Direct generation of functional dopaminergic neurons from mouse and human fibroblasts (original) (raw)

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

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

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

Additional access options:

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
    You can also search for this author inPubMed Google Scholar
  2. Maria Teresa Dell’Anno
    You can also search for this author inPubMed Google Scholar
  3. Elena Dvoretskova
    You can also search for this author inPubMed Google Scholar
  4. Dejan Lazarevic
    You can also search for this author inPubMed Google Scholar
  5. Stefano Taverna
    You can also search for this author inPubMed Google Scholar
  6. Damiana Leo
    You can also search for this author inPubMed Google Scholar
  7. Tatyana D. Sotnikova
    You can also search for this author inPubMed Google Scholar
  8. Andrea Menegon
    You can also search for this author inPubMed Google Scholar
  9. Paola Roncaglia
    You can also search for this author inPubMed Google Scholar
  10. Giorgia Colciago
    You can also search for this author inPubMed Google Scholar
  11. Giovanni Russo
    You can also search for this author inPubMed Google Scholar
  12. Piero Carninci
    You can also search for this author inPubMed Google Scholar
  13. Gianni Pezzoli
    You can also search for this author inPubMed Google Scholar
  14. Raul R. Gainetdinov
    You can also search for this author inPubMed Google Scholar
  15. Stefano Gustincich
    You can also search for this author inPubMed Google Scholar
  16. Alexander Dityatev
    You can also search for this author inPubMed Google Scholar
  17. Vania Broccoli
    You can also search for this author inPubMed 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 toVania 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

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

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.

Associated content