In agreement with the 3Rs41, the rationale for the current study is that each case represents its own control through detailed follow-up of all the periods of the protocol i.e. CTR, MPTP, post-MPTP and Pre- vs. Post-Graft. We published precise and detailed reports for each clinical, behavioral and functional parameter followed as well as for the characterization of NSCs grafted in the present study. These precise and detailed control procedures are described in Extended Data Table 1.
Ethical statements
All procedures were carried out according to the 1986 European Community Council Directives (86/609/EEC) which was the official directive at the time of experiments, the French Commission for animal experimentation, the Department of Veterinary Services (DDSV Lyon, France). Authorization for the present study was delivered by the “Préfet de la Région Rhône Alpes” and the “Directeur départemental de la protection des populations” under Permit Number: #A690290402. All procedures were designed with reference to the recommendations of the Weatherall report, “The use of non-human primates in research”. All monkeys were closely monitored on a regular basis throughout the day, by researchers and animal care staff, in order to ensure that levels of health and welfare were strictly maintained, particularly during the MPTP period. Adaptations to housing and feeding procedures were made in direct response to individual symptoms in the MPTP phase, for example adaptations of water provision to ensure monkeys were able to drink ad libitum. To alleviate physical suffering from motor symptoms progression (such as rigidity) the intoxication procedure was first cautiously stopped when the PMRS-motor score was above ten for two consecutive days following one MPTP injection; further, antalgic and/or anti-inflammatory drugs were delivered to animals experiencing debilitating motor symptoms. Before being sacrificed, animals were first tranquilized with a pre-anesthetic agent (chlorpromazine hydrochloride, Largactil) and anesthesia was induced with Ketamine before deep anesthesia was obtained by means of a large dose of pentobarbital sodium (Vibrac, 100 mg kg− 1, i.p.; lethal dose confirmed by complete loss of corneal reflex).
MPTP-intoxication and study design
Six late middle-aged – 11-13 (13-17) years old at protocol onset (end) female macaque monkeys (Macaca fascicularis, 4-5kg) were intoxicated with low-dose 6-methyl- 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin injections (MPTP, 0.2mg.kg-1, i.m). Animals were housed in a room dedicated to MPTP experiments, with free access to water and received food twice a day. The neurotoxin was delivered according to two different regimes: 1) chronically each 3-4 days and 2) acute intoxication (daily injections) as described previously19,42. In cases 1, 3 and 4, the first session of MPTP injections was suspended as soon as the clinical score reached symptomatic threshold (Extended Data Fig.1), allowing investigation of dopaminergic (DA) neurotransmission after spontaneous behavioral recovery18,19 and thus providing critical parameters to compare with potential recovery following transplantation. For the last-MPTP intoxication i.e. ending with persistent motor-symptoms in all cases, injections were cautiously stopped when the PMRS-motor score was above ten for two consecutive days following one MPTP injection. For results presentation, animals were grouped according to their clinical state after transplantation (see Fig.1) i.e. 1st group – recovered (n=3, cases 1, 4, 5) and, 2nd group – non-recovered (n=2, cases 3 and 6). Delays between last MPTP injection and i) transplantation were 4-23 weeks (n=6, all cases), ii) sham-grafts were 6-16 weeks (n=2, cases 1 and 6). Data are presented according to the following 5 periods of the protocol: 1- MPTP (during MPTP intoxication period); 2- post-MPTP Recovery (following arrest of first MPTP injections, clinical motor score returned below 5 throughout this period i.e. non-symptomatic); 3- post-MPTP Symptomatic (following arrest of last MPTP injections, clinical motor score remained stably above 5 during this period i.e. motor-symptomatic); 4- post-Sham (following sham grafts, details in Surgical procedures); 5- post-Grafts (following transplantation, details in Surgical procedures). Case 4 had a high-score post-MPTP (motor symptoms>15), characterized by tremor at rest and freezing. This motor-symptomatic state persisted during the 4 weeks prior to transplantation (Extended Data Fig.1a). Case 6 had a high-score post-MPTP (motor symptoms>15), characterized by severe rigidity. Musculoskeletal pain is a recognized source of pain syndromes and discomfort in Parkinson’s disease patients. We thus delivered regularly ketoprofen (Profenid), from week 5 post-MPTP, in order to relieve suspected apparent musculoskeletal pain. Subsequently the clinical score for this animal progressively improved, due to amelioration on the rigidity scale and ability to manipulate food (from week5 to week10 post-MPTP, Extended Data Fig.1a), but stabilized at week10 and remained largely symptomatic (score >12) despite continuous treatment and disappearance of apparent musculoskeletal pain syndrome. Case 6 was transplanted at week 23 post-MPTP. Case 2 had per operative brain hemorrhage following NSCs transplantation and was thus excluded from comparative analyses pre- vs. post-Graft.
