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A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Nature Biotechnology volume 26, pages 916–924 (2008)Cite this article

Abstract

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

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Figure 1: Generation of genetically homogeneous cell cultures for epigenetic reprogramming.
Figure 2: Reprogramming kinetics and efficiencies vary between MEFs from distinct iPS cell lines.
Figure 3: Requirement and expression of four-factor transgenes in secondary MEFs.
Figure 4: Reprogramming of intestinal epithelial cells.
Figure 5: Reprogramming of other somatic cell types.

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Acknowledgements

We thank J. Dausman for assistance with animal husbandry. M.W. was supported in part by fellowships from the Human Frontiers Science Organization Program and the Ellison Foundation. C.J.L. was supported by a Ruth L. Kirschstein Fellowship from the US National Institutes of Health. J.H. was supported by a fellowship from the Helen Hay Whitney Foundation. R.J. was supported by grants from the US National Institutes of Health.

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Author notes

  1. Marius Wernig and Christopher J Lengner: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Marius Wernig, Christopher J Lengner, Jacob Hanna, Michael A Lodato, Eveline Steine, Ruth Foreman, Judith Staerk, Styliani Markoulaki & Rudolf Jaenisch

  2. Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, 02139, Massachusetts, USA

    Michael A Lodato, Ruth Foreman & Rudolf Jaenisch

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  1. Marius Wernig
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Contributions

M.W. and R.J. conceived the experiments and M.W. and C.J.L. designed experimental details. M.W. developed the dox-inducible vectors and secondary system, J.H. and R.F. generated the iPS cell lines NGFP1 and 2, M.A.L. helped determine the reprogramming efficiencies, E.S. performed the bisulfite sequencing reaction, J.S. performed the experiment shown in Figure 3f, and S.M. generated NGFP1 chimeras. M.W. and C.J.L. performed all remaining experiments. The manuscript was written by M.W., C.J.L. and R.J.

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Correspondence to Rudolf Jaenisch.

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Competing interests

R.J. is an advisor to Stemgent, which has obtained a license from MIT to distribute some of the reagents developed in this paper.

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Wernig, M., Lengner, C., Hanna, J. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 26, 916–924 (2008). https://doi.org/10.1038/nbt1483

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