Skip to main content

Thank you for visiting 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.

Optogenetics enables functional analysis of human embryonic stem cell–derived grafts in a Parkinson's disease model


Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease1,2,3,4. However, little is known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Figure 1: In vitro immunocytochemical characterization of opsin-expressing hESC lines and dopaminergic progeny.
Figure 2: In vitro physiologic and neurochemical assessment of optogenetic control.
Figure 3: Behavioral, physiological and morphological assessment of graft functional connectivity.


  1. Kirkeby, A. et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Reports 1, 703–714 (2012).

    Article  CAS  Google Scholar 

  2. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).

    Article  CAS  Google Scholar 

  3. Roy, N.S. et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259–1268 (2006).

    Article  CAS  Google Scholar 

  4. Tornero, D. et al. Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery. Brain 136, 3561–3577 (2013).

    Article  Google Scholar 

  5. Dunnett, S.B., Hernandez, T.D., Summerfield, A., Jones, G.H. & Arbuthnott, G. Graft-derived recovery from 6-OHDA lesions: specificity of ventral mesencephalic graft tissues. Exp. Brain Res. 71, 411–424 (1988).

    Article  CAS  Google Scholar 

  6. 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  CAS  Google Scholar 

  7. Cummings, B.J. et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc. Natl. Acad. Sci. USA 102, 14069–14074 (2005).

    Article  CAS  Google Scholar 

  8. Gradinaru, V., Thompson, K.R. & Deisseroth, K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 36, 129–139 (2008).

    Article  Google Scholar 

  9. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  CAS  Google Scholar 

  10. Chaudhury, D. et al. Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature 493, 532–536 (2013).

    Article  CAS  Google Scholar 

  11. Kravitz, A.V. et al. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466, 622–626 (2010).

    Article  CAS  Google Scholar 

  12. Piña-Crespo, J.C. et al. High-frequency hippocampal oscillations activated by optogenetic stimulation of transplanted human ESC-derived neurons. J. Neurosci. 32, 15837–15842 (2012).

    Article  Google Scholar 

  13. Weick, J.P. et al. Functional control of transplantable human ESC-derived neurons via optogenetic targeting. Stem Cells 28, 2008–2016 (2010).

    Article  CAS  Google Scholar 

  14. Johnson, M.A., Weick, J.P., Pearce, R.A. & Zhang, S.C. Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J. Neurosci. 27, 3069–3077 (2007).

    Article  CAS  Google Scholar 

  15. Lindvall, O. & Kokaia, Z. Stem cells in human neurodegenerative disorders–time for clinical translation? J. Clin. Invest. 120, 29–40 (2010).

    Article  CAS  Google Scholar 

  16. Ganat, Y.M. et al. Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment. J. Clin. Invest. 122, 2928–2939 (2012).

    Article  CAS  Google Scholar 

  17. Miller, J.D. et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691–705 (2013).

    Article  CAS  Google Scholar 

  18. Guzman, J.N., Sanchez-Padilla, J., Chan, C.S. & Surmeier, D.J. Robust pacemaking in substantia nigra dopaminergic neurons. J. Neurosci. 29, 11011–11019 (2009).

    Article  CAS  Google Scholar 

  19. Sørensen, A.T. et al. Functional properties and synaptic integration of genetically labelled dopaminergic neurons in intrastriatal grafts. Eur. J. Neurosci. 21, 2793–2799 (2005).

    Article  Google Scholar 

  20. Dowd, E., Monville, C., Torres, E.M. & Dunnett, S.B. The Corridor Task: a simple test of lateralised response selection sensitive to unilateral dopamine deafferentation and graft-derived dopamine replacement in the striatum. Brain Res. Bull. 68, 24–30 (2005).

    Article  CAS  Google Scholar 

  21. Grealish, S., Mattsson, B., Draxler, P. & Bjorklund, A. Characterisation of behavioural and neurodegenerative changes induced by intranigral 6-hydroxydopamine lesions in a mouse model of Parkinson's disease. Eur. J. Neurosci. 31, 2266–2278 (2010).

    Article  Google Scholar 

  22. Fujita, S. et al. Apomorphine-induced modulation of neural activities in the ventrolateral striatum of rats. Synapse 67, 363–373 (2013).

    Article  CAS  Google Scholar 

  23. Kish, L.J., Palmer, M.R. & Gerhardt, G.A. Multiple single-unit recordings in the striatum of freely moving animals: effects of apomorphine and D-amphetamine in normal and unilateral 6-hydroxydopamine-lesioned rats. Brain Res. 833, 58–70 (1999).

    Article  CAS  Google Scholar 

  24. Cenci, M.A., Kalen, P., Mandel, R.J., Wictorin, K. & Bjorklund, A. Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum. Neuroscience 46, 943–957 (1992).

    Article  CAS  Google Scholar 

  25. Rylander, D. et al. Region-specific restoration of striatal synaptic plasticity by dopamine grafts in experimental parkinsonism. Proc. Natl. Acad. Sci. USA 110, E4375–E4384 (2013).

    Article  CAS  Google Scholar 

  26. Nambu, A. Seven problems on the basal ganglia. Curr. Opin. Neurobiol. 18, 595–604 (2008).

    Article  CAS  Google Scholar 

  27. Surmeier, D.J., Ding, J., Day, M., Wang, Z. & Shen, W. D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons. Trends Neurosci. 30, 228–235 (2007).

    Article  CAS  Google Scholar 

  28. Cepeda, C., Buchwald, N.A. & Levine, M.S. Neuromodulatory actions of dopamine in the neostriatum are dependent upon the excitatory amino acid receptor subtypes activated. Proc. Natl. Acad. Sci. USA 90, 9576–9580 (1993).

    Article  CAS  Google Scholar 

  29. Paillé, V. et al. Distinct levels of dopamine denervation differentially alter striatal synaptic plasticity and NMDA receptor subunit composition. J. Neurosci. 30, 14182–14193 (2010).

    Article  Google Scholar 

  30. Steinbeck, J.A., Koch, P., Derouiche, A. & Brustle, O. Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain. Cell. Mol. Life Sci. 69, 461–470 (2012).

    Article  CAS  Google Scholar 

  31. Battista, D., Ganat, Y., El Maarouf, A., Studer, L. & Rutishauser, U. Enhancement of polysialic acid expression improves function of embryonic stem-derived dopamine neuron grafts in Parkinsonian mice. Stem Cells Transl. Med. 3, 108–113 (2014).

    Article  CAS  Google Scholar 

  32. Chan, W.S. et al. Differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate glutamate receptors in the rat striatum during postnatal development. Neurosignals 12, 302–309 (2003).

    Article  CAS  Google Scholar 

  33. Price, C.J., Kim, P. & Raymond, L.A. D1 dopamine receptor-induced cyclic AMP-dependent protein kinase phosphorylation and potentiation of striatal glutamate receptors. J. Neurochem. 73, 2441–2446 (1999).

    Article  CAS  Google Scholar 

  34. Yan, Z. et al. Protein phosphatase 1 modulation of neostriatal AMPA channels: regulation by DARPP-32 and spinophilin. Nat. Neurosci. 2, 13–17 (1999).

    Article  CAS  Google Scholar 

  35. André, V.M. et al. Dopamine modulation of excitatory currents in the striatum is dictated by the expression of D1 or D2 receptors and modified by endocannabinoids. Eur. J. Neurosci. 31, 14–28 (2010).

    Article  Google Scholar 

  36. Pothos, E., Desmond, M. & 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 

  37. Snyder, G.L. et al. Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo . J. Neurosci. 20, 4480–4488 (2000).

    Article  CAS  Google Scholar 

  38. Song, R.S. et al. ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion. Proc. Natl. Acad. Sci. USA 110, 15437–15442 (2013).

    Article  CAS  Google Scholar 

  39. Tukey, D.S. & Ziff, E.B. Ca2+-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and dopamine D1 receptors regulate GluA1 trafficking in striatal neurons. J. Biol. Chem. 288, 35297–35306 (2013).

    Article  CAS  Google Scholar 

  40. Umemiya, M. & Raymond, L.A. Dopaminergic modulation of excitatory postsynaptic currents in rat neostriatal neurons. J. Neurophysiol. 78, 1248–1255 (1997).

    Article  CAS  Google Scholar 

  41. Bamford, N.S. et al. Repeated exposure to methamphetamine causes long-lasting presynaptic corticostriatal depression that is renormalized with drug readministration. Neuron 58, 89–103 (2008).

    Article  CAS  Google Scholar 

  42. Dell'Anno, M.T. et al. Remote control of induced dopaminergic neurons in parkinsonian rats. J. Clin. Invest. 124, 3215–3229 (2014).

    Article  CAS  Google Scholar 

  43. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kim, H. et al. miR-371–3 expression predicts neural differentiation propensity in human pluripotent stem cells. Cell Stem Cell 8, 695–706 (2011).

    Article  CAS  Google Scholar 

  45. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  46. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  47. Barreto-Chang, O.L. & Dolmetsch, R.E. Calcium imaging of cortical neurons using Fura-2 AM. J. Vis. Exp., 10.3791/1067 (2009).

  48. Akopian, G. & Walsh, J.P. Corticostriatal paired-pulse potentiation produced by voltage-dependent activation of NMDA receptors and L-type Ca(2+) channels. J. Neurophysiol. 87, 157–165 (2002).

    Article  CAS  Google Scholar 

Download references


We thank S. Oh and K. Manova (MSKCC molecular cytology core), and M. Tomishima (SKI stem cell core) for excellent technical support. We further thank Y. Schmitz (Sulzer laboratory) for advice on the corridor test. J.A.S. was supported by a Deutsche Forschungsgemeinschaft fellowship. The work was supported in part by US National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) grant NS052671 and NYSTEM contract C028503 to L.S. and by the NINDS/NIH grant NS075222 to E.V.M. This work was further supported by grants from the Parkinson's disease and Jeffry and Barbara Picower Foundations and the Udall Center of Excellence to D.S.

Author information

Authors and Affiliations



J.A.S.: conception and study design, hESC manipulation, differentiation and characterization, calcium imaging, animal lesioning and transplantation, histological and behavioral assays, data analysis and interpretation and writing of manuscript. S.J.C. and A.M.: slice physiology and data analysis, writing of manuscript. Y.G.: animal lesioning and transplantation assays. K.D.: study design and provision of materials. D.S.: study design and data interpretation. E.V.M.: study design, neurochemical assays, data analysis and interpretation and writing of manuscript. L.S.: conception and study design, data analysis and interpretation, writing of manuscript.

Corresponding authors

Correspondence to Eugene V Mosharov or Lorenz Studer.

Ethics declarations

Competing interests

L.S. is inventor on a patent application (WO 2013067362: Midbrain dopamine neurons for engraftment) partly related to the work. The authors declare no other competing financial interest.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–3 (PDF 9138 kb)

Ratiometric calcium imaging of a HALO expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame. (MOV 2459 kb)

Ratiometric calcium imaging of a HALO expressing D90 culture during continuous glutamate stimulation (50 μM). Three inactivating 550 nm light pulses are applied, which produced a decrease in calcium levels. 1 s/frame. (MOV 7677 kb)

Ratiometric calcium imaging of an EYFP expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame. (MOV 2522 kb)

Ratiometric calcium imaging of an EYFP expressing D90 culture during continuous glutamate stimulation (50 μM). Four 550 nm light pulses were applied without producing any change in the calcium response. 1 s/frame. (MOV 2188 kb)

Corridor test. Representative recording of a CTRL animal (unlesioned and non-grafted) during optogenetic illumination showing unbiased exploration and food retrieval from both sides of the corridor. (MOV 15200 kb)

Corridor test. Representative section of a Lesion-only animal showing lateralized exploration and food retrieval ipsilateral to the lesion (right side). (MOV 20718 kb)

Corridor test. Representative recording of a Lesion-only animal (same animal as in video S5) injected with apomorphine showing lateralized exploration and food retrieval contralateral to the lesion (left side). (MOV 22243 kb)

Corridor test. Representative recording of a recovered EYFP animal (lesioned + EYFP graft) during optogenetic illumination showing unbiased exploration and food retrieval from both sides of the corridor. (MOV 17180 kb)

Corridor test. Representative recording of a recovered HALO animal (lesioned + HALO graft) during optogenetic graft silencing, showing reversion to lateralized behavior (exploration and food retrieval from right side of the corridor). (MOV 16045 kb)

Corridor test. Representative recording of a recovered HALO animal (same animal as in video S9, lesioned + HALO graft + APO) during optogenetic graft silencing injected with apomorphine, showing no reversion to lateralized behavior and no contralateral overshoot. (MOV 13438 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Steinbeck, J., Choi, S., Mrejeru, A. et al. Optogenetics enables functional analysis of human embryonic stem cell–derived grafts in a Parkinson's disease model. Nat Biotechnol 33, 204–209 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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