Clinical motor scale – PMRS
Clinical scoring was done independently by three trained experimenters at separate times throughout the day, 3-5 days per week using the Parkinsonian Monkey Rating Scale18,19. The PMRS contains a motor component, employing a previously published scale43 used to define the onset of the motor symptomatic period. The total score for freezing (0–3), resting tremor (0–3, for right and left side), rigidity (0–3), posture (0–2), bradykinesia (0–3), and ability to manipulate food (0–3, for right and left side) was used to frame injections (maximum total score of 23): a score below 5 defined the non-symptomatic state in premotor and recovery periods, and a score above 5 defined symptomatic periods.
Cognitive behavior task – detour task
Cognitive performance was monitored 3-5 days a week using a previously described behavioral test18,19. Briefly, performance on the ‘detour task’ was evaluated by the percent of successes (retrieval of reward on the first reach, over the total number of trials) and errors (barrier hits, over the total number of responses observed - there could be several responses per trial except in the case of success). Errors due to motor impairments were classified as such and further discarded from the performance evaluation, so as to avoid confounds due to the symptomatic state of the animal. The performance on the ‘detour task’ depends on the integrity of frontal cortex, the dopaminergic system and DA innervation of frontal cortex44 and has been used to assess the impact of lesion of the nigrostriatal axis on cognitive performance as well as potential therapy for cognitive impairments45-47. For inter-individual comparison, percent errors and success were, for each case, first converted in percent change by the average control value and normalized between 0 and 1 by overall maximum and minimum score respectively.
Circadian rhythm follow-up
Animals were maintained under an LD cycle (12h light: 12h dark) of approximately 450–500 lux during the light phase and 0 lux during the dark phase. Locomotor activity was continuously recorded in 1 min intervals throughout the entire duration of the study using passive-infrared motion detectors mounted above each animal's cage. The motion captors were connected to a computerized data acquisition system (Circadian Activity Monitoring, INSERM, France48). Data were analyzed using the Clocklab software package (Actimetrics, Evanston, IL, USA).
We previously used Chi-squared periodogram method (Extended Data Table 1) to calculate the period of behavioral rhythm which is defined as the time elapsed for one complete oscillation or cycle (the distance in time between two consecutive peaks or troughs of a recurring rhythm). The stability and the consistency of the rhythm are reflected by the value of the periodogram amplitude which corresponds to the zenith of a periodogram curve. The subjective day and the subjective night are defined as the segment of a circadian cycle during the free-running state that corresponds to, respectively, the light and dark segments during entrainment by a LD cycle.
Non-parametric circadian rhythm analyses (NPCRA) were used to estimate the strength and fragmentation of rest-activity rhythms12,18. These included measurements of inter-daily stability (calculated as the ratio between the variance of the average 24-hour pattern around the mean and the overall variance and gives an indication of the consistency of day to day activity or the strength of coupling to the LD cycle), intra-daily variability (calculated as the ratio of the mean squares of the difference between successive hours and the mean squares around the grand mean and gives an indication of the frequency of transitions between rest and activity periods, corresponding to the fragmentation of the rhythm) and, relative amplitude (ratio between acrophase and nadir of the rhythm, representing the ratio between activity amplitude in light and dark phases). When the rest-activity rhythm is stable, inter-daily stability is high, the intra-daily variability is low and relative amplitude is high.
