESCs maintained an intermediate state between somatic fibroblasts TIG1 and pluripotent iPSCs (Fig. 3A). In more detail, scatter plot analyses showed that, in iESCs, expression of somatic marker genes MAB21L1 and NR2F2 was high to iPSCs, whereas expression of pluripotent marker genes TDGF1, NANOG, ZIC2, and TPD52 similar to iPSCs (Fig. 3A). Similar to iESCs, 1st iCSCs were characterized by expression of some somatic marker genes. RT-PCR analyses verified that the expression of endogenous pluripotent marker genes, OCT4, SOX2, NANOG, REX1, LIN28, was obviously low, while somatic marker genes, EMP1, PPARc, Figure 1. Generation of iPSC, induced epithelial stem cell (iESC), and induced cancer stem-like cell (iCSC) lines. (A) Generation of iPSCs and iESCs through direct reprogramming of primary fetal fibroblasts (TIG1). (B) Generation of the first (1st) iCSCs by picking a cell clump (yellow circle) through iESCs-derived embryoid bodies (iESC EB) formation. (C) Generation of the second (2nd) iCSCs by picking a cell clump (yellow circle) through serial transplantation of the 1st iCSCs. doi:10.1371/journal.pone.0048699.g001 PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e48699