Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

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.

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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.

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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).

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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.).

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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.

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Correspondence to Vania Broccoli.

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

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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

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