Induction of human neuronal cells by defined transcription factors (original) (raw)

Nature volume 476, pages 220–223 (2011)Cite this article

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Abstract

Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types1,2,3,4,5,6,7,8,9,10,11,12. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells13. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix–loop–helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

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References

  1. Blau, H. M. et al. Plasticity of the differentiated state. Science 230, 758–766 (1985)
    Article ADS CAS Google Scholar
  2. Gurdon, J. B. From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation. Annu. Rev. Cell Dev. Biol. 22, 1–22 (2006)
    Article ADS CAS Google Scholar
  3. Heins, N. et al. Glial cells generate neurons: the role of the transcription factor Pax6. Nature Neurosci. 5, 308–315 (2002)
    Article CAS Google Scholar
  4. Ieda, M. et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375–386 (2010)
    Article CAS Google Scholar
  5. Shen, C.-N., Slack, J. M. W. & Tosh, D. Molecular basis of transdifferentiation of pancreas to liver. Nature Cell Biol. 2, 879–887 (2000)
    Article CAS Google Scholar
  6. Tada, M., Takahama, Y., Abe, K., Nakatsuji, N. & Tada, T. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558 (2001)
    Article CAS Google Scholar
  7. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)
    Article CAS Google Scholar
  8. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. & Campbell, K. H. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997)
    Article ADS CAS Google Scholar
  9. Xie, H., Ye, M., Feng, R. & Graf, T. Stepwise reprogramming of B cells into macrophages. Cell 117, 663–676 (2004)
    Article CAS Google Scholar
  10. Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455, 627–632 (2008)
    Article ADS CAS Google Scholar
  11. Graf, T. & Enver, T. Forcing cells to change lineages. Nature 462, 587–594 (2009)
    Article ADS CAS Google Scholar
  12. Zhou, Q. & Melton, D. A. Extreme makeover: converting one cell into another. Cell Stem Cell 3, 382–388 (2008)
    Article CAS Google Scholar
  13. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010)
    Article ADS CAS Google Scholar
  14. Hansen, D. V., Lui, J. H., Parker, P. R. & Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010)
    Article ADS CAS Google Scholar
  15. Kriegstein, A., Noctor, S. & Martinez-Cerdeno, V. Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion. Nature Rev. Neurosci. 7, 883–890 (2006)
    Article CAS Google Scholar
  16. Zhang, X. et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90–100 (2010)
    Article CAS Google Scholar
  17. Bottenstein, J. E. & Sato, G. H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl Acad. Sci. USA 76, 514–517 (1979)
    Article ADS CAS Google Scholar
  18. Guo, G. et al. Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 (2010)
    Article CAS Google Scholar
  19. Johnson, M. A., Weick, J. P., Pearce, R. A. & Zhang, S. C. Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J. Neurosci. 27, 3069–3077 (2007)
    Article CAS Google Scholar
  20. Wu, H. et al. Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines. Proc. Natl Acad. Sci. USA 104, 13821–13826 (2007)
    Article ADS CAS Google Scholar
  21. Koch, P., Opitz, T., Steinbeck, J. A., Ladewig, J. & Brustle, O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc. Natl Acad. Sci. USA 106, 3225–3230 (2009)
    Article ADS CAS Google Scholar
  22. Marchetto, M. C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010)
    Article CAS Google Scholar
  23. Hu, B. Y. et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl Acad. Sci. USA 107, 4335–4340 (2010)
    Article ADS CAS Google Scholar
  24. Xu, Y. et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl Acad. Sci. USA 107, 8129–8134 (2010)
    Article ADS CAS Google Scholar
  25. Maximov, A., Pang, Z. P., Tervo, D. G. & Sudhof, T. C. Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation. J. Neurosci. Methods 161, 75–87 (2007)
    Article Google Scholar
  26. Wong, C. C. et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nature Biotechnol. 28, 1115–1121 (2010)
    Article CAS Google Scholar
  27. Somers, A. et al. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28, 1728–1740 (2010)
    Article CAS Google Scholar

