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Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT–MET mechanism for optimal reprogramming

Nature Cell Biology volume 15, pages 829–838 (2013)Cite this article

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Abstract

Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4–Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-β treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-β antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT–MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT–MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.

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Figure 1: Sequential delivery of Yamanaka factors improves reprogramming efficiency in the Vc-free system.
Figure 2: The sequential delivery of OK+M+S outperforms the simultaneous OKMS in the Vc system.
Figure 3: OK+M+S activates an early EMT and delays the MET during reprogramming.
Figure 4: Expression of Slug and E-cadherin in MEFs infected with individual Yamanaka factors.
Figure 5: Modulation of reprogramming efficiency by inducing or inhibiting sequential EMT–MET in the sequential or simultaneous protocol.
Figure 6: Sequential EMT–MET promotes reprogramming in other cell lines.
Figure 7: Cell states determine the ability of OK+M+S to promote reprogramming.
Figure 8: The sequential EMT–MET model.

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Acknowledgements

This work has been supported in part by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020302, XDA01020401), the Guangzhou International Science and Technology Cooperation Projects from the Bureau of Science and Information Technology of the Guangzhou Municipal Government (2012J5100007), the Guangdong Natural Science Foundation (S2012010010087), the National Natural Science Foundation of China (31100773, 30900854) and the Major New Drugs Innovation of Major National Scientific and Technological Project (2011ZX09102-010-01).

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  1. Hao Sun, Jing Qi and Linli Wang: These authors contributed equally to this work

Authors and Affiliations

  1. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

    Xiaopeng Liu, Hao Sun, Jing Qi, Linli Wang, Songwei He, Jing Liu, Chengqian Feng, Chunlan Chen, Wen Li, Dajiang Qin, Guangjin Pan, Jiekai Chen, Duanqing Pei & Hui Zheng

  2. College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China

    Hao Sun & Yunqian Guo

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Contributions

H.Z. and D.P. conceived and supervised the study and wrote the manuscript. H.Z. and X.L. designed the experiments and analysed the data. All authors except H.Z. and D.P. performed the experiments.

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Correspondence to Duanqing Pei or Hui Zheng.

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Liu, X., Sun, H., Qi, J. et al. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT–MET mechanism for optimal reprogramming. Nat Cell Biol 15, 829–838 (2013). https://doi.org/10.1038/ncb2765

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