Reprogramming into Cancer Stem Cell Figure 2. Pluripotent tumorigenicity of iESCs and iCSCs. (A) Expression of exogenous, pluripotent marker, and lineage marker genes in embryoid bodies (EBs) formed with iESCs and iPSCs by RT-PCR analyses. (B) Paraffin sections of teratomas, which were generated by iESCs, 1st iCSCs, and 2nd iCSCs implantation, were stained with hematoxylin and eosin. NE, neuroepithelium (ectoderm); CA, cartilage (ectoderm); MU, muscle (mesoderm); RE, respiratory epithelium (endoderm). doi:10.1371/journal.pone.0048699.g002 FOXF2, and NR2F2 were highly expressed in the 1st and 2nd iCSCs (Fig. 3A and 3B), indicating that epigenetic reprogramming went halfway to erasing the somatic memory and establishing a pluripotent transcription network. Low expression of NANOG was detected by immunocytochemistry in the 1st and 2nd iCSCs, while high expression of endogenous OCT4/exogenous Oct4 and endogenous SOX2/exogenous Sox2 was detected (Fig. 3C). Immunocytochemistry analyses demonstrated that pluripotent markers CDH1 and SSEA4 were weakly expressed in iESCs and iCSCs, while highly in iPSCs. Furthermore, expression of TRA-1-60 was detected in iPSCs, but not iESCs and iCSCs (Fig. S3). Collectively, iESCs and iCSCs retained a cellular state between TIG1 and iPSCs. Acquisision of pluripotency prior to establishment of iPSC identity In the process of reprogramming somatic cells into iPSCs, cells progressively change their expression patterns and morphology. Thus, to compare gene expression profiles of different types of reprogramming cells, an integrated analysis system for expression profiles, in terms of the kinetics of modules, is required. Three ES cell modules, Core, PRC, and Myc (CPM), which are functionally separable, have been defined [9]. The Core module includes known factors in core regulatory circuitry, such as NANOG, OCT4, SOX2, TCF3, and REX1. The PRC module includes gene generally repressed in ES cells, including HOX cluster genes. The Myc module is composed of genes that are common targets of seven factors, MYC, MAX, NMYC, DMAP1, E2F1, E2F4, and ZFX. In addition, the ES cell-like module was defined to distinguish among ES cells, adult tissue stem cells, and human cancers [13]. Here, we demonstrate data with CPM modules, since analysis with data of the ES cell-like module was comparable to data of the Core module of CPM modules, which included pluripotent marker genes. To reprogram TIG1 into iESCs, up-regulation of the Core and Myc modules was required (Fig. 4 and Fig. S4). iESCs and iCSCs were characterized by the high activity of the PRC and Myc modules, while the Core module was high in iESCs and low in iCSCs. It was crucial to impose a barrier for erasing somatic memory that the PRC module retained high activity in all TIG1, iESCs, and iCSCs as shown by FOXF2 and NR2F2 (Fig. 3A). To be fully reprogrammed into iPSCs from TIG1, induction of the down-regulation of the PRC module was necessary (Fig. 4 and Fig. S4), indicating that completion of the establishment of the iPSC-transcriptional network is associated with down-regulation of the PRC module. Stable cell lines, TIG1, iCSC, and iPSC, were characterized by the reciprocal activity of the Core and PRC modules, while unstable iESCs showed simultaneous up-regulation of the two modules, suggesting that genes categorized into the two modules functioned competitively in reprogramming. Notably, acquisition of self-renewal and pluripotency was linked with up-regulation of the Core and Myc, but not PRC, modules (Fig. 4). These data indicated that the property of self-renewal and pluripotency could be conferred on somatic nuclei prior to full reprogramming into iPSCs. The finding that a certain cell population of iESCs was reprogrammed into iPSCs spontaneously supported this concept (Fig. S1B). The criteria of self-renewal and pluripotency are widely used for defining human iPSCs. However, the property of self- renewal and pluripotency can be conferred on a variety of cell PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e48699