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Tbx3 improves the germ-line competency of induced pluripotent stem cells

Abstract

Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells1. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

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Figure 1: Global gene expression profiling reveals Tbx3 aids cell fusion-mediated reprogramming.
Figure 2: Generation of iPS cells with Oct4, Sox2, Klf4 and Tbx3 retroviral transduction.
Figure 3: Transcriptome of iPS cell clones generated with different combinations of reprogramming factors.
Figure 4: OSKT iPS cells show enhanced germ-line contribution and transmission.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

All microarray and sequencing data are available from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession codes GSE19164 and GSE19219.

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Acknowledgements

This work is supported by the Agency for Science, Technology and Research (Singapore) and the Singapore Stem Cell Consortium grant (SSCC-06-003). The work is also partially supported by National Institutes of Health (NIH) grants to B.L. (DK047636 and AI54973). W.-L.T. is supported by the A*STAR Post-doctoral Fellowship. We acknowledge the Genome Technology and Biology group for technical support. We are grateful to T. Huber, P. Robson and L. Stanton for critical comments, V. Cacheux-Rataboul for karyotype analyses, Y. Loon Lee, P. Gaughwin and colleagues from the Stem Cell and Developmental Biology group for technical assistance. We also thank T. Wakayama and N. Li for imparting mouse embryo manipulation techniques.

Author Contributions J.H., W.-L.T. and B.L. conceptualized and designed the experiments; J.H. and W.L.T. performed most of the experiments and analysed the data; P.Y. and J.Z. performed molecular cloning and ChIP experiments; S.L.L. performed Southern hybridizations; J.T., P.L. and S.C. assisted with cell culture and mouse embryo manipulation; B.S.S. performed microarray and quantitative PCR measurements; H.Y. and Y.L.O. performed bioinformatics analyses; T.L. and H.-H.N. analysed the data and commented on the manuscript. W.L.T. and B.L. conceived the study and wrote the manuscript.

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Correspondence to Wai-Leong Tam or Bing Lim.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-19 with Legends, Supplementary Methods and Supplementary References. (PDF 4575 kb)

Supplementary Table 1

This table shows genes upregulated in Nanog over-expressing ESCs. (XLS 1967 kb)

Supplementary Table 2

This table shows genes upregulated in Tcf3 RNAi ESCs. (XLS 2269 kb)

Supplementary Table 3

This table shows genes downregulated in Tbx3 RNAi ESCs. (XLS 11573 kb)

Supplementary Table 4

This table shows genes upregulated in Nanog OE ESC/MEF hybrids. (XLS 1455 kb)

Supplementary Table 5

This table shows genes upregulated in Tcf3 RNAi ESC/MEF hybrids. (XLS 2247 kb)

Supplementary Table 6

This table shows Tbx3 bound sites obtained by Solexa sequencing (of tags 7 and above). (XLS 273 kb)

Supplementary Table 7

This table shows summary of iPS clone ID and analyses performed on these clones. (XLS 33 kb)

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Han, J., Yuan, P., Yang, H. et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463, 1096–1100 (2010). https://doi.org/10.1038/nature08735

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