Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

Nature volume 476, pages 224–227 (2011)Cite this article

Subjects

Abstract

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson’s disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons1,2. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency3,4. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled5. Here we identified a minimal set of three transcription factors—Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a—that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson’s disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mash1, Nurr1 and Lmx1a reprogram mouse fibroblasts into iDA cells.
Figure 2: Mouse iDA cells expression profiling.
Figure 3: Functional characterization of mouse iDA cells.
Figure 4: Characterization of human fibroblasts reprogrammed into iDA cells.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE27174 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE27174).

References

  1. Lindvall, O. & Björklund, A. Cell therapy in Parkinson’s disease. NeuroRx 1, 382–393 (2004)

    Article  Google Scholar 

  2. Politis, M. et al. Serotonergic neurons mediate dyskinesia side effects in Parkinson’s patients with neural transplants. Sci. Transl. Med. 2, 38–46 (2010)

    Article  Google Scholar 

  3. Kim, J. H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50–56 (2002)

    Article  ADS  CAS  Google Scholar 

  4. Barberi, T. et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nature Biotechnol. 21, 1200–1207 (2003)

    Article  CAS  Google Scholar 

  5. Wernig, M. et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc. Natl Acad. Sci. USA 105, 5856–5861 (2008)

    Article  ADS  CAS  Google Scholar 

  6. 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 

  7. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010)

    Article  ADS  CAS  Google Scholar 

  8. Ang, S.-L. Transcriptional control of midbrain dopaminergic neuron development. Development 133, 3499–3506 (2006)

    Article  CAS  Google Scholar 

  9. Smidt, M. P. & Burbach, J. P. How to make a mesodiencephalic dopaminergic neuron. Nature Rev. Neurosci. 8, 21–32 (2007)

    Article  CAS  Google Scholar 

  10. Sawamoto, K. et al. Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons. Proc. Natl Acad. Sci. USA 98, 6423–6428 (2001)

    Article  ADS  CAS  Google Scholar 

  11. Perlmann, T. & Wallén-Mackenzie, A. Nurr1, an orphan nuclear receptor with essential functions in developing dopamine cells. Cell Tissue Res. 318, 45–52 (2004)

    Article  CAS  Google Scholar 

  12. Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008)

    Article  CAS  Google Scholar 

  13. Grace, A. A. & Bunney, B. S. The control of firing pattern in nigral dopamine neurons: single spike firing. J. Neurosci. 4, 2866–2876 (1984)

    Article  CAS  Google Scholar 

  14. Grace, A. A. & Onn, S. P. Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro . J. Neurosci. 9, 3463–3481 (1989)

    Article  CAS  Google Scholar 

  15. Pothos, E. N., Davila, V. & Sulzer, D. Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size. J. Neurosci. 18, 4106–4118 (1998)

    Article  CAS  Google Scholar 

  16. Staal, R. G. W., Mosharov, E. V. & Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nature Neurosci. 7, 341–346 (2004)

    Article  CAS  Google Scholar 

  17. Simeone, A. Genetic control of dopaminergic neuron differentiation. Trends Neurosci. 28, 62–65 (2005)

    Article  CAS  Google Scholar 

  18. Zappone, M. V. et al. Sox2 regulatory sequences direct expression of a β-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. Development 127, 2367–2382 (2000)

    CAS  PubMed  Google Scholar 

  19. Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature advance online publication,. 10.1038/nature10202 (26 May 2011)

  20. Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl Acad. Sci. USA 10.1073/pnas.1105135108 (6 June 2011)

  21. Amariglio, N. et al. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 6, e1000029 (2009)

    Article  Google Scholar 

  22. Sironi, F. et al. α-Synuclein multiplication analysis in Italian familial Parkinson disease. Parkinsonism Relat. Disord. 16, 228–231 (2010)

    Article  Google Scholar 

  23. Sironi, F. et al. Parkin analysis in early onset Parkinson’s disease. Parkinsonism Relat. Disord. 14, 326–333 (2008)

    Article  Google Scholar 

  24. Pruszak, J. et al. Isolation and culture of ventral mesencephalic precursor cells and dopaminergic neurons from rodent brain. Curr. Prot. Stem Cell Biol. Chapter 2,. 2D.5.1–2D.5.21 (2009)

  25. Broccoli, V. et al. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 401, 164–168 (1999)

    Article  ADS  CAS  Google Scholar 

  26. Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31, e15 (2003)

    Article  Google Scholar 

  27. Smyth, G. K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 3. (2004)

    Article  MathSciNet  Google Scholar 

  28. Hochberg, Y. & Benjamini, Y. More powerful procedures for multiple significance testing. Stat. Med. 9, 811–818 (1990)

    Article  CAS  Google Scholar 

  29. Huang, D. W. et al. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009)

    Article  Google Scholar 

  30. Biagioli, M. et al. Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells. Proc. Natl Acad. USA 106, 15454–15459 (2009)

    Article  ADS  CAS  Google Scholar 

  31. Pothos, E., Desmond, D. & Sulzer, D. l-3,4-Dihydroxyphenylalanine increases the quantal size of exocytotic dopamine release in vitro. J. Neurochem. 66, 629–636 (1996)