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Acknowledgements

We would like to thank Y. Kokubu for technical assistance in molecular cloning and Y. Zhang for assistance in iPS cell induced neuron culture. We also thank Y. Sun for providing the microRNAs expression lentiviral vectors and S. Majumder for the REST-VP16 construct. This work was enabled by start-up funds from the Institute for Stem Cell Biology and Regenerative Medicine at Stanford (M.W.), the Ellison Medical Foundation (M.W.), the Stinehard-Reed Foundation (M.W.), the Donald E. and Delia B. Baxter Foundation (M.W.), the NIH grants 1R01MH092931 (M.W. and T.C.S.) and RC4 NS073015 (M.W.), and a Robertson Investigator Award from the New York Stem Cell Foundation. Z.P.P. is supported by 2008 and 2010 NARSAD Young Investigator Awards. T.V. is supported by the Ruth and Robert Halperin Stanford Graduate Fellowship. A.C. is supported by the AXA research fund and D.R.F. is supported by BioX Undergraduate Fellowship.

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Author notes

  1. Zhiping P. Pang, Nan Yang and Thomas Vierbuchen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 265 Campus Drive, Stanford, 94305, California, USA
    Zhiping P. Pang & Thomas C. Südhof
  2. Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, 94305, California, USA
    Nan Yang, Thomas Vierbuchen, Austin Ostermeier, Daniel R. Fuentes, Troy Q. Yang, Vittorio Sebastiano, Samuele Marro & Marius Wernig
  3. Program in Cancer Biology, Stanford University School of Medicine, 265 Campus Drive, Stanford, 94305, California, USA
    Thomas Vierbuchen, Austin Ostermeier & Marius Wernig
  4. Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford, 94305, California, USA
    Ami Citri
  5. Howard Hughes Medical Institute, Stanford University School of Medicine, 265 Campus Drive, Stanford, 94305, California, USA
    Thomas C. Südhof

Authors

  1. Zhiping P. Pang
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  2. Nan Yang
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  3. Thomas Vierbuchen
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  4. Austin Ostermeier
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  5. Daniel R. Fuentes
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  6. Troy Q. Yang
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  7. Ami Citri
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  8. Vittorio Sebastiano
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  9. Samuele Marro
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  10. Thomas C. Südhof
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  11. Marius Wernig
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Contributions

Z.P.P., N.Y., T.V., A.O., T.C.S. and M.W. designed the experiments and analysed the data. D.R.F. and T.Q.Y. helped with lentiviral production. A.C., V.S. and S.M. helped to provide experimental material and helped with the analyses. Z.P.P., N.Y., T.V., T.C.S. and M.W. wrote the paper.

Corresponding author

Correspondence toMarius Wernig.

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The authors declare no competing financial interests.

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Pang, Z., Yang, N., Vierbuchen, T. et al. Induction of human neuronal cells by defined transcription factors.Nature 476, 220–223 (2011). https://doi.org/10.1038/nature10202

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Editorial Summary

Neurons from fibroblasts

Three papers in this issue demonstrate the production of functional induced neuronal (iN) cells from human fibroblasts, a procedure that holds great promise for regenerative medicine. Pang et al. show that a combination of the three transcription factors Ascl1 (also known as Mash1), Brn2 (or Pou3f2) and Myt1l greatly enhances the neuronal differentiation of human embryonic stem cells. When combined with the basic helix–loop–helix transcription factor NeuroD1, these factors can also convert fetal and postnatal human fibroblasts into iN cells. Caiazzo et al. use a cocktail of three transcription factors to convert prenatal and adult mouse and human fibroblasts into functional dopaminergic neurons. The three are Mash1, Nurr1 (or Nr4a2) and Lmx1a. Conversion is direct with no reversion to a progenitor cell stage, and it occurs in cells from Parkinson's disease patients as well as from healthy donors. Yoo et al. use an alternative approach. They show that microRNAs can have an instructive role in neural fate determination. Expression of miR-9/9* and miR-124 in human fibroblasts induces their conversion into functional neurons, and the process is facilitated by the addition of some neurogenic transcription factors.

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