Self-Renewal and Pluripotency Acquired through Somatic Reprogramming to Human Cancer Stem Cells Shogo Nagata, Kunio Hirano, Michele Kanemori, Liang-Tso Sun, Takashi Tada* Department of Stem Cell Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Abstract Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re- plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARc. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc- related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor. Citation: Nagata S, Hirano K, Kanemori M, Sun L-T, Tada T (2012) Self-Renewal and Pluripotency Acquired through Somatic Reprogramming to Human Cancer Stem Cells. PLoS ONE 7(11): e48699. doi:10.1371/journal.pone.0048699 Editor: Martin Pera, University of Melbourne, Australia Received July 18, 2012; Accepted September 28, 2012; Published November 8, 2012 Copyright: 2012 Nagata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by the JSPS Kakenhi, grant number 23390067. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ttada@frontier.kyoto-u.ac.jp Introduction Cancer stem cells (CSCs), which are subpopulations of tumor cells, function in maintaining cancers through initiation and propagation of perpetuating tumor growth [1]. CSCs are thought to be key targets of cancer therapies, but the details of their genetic and epigenetic signatures are unclear. As a gold standard to define CSC properties, a serial transplantation assay based on the ability to self-renew and generate tumors has been widely used [2]. A crucial event in initiating cancers is activation of the self-renewal machinery, which is normally limited to stem cells. Therefore, it is likely that CSCs share several gene expression signatures detected in pluripotent stem cells. In fact, pluripotent marker genes, Oct4 and Nanog, are expressed in some cancers [3,4], and oncogene Myc is involved in the generation of many cancers [5]. Forced expression of a combination of transcriptional factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), can promote direct reprogramming of human and mouse somatic cells into induced pluripotent stem cells (iPSCs) [6,7]. In direct reprogramming, Oct4 and Sox2, which are core pluripotency factors, function as regulators of developmental and transcription-associated processes [8], Myc targets genes predominantly involved in cellular metabolism, cell cycle, and protein synthesis pathways [9]. Furthermore, Myc functions to increase efficiency by regulating the p53 pathway [10]. This evidence indicates that common pathways could be used both in the acquisition of pluripotency and tumorigenesis. In humans, CSC-like cells were transformed from primary skin fibroblasts by the stable expression of hTERT, H- RasV12, and SV40 LT and ST antigens [11]. In mice, CSCs were generated from mouse induced pluripotent stem cells (iPSCs) by culture with a conditioned medium of cancer cell lines, which was a mimic of the carcinoma microenvironment [12]. Thus, global change of the transcription signature through direct reprogram- ming or alternative culture conditions could promote the trans- formation to CSCs. In this context, it is possible that forced expression of OSKM in somatic cells induces direct reprogram- ming into CSCs. To address the molecular mechanisms involved in embryonic stem (ES) cells and CSCs, three functionally different gene sets, called the Core (core pluripotency factors), PRC (polycomb repressive complex factors), and Myc (Myc-related factors) modules, proposed recently were used for comparative analyses of global gene activity between different types of cells [9]. Here, in order to address questions of whether human induced cancer stem-like cells (iCSCs) can be generated by somatic reprogramming through conventional OSKM viral induction, and how human iCSCs, but not iPSCs, are generated, first we isolated iCSCs from cell populations, which acquired the ability to self-renew after forced expression of exogenous OSKM in human somatic fibroblasts TIG1. iCSCs have the property of pluripotency as verified by teratoma formation through serial transplantation to immunodeficient mice. Notably, the gene expression signature PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e48699