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Generation of germline-competent induced pluripotent stem cells

Abstract

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.

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Figure 1: Nanog-GFP-IRES-Puro r transgenic mice.
Figure 2: Generation of iPS cells from MEFs of Nanog-GFP-IRES-Puro r transgenic mice.
Figure 3: Characterization of Nanog iPS cells.
Figure 4: Gene expression in Nanog iPS cells.
Figure 5: DNA methylation of ES-cell-specific genes and imprinting genes.
Figure 6: Germline chimaeras from Nanog iPS cells.

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Acknowledgements

We thank K. Takahashi, M. Nakagawa and T. Aoi for scientific discussion; M. Maeda for histological analyses; M. Narita, J. Iida, H. Miyachi and S. Kitano for technical assistance; and R. Kato, R. Iyama and Y. Ohuchi for administrative assistance. We also thank T. Kitamura for Plat-E cells and pMXs retroviral vectors, and R. Farese for RF8 ES cells. This study was supported in part by a grant from the Uehara Memorial Foundation, the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, a grant from the Leading Project of MEXT, and Grants-in-Aid for Scientific Research of JSPS and MEXT (to S.Y.). K.O. is a JSPS research fellow. The microarray data are deposited in GEO under accession number GSE7841.

Author Contributions K.O. conducted most of the experiments in this study. T.I. performed manipulation of mouse embryos to generate Nanog-GFP transgenic mice. T.I. also maintained the mouse lines. S.Y. designed and supervised the study, and prepared the manuscript. S.Y. also performed computer analyses of DNA microarray data.

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Correspondence to Shinya Yamanaka.

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The microarray data are deposited in GEO under accession number GSE7841. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13 with Legends and Supplementary Table 1, which show detailed characterization of Nanog-iPS cells. (PDF 1076 kb)

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Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007). https://doi.org/10.1038/nature05934

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