DA function imaging – [11C]-PE2I and [18F]-FDOPA
To evaluate the in-vivo DA function, we acquired images from Positron Emission Tomography (PET) scans using (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)-nortropane labeled with carbon 11 ([11C]-PE2I) at different intervals throughout the protocol as described previously19 in cases 1,2,3 and 6 and after grafts at different delays (see Extended Data Figs.1, 5). [11C]-PE2I specifically binds with high affinity and selectivity to DA transporters (DAT, Ki = 17nM) and is considered to provide an index of the integrity of the DA pathway that has been used for PD diagnosis, see49-51 for a review. Additionally, we used images from PET scans using L-3,4-dihydroxy-6-(18F)fluorophenylalanine ([18F]-FDOPA, an analog of the DA precursor L-DOPA) to evaluate the central dopaminergic function of pre-synaptic neurons, i) just before grafts (during stable expression of typical motor parkinsonian symptoms i.e. symptomatic period) and, ii) after grafts at different delays (see Extended Data Fig.1).
We performed [11C]-PE2I and [18F]-FDOPA PET scans with an ECAT Exact HR+ tomograph (Siemens CTI), in 3D acquisition mode, covering an axial distance of 15.2cm. The transaxial resolution of the reconstructed images was about 4.1mm full-width and half maximum in the center. We acquired transmission scans with three rotating 68Ge sources to correct emission scans for the attenuation of 511 keV photonrays through tissue and head support. Full procedure for PET-scans acquisition, modeling and ROIs definition for regional assessment of [11C]-PE2I changes have been published19. Fluoro-DOPA was labeled with 18F-fluoride (cyclotron–produced isotope, half-life = 109 min). Specific radioactivity was 3.27 ± 1.3 mCi/μmol. Radiochemical and chemical purity of produced [18F]-FDOPA (as determined by HPLC) was above 99%. After anesthesia was induced and head secured with a MRI-compatible stereotaxic frame (Kopf, CA, USA) to reduce variability in the measure, a cannula was inserted in the femoral vein. [18F]-FDOPA was injected as a bolus over a 4 s period followed by a saline flush. Radioactivity was measured in a series of 26 sequential time frames of increasing duration (from 30 sec to 5 min; total time 90 min).
PET modeling and regional assignment of PET changes were done as previously described19 and applied to F-DOPA measurement52. Statistical positive (shown in Fig.1d) and negative (shown in Extended Data Fig.6c, right) difference were determined from 2.5th and 97.5th percentile boundaries of voxel-level distribution statistics of all brain ROIs as described previously19 and illustrated in Extended Data Fig.6c, left.
Transplanted cells and Surgical procedures
We derived neural stem cells (NSCs) from a rhesus embryonic stem cell line stably expressing tau-GFP (LYON-ESC line)21. NSCs were obtained either i) as described in53 where multipotent NSCs were amplified by mild trypsinization and cultured in the presence of EGF and FGF2 (Extended Data Fig.2a, c), or ii) after MS5 induced-neural differentiation followed by early midbrain differentiation, as described in54 (Extended Data Fig.2b, c, f). The latter cell type corresponds to early DA midbrain neural precursors. For simplicity and because the two cell types lead to similar recovery when grafted in different hemispheres (Extended Data Fig.6), we used the generic term of NSCs. NSCs express NSC specific markers (Extended Data Figs.2a-c, 3a) and showed in-vitro potential to fully differentiate along glial and neuronal pathways (Extended Data Fig.2e) including mature DA neurons (Extended Data Fig.2f). Grafted NSCs retained the capacity to differentiate into glial and neurons in vivo, depending on the local environment of the graft (Figs.2b-e, i, 3 and Extended Data Fig.4a).
