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Direct conversion of fibroblasts to functional neurons by defined factors

Nature volume 463, pages 1035–1041 (2010)Cite this article

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Abstract

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

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Figure 1: A screen for neuronal-fate-inducing factors and characterization of MEF-derived iN cells
Figure 2: Efficient induction of neurons from perinatal tail-tip fibroblasts
Figure 3: The 5F-pool-induced conversion is rapid and efficient
Figure 4: MEF-derived iN cells show functional synaptic properties.
Figure 5: Defining a minimal pool for efficient induction of functional iN cells

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References

  1. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001)

    Article  CAS  Google Scholar 

  2. Bernstein, B. E., Meissner, A. & Lander, E. S. The mammalian epigenome. Cell 128, 669–681 (2007)

    Article  CAS  Google Scholar 

  3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

    Article  CAS  Google Scholar 

  4. Briggs, R. & King, T. J. Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl Acad. Sci. USA 38, 455–463 (1952)

    Article  ADS  CAS  Google Scholar 

  5. Gurdon, J. B., Elsdale, T. R. & Fischberg, M. Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182, 64–65 (1958)

    Article  ADS  CAS  Google Scholar 

  6. Campbell, K. H., McWhir, J., Ritchie, W. A. & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66 (1996)

    Article  ADS  CAS  Google Scholar 

  7. 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 

  8. Do, J. T. & Scholer, H. R. Nuclei of embryonic stem cells reprogram somatic cells. Stem Cells 22, 941–949 (2004)

    Article  CAS  Google Scholar 

  9. Cowan, C. A., Atienza, J., Melton, D. A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005)

    Article  ADS  CAS  Google Scholar 

  10. Silva, J. & Smith, A. Capturing pluripotency. Cell 132, 532–536 (2008)

    Article  CAS  Google Scholar 

  11. Blau, H. M. How fixed is the differentiated state? Lessons from heterokaryons. Trends Genet. 5, 268–272 (1989)

    Article  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. Davis, R. L., Weintraub, H. & Lassar, A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987–1000 (1987)

    Article  CAS  Google Scholar 

  14. Schäfer, B. W., Blakely, B. T., Darlington, G. J. & Blau, H. M. Effect of cell history on response to helix-loop-helix family of myogenic regulators. Nature 344, 454–458 (1990)

    Article  ADS  Google Scholar 

  15. Kondo, M. et al. Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature 407, 383–386 (2000)

    Article  ADS  CAS  Google Scholar 

  16. Bussmann, L. H. et al. A robust and highly efficient immune cell reprogramming system. Cell Stem Cell 5, 554–566 (2009)

    Article  CAS  Google Scholar 

  17. Feng, R. et al. PU.1 and C/EBPα/β convert fibroblasts into macrophage-like cells. Proc. Natl Acad. Sci. USA 105, 6057–6062 (2008)

    Article  ADS  CAS  Google Scholar 

  18. Xie, H., Ye, M., Feng, R. & Graf, T. Stepwise reprogramming of B cells into macrophages. Cell 117, 663–676 (2004)

    Article  CAS  Google Scholar 

  19. Cobaleda, C., Jochum, W. & Busslinger, M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature 449, 473–477 (2007)

    Article  ADS  CAS  Google Scholar 

  20. 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 

  21. Tucker, K. L., Meyer, M. & Barde, Y. A. Neurotrophins are required for nerve growth during development. Nature Neurosci. 4, 29–37 (2001)

    Article  CAS  Google Scholar 

  22. Wernig, M. et al. Tau EGFP embryonic stem cells: an efficient tool for neuronal lineage selection and transplantation. J. Neurosci. Res. 69, 918–924 (2002)

    Article  CAS  Google Scholar 

  23. Lee, J. E. et al. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268, 836–844 (1995)

    Article  ADS  CAS  Google Scholar 

  24. Guillemot, F. et al. Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463–476 (1993)

    Article  CAS  Google Scholar 

  25. Farah, M. H. et al. Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127, 693–702 (2000)

    CAS  PubMed  Google Scholar 

  26. Guillemot, F. Cellular and molecular control of neurogenesis in the mammalian telencephalon. Curr. Opin. Cell Biol. 17, 639–647 (2005)

    Article  CAS  Google Scholar 

  27. Escurat, M., Djabali, K., Gumpel, M., Gros, F. & Portier, M. M. Differential expression of two neuronal intermediate-filament proteins, peripherin and the low-molecular-mass neurofilament protein (NF-L), during the development of the rat. J. Neurosci. 10, 764–784 (1990)

    Article  CAS  Google Scholar 

  28. Beard, C., Hochedlinger, K., Plath, K., Wutz, A. & Jaenisch, R. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23–28 (2006)

    Article  CAS  Google Scholar 

  29. Christopherson, K. S. et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120, 421–433 (2005)

    Article  CAS  Google Scholar 

  30. 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 

  31. Hochedlinger, K. & Jaenisch, R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 415, 1035–1038 (2002)

    Article  ADS  CAS  Google Scholar 

  32. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008)

    Article  CAS  Google Scholar 

  33. Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008)

    Article  CAS  Google Scholar 

  34. Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49–52 (2009)

    Article  ADS  CAS  Google Scholar 

  35. Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnol. 26, 916–924 (2008)

    Article  CAS  Google Scholar 

  36. 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 

  37. Maximov, A. & Sudhof, T. C. Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48, 547–554 (2005)

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank S. Marro and P. Lovelace for help with FACS sorting, S. Hafeez and Y. Huh for assistance with molecular cloning and mouse husbandry, and K. Jann for assistance with the diagram in Fig. 1. We would also like to thank I. Graef, R. Bajpai, J. Wysocka, J.-R. Lin and J.-Y. Chen for contributing reagents and help with analysis. This work was supported by start-up funds from the Institute for Stem Cell Biology and Regenerative Medicine at Stanford (M.W.), the Donald E. and Delia B. Baxter Foundation (M.W.), an award from William Stinehart Jr and the Reed Foundation (M.W.), the National Institute of Health Training Grant 1018438-142-PABCA (A.O.) and the Ruth and Robert Halperin Stanford Graduate Fellowship (T.V.). Z.P.P. is supported by NARSAD Young Investigator Award and NIH/NINDS Epilepsy Training Grant 5T32NS007280.

Author Contributions T.V., A.O. and M.W. designed and conceived the experiments. T.V., Y.K. and M.W. produced the lentiviral vectors. T.V. and A.O. performed the lentiviral infections, isolated the fibroblasts and completed the molecular characterization of the iN cells. Z.P.P. and T.C.S. designed, performed and analysed the electrophysiological assays. T.V., A.O., Z.P.P., T.C.S. and M.W. wrote and edited the manuscript and produced the figures.

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Authors and Affiliations

  1. Department of Pathology,, Institute for Stem Cell Biology and Regenerative Medicine,

    Thomas Vierbuchen, Austin Ostermeier, Yuko Kokubu & Marius Wernig

  2. Program in Cancer Biology,,

    Thomas Vierbuchen, Austin Ostermeier & Marius Wernig

  3. Department of Molecular and Cellular Physiology,,

    Zhiping P. Pang & Thomas C. Südhof

  4. Howard Hughes Medical Institute, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA ,

    Thomas C. Südhof

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  1. Thomas Vierbuchen
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  2. Austin Ostermeier
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Correspondence to Marius Wernig.

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Vierbuchen, T., Ostermeier, A., Pang, Z. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010). https://doi.org/10.1038/nature08797

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