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

Journal name:
Nature Biotechnology
Volume:
33,
Pages:
204–209
Year published:
DOI:
doi:10.1038/nbt.3124
Received
Accepted
Published online

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.

At a glance

Figures

  1. In vitro immunocytochemical characterization of opsin-expressing hESC lines and dopaminergic progeny.
    Figure 1: In vitro immunocytochemical characterization of opsin-expressing hESC lines and dopaminergic progeny.

    Upper panels, hSyn-eNpHR3.0-EYFP (HALO) line, lower panels, hSyn-EYFP (EYFP) line. (a) Transgene harboring clonal hESC lines expressed OCT4 (red). (b) By day (D) 30, >98% of all TUJ+ neurons (red) expressed HALO/EYFP. (c) At D30, >98% of all TH+ (red)/NURR1+ (blue) neurons expressed HALO/EYFP. (d) Confocal imaging shows HALO localization in TH+/NURR1+ neurons largely confined to membranes and processes. (e) Quantification and comparison of transgene expression in all neurons (t = 0.18, P = 0.86, N.S.) and TH+ neurons (t = 0.32, P = 0.76, N.S.) between the HALO and EYFP line. Scale bar, 50 μm. Error bars represent s.e.m.

  2. In vitro physiologic and neurochemical assessment of optogenetic control.
    Figure 2: In vitro physiologic and neurochemical assessment of optogenetic control.

    (a) Representative ratiometric image of a D90, HALO-expressing, mesDA-rich culture after incubation with Fura-2. (b,c) A glutamate pulse (GLU, 100 μM) generates a calcium response (b), quantified in c. (df) During continuous glutamate perfusion (GLU, 50 μM, d) 550-nm light pulses (e) generate inactivating calcium signals in soma and dendrites of neurons, quantified in f. (g,h) Bright-field (g) and eNpHR3.0-EYFP expression (h) of the same region. Scale bar, 20 μm. (i) HPLC measurements of dopamine (DA) release from 90- to 100-day-old cultures after exposure to various stimuli and in the presence or absence of light-induced neuronal silencing. KCl (55 mM) and glutamate (GLU, 100 μM) exposure increase dopamine release in both EYFP- (black bars) and HALO- (green bars) expressing dopamine neurons by about twofold. Optogenetic inhibition at 543 nm (bars with bright green border) reduced dopamine release from HALO-expressing cultures only, in the presence and absence of glutamate stimulation. *P < 0.05, **P < 0.01, ****P < 0.0001.

  3. Behavioral, physiological and morphological assessment of graft functional connectivity.
    Figure 3: Behavioral, physiological and morphological assessment of graft functional connectivity.

    (a) Amphetamine-induced rotation assay demonstrates behavioral recovery of grafted animals. (b) Characteristic ~3-Hz pacemaking activity is optogenetically silenced in HALO-expressing neurons only. (c) Evoked postsynaptic potentials (EPSPs) in grafted HALO-expressing neurons following local electrical stimulation are blocked by AMPA-receptor-antagonist NBQX. (d) In the corridor test, unlesioned and nongrafted animals (CTRL, blue bars) did not show lateralized behavior regardless of illumination (green border). Before transplantation, lesioned animals (LES.) showed a strong preference for food retrieval on the side ipsilateral to the lesion, which was inverted by the dopamine agonist apomorphine (LES. + APO, green bar). Animals with EYFP grafts (black bars POST TX) recovered from lesion-induced motor deficiency and were insensitive to illumination or apomorphine. Animals with HALO grafts (green bars POST TX) also recovered from the lesion but reverted to lateralized behavior during optogenetic graft silencing, which was reversed by prior apomorphine injection. Significance was calculated using one-way ANOVA (F (10, 44) = 29.99, P < 0.0001). Adjusted P values indicated in the graph are calculated using Sidak's multiple comparisons test. (e,f) Confocal analysis of human axons staining for human neurofilament (hNF) and human synaptophysin (hSyn) extend from HALO and EYFP grafts into the host striatum and are in close contact with host DARPP32+ MSNs. Scale bar, 10 μm. (g) Representative examples of electrically evoked AMPA EPSPs recorded from a MSN before and during optogenetic graft silencing in a HALO animal. (h) EPSP amplitudes in MSNs were modulated by optogenetic inhibition of HALO- but not EYFP-expressing grafted neurons or in CTRL animals. (i) EPSPs recorded from MSNs in CTRL, lesioned, EYFP or HALO striata in the presence of optogenetic graft silencing or D1 receptor blockade with 2 μM SCH39166. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Videos

  1. Ratiometric calcium imaging of a HALO expressing D90 culture during glutamate stimulation (100 M, 15 s pulse). 5 s/frame.
    Video 1: Ratiometric calcium imaging of a HALO expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame.
  2. Ratiometric calcium imaging of a HALO expressing D90 culture during continuous glutamate stimulation (50 M). Three inactivating 550 nm light pulses are applied, which produced a decrease in calcium levels. 1 s/frame.
    Video 2: 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.
  3. Ratiometric calcium imaging of an EYFP expressing D90 culture during glutamate stimulation (100 M, 15 s pulse). 5 s/frame.
    Video 3: Ratiometric calcium imaging of an EYFP expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame.
  4. Ratiometric calcium imaging of an EYFP expressing D90 culture during continuous glutamate stimulation (50 M). Four 550 nm light pulses were applied without producing any change in the calcium response. 1 s/frame.
    Video 4: 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.
  5. 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.
    Video 5: 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.
  6. Corridor test. Representative section of a Lesion-only animal showing lateralized exploration and food retrieval ipsilateral to the lesion (right side).
    Video 6: Corridor test. Representative section of a Lesion-only animal showing lateralized exploration and food retrieval ipsilateral to the lesion (right side).
  7. 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).
    Video 7: 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).
  8. 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.
    Video 8: 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.
  9. 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).
    Video 9: 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).
  10. 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.
    Video 10: 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.

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Author information

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

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.

Competing financial 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.

Corresponding authors

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Supplementary information

Video

  1. Video 1: Ratiometric calcium imaging of a HALO expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame. (2.4 MB, Download)
  2. Video 2: 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. (7.49 MB, Download)
  3. Video 3: Ratiometric calcium imaging of an EYFP expressing D90 culture during glutamate stimulation (100 μM, 15 s pulse). 5 s/frame. (2.46 MB, Download)
  4. Video 4: 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. (2.13 MB, Download)
  5. Video 5: 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. (14.84 MB, Download)
  6. Video 6: Corridor test. Representative section of a Lesion-only animal showing lateralized exploration and food retrieval ipsilateral to the lesion (right side). (20.23 MB, Download)
  7. Video 7: 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). (21.72 MB, Download)
  8. Video 8: 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. (16.77 MB, Download)
  9. Video 9: 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). (15.66 MB, Download)
  10. Video 10: 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. (13.12 MB, Download)

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