    Article  CAS  Google Scholar 

  32. Mundroff, M. L. & Wightman, R. M. Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells. Curr. Protoc. Neurosci. Chapter 6,. 6.14.1–6.14.22 (2002)

  33. Menegon, A. et al. Protein kinase A-mediated synapsin I phosphorylation is a central modulator of Ca2+-dependent synaptic activity. J. Neurosci. 26, 11670–11681 (2006)

    Article  CAS  Google Scholar 

  34. Colasante, G. et al. Arx is a direct target of Dlx2 and thereby contributes to the tangential migration of GABAergic interneurons. J. Neurosci. 28, 10674–10686 (2008)

    Article  CAS  Google Scholar 

  35. West, M. J. et al. Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231, 482–497 (1991)

    Article  CAS  Google Scholar 

  36. Bacigaluppi, M. et al. Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms. Brain 132, 2239–2251 (2009)

    Article  Google Scholar 

Download references

Acknowledgements

We are thankful to D. Bonanomi, S.-L. Ang, S. El Mestikawy, M. P. Smidt, M. German and F. Valtorta for providing valuable antibodies. We thank A. Sessa and V.B. laboratory members for helpful discussion. M. Wernig is acknowledged for providing the iN-inducing lentiviral vectors. We are thankful to S. Nicolis for sharing Sox2β-geo mice. L. Muzio, C. Laterza and G. Martino are acknowledged for the generation of Sox2β-geo induced pluripotent stem cells. M. Bacigaluppi is acknowledged for advice on stereological countings. We thank the “Cell Line and DNA Biobank” (G. Gaslini Institute) and “Human Genetic Bank of Patients affected by Parkinson Disease and parkinsonism” (Parkinson Institute of Milan) of the Telethon Genetic Biobank Network for human fibroblast samples. This study was supported by the “Fondazione Grigioni per il Morbo di Parkinson” (grant no. FGBRCVNI10310-001-V.B.), Eranet Neuron (V.B.), Cariplo Foundation (V.B.), Ministry of Health (Giovani ricercatori Award) (V.B.) and Italian Institute of Technology (V.B., A.D., S.G., T.S., R.G.).

Author information

Author notes

  1. Maria Teresa Dell’Anno and Elena Dvoretskova: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Neuroscience, Stem Cells and Neurogenesis Unit, San Raffaele Scientific Institute, 20132 Milan, Italy,

    Massimiliano Caiazzo, Maria Teresa Dell’Anno, Giorgia Colciago & Vania Broccoli

  2. Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genoa, Italy,

    Elena Dvoretskova, Stefano Taverna, Damiana Leo, Tatyana D. Sotnikova, Giovanni Russo, Raul R. Gainetdinov & Alexander Dityatev

  3. CBM Srl. Area Science park, Basovizza, SS14, km165, 34149 Trieste, Italy ,

    Dejan Lazarevic

  4. Sector of Neurobiology, International School for Advanced Studies (SISSA), via Bonomea, 265, 34136 Trieste, Italy ,

    Dejan Lazarevic, Paola Roncaglia & Stefano Gustincich

  5. Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, 20132 Milan, Italy ,

    Andrea Menegon

  6. Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-chô, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan ,

    Piero Carninci

  7. Parkinson Institute, Istituti Clinici di Perfezionamento, 20126 Milan, Italy ,

    Gianni Pezzoli

  8. The Giovanni Armenise-Harvard Foundation Laboratory, 34136 Trieste, Italy ,

    Stefano Gustincich

Authors
  1. Massimiliano Caiazzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Teresa Dell’Anno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elena Dvoretskova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dejan Lazarevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefano Taverna
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Damiana Leo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tatyana D. Sotnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrea Menegon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Paola Roncaglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Giorgia Colciago
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Giovanni Russo
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Piero Carninci
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Gianni Pezzoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Raul R. Gainetdinov
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Stefano Gustincich
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Alexander Dityatev
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Vania Broccoli
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.C. and V.B. designed and conceived the experiments. M.C., M.T.D. and G.C. performed the lentiviral infections, characterized reprogrammed cells and analysed their fate after in vivo transplantation. E.D. and A.D. designed, performed and analysed all electrophysiological experiments. P.R., D.L., P.C. and S.G. performed the microarray gene expression profiling and analysed the data. D.L., A.D. and R.R.G. designed andD.L. andE.D. performed theamperometric experiments. R.R.G. and T.D.S. designed the protocol and performed the assessment of dopamine levels. S.T and G.R. performed patch-clamp recording on brain slices. A.M. performed the functional analysis of synaptic activity. G.P. supervised the selection of the Parkinson's disease patients and the isolation of the primary fibroblasts. V.B. and A.D. should be considered as co-senior authors and wrote the manuscript.

Corresponding author

Correspondence to Vania Broccoli.

Ethics declarations

Competing interests

A patent application including the results of this manuscript has been filed, but has not been published yet.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13 with legends, and legends for Supplementary Tables 1-6. (PDF 10440 kb)

Supplementary Tables

This file contains Supplementary Tables 1-6 (see Supplementary Information file for legends). (ZIP 321 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Caiazzo, M., Dell’Anno, M., Dvoretskova, E. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227 (2011). https://doi.org/10.1038/nature10284

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10284

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing