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

Nature Biotechnology volume 33, pages 204–209 (2015)Cite this article

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Abstract

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.

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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.

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Acknowledgements

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

  1. Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, New York, USA

    Julius A Steinbeck, Yosif Ganat & Lorenz Studer

  2. Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, New York, USA

    Julius A Steinbeck, Yosif Ganat & Lorenz Studer

  3. Department of Neurology, Columbia University Medical Center, New York, New York, USA

    Se Joon Choi, Ana Mrejeru, David Sulzer & Eugene V Mosharov

  4. Department of Bioengineering, Stanford University, Stanford, California, USA

    Karl Deisseroth

  5. Department of Psychiatry, Stanford University, Stanford, California, USA

    Karl Deisseroth

  6. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Karl Deisseroth

  7. Department of Psychiatry, Columbia University Medical Center, New York, New York, USA

    David Sulzer

  8. Department of Pharmacology, Columbia University Medical Center, New York, New York, USA

    David Sulzer

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  1. Julius A Steinbeck
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  7. Eugene V Mosharov
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Contributions

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.

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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)

41587_2015_BFnbt3124_MOESM24_ESM.mov

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

41587_2015_BFnbt3124_MOESM25_ESM.mov

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)

41587_2015_BFnbt3124_MOESM26_ESM.mov

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

41587_2015_BFnbt3124_MOESM27_ESM.mov

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)

41587_2015_BFnbt3124_MOESM28_ESM.mov

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)

41587_2015_BFnbt3124_MOESM29_ESM.mov

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

41587_2015_BFnbt3124_MOESM30_ESM.mov

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)

41587_2015_BFnbt3124_MOESM31_ESM.mov

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)

41587_2015_BFnbt3124_MOESM32_ESM.mov

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)

41587_2015_BFnbt3124_MOESM33_ESM.mov

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)

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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). https://doi.org/10.1038/nbt.3124

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