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Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model

Nature Biotechnology volume 35, pages 444–452 (2017)Cite this article

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Abstract

Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.

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Figure 1: Human astrocytes can be reprogrammed with NeAL218.
Figure 2: Small molecules increase maturation of human-astrocyte-derived iDANs.
Figure 3: NeAL218 reprograms astrocytes into iDANs in vivo.
Figure 4: Reprogramming of adult striatal astrocytes into functional iDANs by NeAL218 in vivo.
Figure 5: iDANsNeAL218 induce behavioral recovery in a mouse model of Parkinson's disease.

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  • 19 April 2017

    In the version of this article initially published, in the paragraph before the Discussion, the sentence that read in part “…the very high levels of DA that are required in the synapses to induce circling behavior…” should have read “…the very high levels of DA that are required in the synapses to reduce circling behavior…” The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank the members of the Arenas laboratory for help and suggestions; J. Söderlund and A. Nanni for technical and secretarial assistance; and the SciLife National Genomic Infrastructure (Stockholm) for RNA sequencing. This work was supported by grants from the Swedish Research Council (VR: DBRM, 2011-3116/3318 and 2016-01526), Swedish Foundation for Strategic Research (SRL), EU (NeuroStemcellRepair and DDPDgenes), Karolinska Institutet, Strat Regen, Hjärnfonden (FO2013:0108, FO2015:0202) and Cancerfonden (CAN 2016/572) to E.A.; VR (2012-13482 and 2015-02886), StratNeuro, Parkinsonfonden, Hjärnfonden and KI/NIH to G.F.; VR (2013-3080), EU (PAINCAGE), Hjärnfonden, NovoNordisk Foundation and the European Research Council (“Secret Cells”) to T.H.; and New York Stem Cell Foundation, NIH and CIRM to M.W. Support to P.R.d.V.C. was provided by VR (524-2011-962) and EMBO (ALTF583-2011); to R.A.R. by EMBO (ALTF596-2014) and Marie Curie (EMBOCOFUND2012, GA-2012-600394); to D.M. by KI and by the Brasilian Ministry of Education (CAPES) and to E.M.-M. by the Spanish Ministry of Education (José Castillejo). The authors acknowledge support from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, the National Genomics Infrastructure funded by the Swedish Research Council, and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure.

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Authors and Affiliations

  1. Department of Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology, Karolinska Institutet, Stockholm, Sweden

    Pia Rivetti di Val Cervo, Elisa Martín-Montañez, Enrique M Toledo, Gioele La Manno, Sara Padrell Sánchez, Sten Linnarsson & Ernest Arenas

  2. Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria

    Roman A Romanov, Christian Pifl & Tibor Harkany

  3. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Roman A Romanov, Giada Spigolon, Débora Masini, Michael Feyder, Tibor Harkany & Gilberto Fisone

  4. Department of Pharmacology, Faculty of Medicine, Biomedical Research Institute of Malaga (IBIMA), Malaga University, Malaga, Spain

    Elisa Martín-Montañez

  5. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA

    Yi-Han Ng & Marius Wernig

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Contributions

E.A. and P.R.d.V.C. conceived the experiments and wrote the manuscript; P.R.d.V.C., R.A.R., G.S., D.M., E.M.-M., E.M.T., G.L.M., M.F., C.P., Y.-H.N. and S.P.S. performed the experiments; S. L., M.W., T.H., G.F. and E.A. provided expertise and funding.

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Correspondence to Ernest Arenas.

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Integrated supplementary information

Supplementary Figure 1 Characterization of hIA cultures and midbrain markers after iDAN reprogramming.

a. Uninfected hIAs do not spontaneously become TH+ in MP media at day 17. b. hIAs infected with GFP show a high infection efficiency in MP media at day 17 c. Gene expression analysis by real-time q-PCR revealed an increase in multiple mDA markers in hIA cultures treated with AL-MP, AL218-MP or NeAL218-MP at day 10. Data are shown as fold increase over control cells. * p<0.05 and ** p<0.01, n≥3, n.d.: not detectable

Supplementary Figure 2 Properties of hIA-derived iDANsNeAL218-RTMP cells.

a. Percentage of hIAs that become TH+ after infection with NeAL218 in RTMP conditions at day 4(n=3) and day 10(n=3). b. hIAs infected with NeAL218 in RTMP conditions at day 4 (scale bar 50μm) c. Illustration of the experiment for doxycycline withdrawal. d. hIAs infected with NeAL218 and cultured in RTMP for 13 days, but with only 7 or 10 days of doxycycline supplementation (scale bars 50μm). e. Bright field image of a representative hIA infected with NeAL218 and treated with RTMP media, probed by patch-clamp electrophysiology with its current profile (f) depicted in voltage-clamp mode. g. Current clamp recordings show the presence of hIA-derived iDANsNeAL218-RTMP capable of generating single action potential upon current injection (5, 20 and 30 pA). h. Vertical box plots, reflecting amplitudes of outward and inward currents in control (n=16) and NeAL218-infected hIAs cells with RTMP protocol (n=12). Whitney Rank Sum Test for outward currents p = 0,043; for inward currents p < 0,001. i. Pie chart depicting the fraction of hIA-derived iDANsNeAL218-RTMP generating single action potentials among the 12 cells probed * p<0.05 and ** p<0.01.

Supplementary Figure 3 Properties of hPA-derived control and iDANsNeAL218-RTMP cells

a. Efficient expression of GFP by hPAs in RTMP media at day 17. b. Uninfected hPAs do not become TH+ in RTMP media at day 17 (scale bar 50μm). c. Representative voltage-clamp (top) and current-clamp (bottom) recordings from control-RTMP hPAs cells. Control cells did not generate any action potential (AP). d. Comparison of gene expression by real-time q-PCR of hPAs treated with control-RTMP or NeAL218-RTMP at day 10. Data are mean ± s.d. FOXA2 was compared to untreated cells at day 0, as Foxa2 mRNA was undetectable in control-RTMP cells at day 10.

Supplementary Figure 4 Characterization of the 6-OHDA lesion model, transduction of the GFAP-tTA mice and reporter activity of the GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice.

a. Ventral midbrain cryosection of GFAP-tTA adult mice infected in the striatum with NeAL218 for 13 weeks (scale bar 50μm). Comparison of the right and left substantia nigra showing the effect of the 6-OHDA injection in the ipsilateral medial forebrain bundle. b. Striatal cryosections of GFAP-tTA adult mice 2 weeks after unilateral 6-OHDA (scale bar 100μm) showing loss of TH and increase in GFAP in the ipsilateral striatum. c. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 2 weeks (scale bar 25μm). d. Striatal cryosections of GFAP-tTA adult mice infected with GFP for 13 weeks (scale bar 10μm). e. Striatal cryosections of GFAP- tTa;SLC6A3cre/+;Rosa26RTomato adult mice infected with GFP for 5 weeks (scale bar 10μm). f. Cryosection of adult GFAP-tTa;SLC6A3cre/+;Rosa26RTomato mice showing the functionality of the reporter mice in the intact contralateral side of 6-OHDA lesioned mice infected with GFP or NeAL218 for 5 weeks (scale bar 10μm).

Supplementary Figure 5 Characterization of in vivo iDAN reprogramming by NeAL218.

a. Diagram depicting am alternative strategy used to visualize in vivo reprogrammed mature iDANs: GFAP-tTA; SLC6A3Cre/+ mice and flex-tdTomato virus. b. Time-lapse microphotographs showing release of a fluorescent dopamine derivative, FFN 206, upon KCl-induced depolarization in acute brain slices of GFAP-tTA adult mice, 5 weeks after NeAL218 or GFP injection (scale bar 25μm). c-e. Electrophysiological recordings at 5 weeks showing NeAL218-induced SLC6A3Tomato+ cells unable to generate action potentials in voltage clamp mode (c: voltage clamp profile, n=19 out of 28) or able to generate single action potentials upon somatic current injection (n=4 out of 28 in d and e, showing voltage-clamp and current clamp profiles, respectively). f. Control GFP+ cells recorded in voltage-clamp configuration (polarization for 100 ms from -100 to + 50 mV, n=9; 2 examples are presented) showed outward voltage-gated currents but no inward voltage gated (sodium/calcium) currents. g. Net rotations after systemic amphetamine administration to control and 6-OHDA lesioned GFAP-tTA mice ± intrastriatatal GFP or NeAL218 for 5, 9 or 13 weeks. GFP n = 6; NeAL218, n = 7. Two Way RM ANOVA with time effect F(4,44) = 34,75; p < 0,0001 and subject matching F(11, 44) = 7,908; p < 0,0001 followed by Fisher’s comparison (for GFP: naïve vs lesion p = 0,0003; W5 vs lesion p = 0,0125; W9 vs lesion p = 0,0039 and W13 vs lesion p = 0,0122 and for NeAL218: naïve vs lesion p < 0,0001; W5 vs lesion p = 0,0813; W9 vs lesion p = 0,0011 and W13 vs lesion p = 0,0649).

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Rivetti di Val Cervo, P., Romanov, R., Spigolon, G. et al. Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model. Nat Biotechnol 35, 444–452 (2017). https://doi.org/10.1038/nbt.3835

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