We performed all transplantations bilaterally using 5µL Hamilton syringe and small trepanations over target sites. Stereotaxic coordinates were calculated from individuals T1 and T2 structural MRI images. We undertook sham-grafts (trepanation and injection of 5µL PBS without NSCs) in cases 1 and 6 in order to control for behavioral and clinical effects due to the surgical act in itself. We placed sham-grafts (PBS-only) in posterior caudate and anterior putamen, using the same protocol as for NSCs transplantation. The amount of injected NSCs varied between 3.105-106 cells for SN sites and 1.5.105-106 for striatum sites (Extended Data Table 2). The syringe was lowered 500µm further than the target position and then retracted to target; cells were then injected at a rate of 1µL per min after a 5min delay. After an additional 3 minute delay after injection completion, the syringe was retracted slowly by 2mm; another 2 minute delay was observed before full retraction. We assessed cell viability by trypan blue exclusion before injection and counted the remaining cells after injection. Viability at both time points was found to be between 95 and 100%. Cell mortality due to syringe uptake was estimated at 2%.
Immuno-histological procedures
Deep anesthesia was induced with Ketamine after premedication with chlorpromazine hydrochloride (Largactil) followed by a lethal dose of pentobarbital sodium (Vibrac, 100 mg.kg-1, i.p.; confirmed by complete loss of corneal reflex) before animals were transcardially perfused with saline (0.9% with procaine), 4% paraformaldehyde and 0.05%-glutaraldehyde in phosphate buffer. Cryoprotection was ensured by sucrose gradients (10-30%) perfusion post-fixation. Brains were removed, kept in cryoprotecting liquid overnight and coronal 50µm thick sections cut on a freezing microtome and collected serially. Sections were processed together with equivalent sections from control animals for Tyrosine Hydroxylase (TH; Millipore, #MAB318) and DAT (Millipore, #MAB369) immunochemistry visualized with 3,3-diaminobenzidine, in Ni²+H202 (0.5-1.0%). All sections were washed, mounted, dried, and dehydrated in increasing gradients of ethanol, cleared in toluene, mounted with mounting medium and cover slipped.
For immunofluorescent staining on free-floating brain slices, sections were washed six to ten times in Tris buffered saline (TBS) and permeabilized in Triton X-100 (0,5%; SIGMA, #T9284). Nonspecific binding was blocked with 10% normal goat serum or donkey serum (Goat: Invitrogen; #16210064; Donkey: SIGMA #D9663) for 45 min at room temperature. Sections were incubated for three days at 4°C, with primary antibodies diluted in Dako antibody diluent (Dako #S3022) supplemented with 0,5% Triton X-100. They were then exposed to secondary antibodies and DAPI (1/10 000; Invitrogen #D1306) for 2 hours at room temperature. Sections were mounted on slides and examined using a confocal microscope (Leica TCS SP5). Sections were regularly (every five sections i.e. every 250µm) processed for GFP immunostaining to detect grafted sites extent.
We determined the length of processes emanating from the graft core by measuring the length of tau-GFP positive projections using the ImageJ software (NeuronJ plugins, http://imagej.nih.gov/ij/), presented in Extended Data Fig.5d. We estimated the graft volume through a 3D reconstruction process using contours traced and compiled with the Mercator software (ExploraNova, http://www.exploranova.com/products/mercator/), presented in Extended Data Table 3, Fig.2f-h, Extended Data Figs.4, 5a-d. We determined the average cell density by (i) counting all cells present in four random locations within each confocal optical section in several slices at different levels of the graft core to return cell density per mm3 and (2) by scaling those counts to the estimated graft volume in mm3 to return estimated cell number per graft. Finally, we calculated survival rate according to the number of transplanted cells per site (Extended Data Tables 2-4).
Immunostaining and RT-PCR analyses of cultured cells were done according to previously published protocols21.
Semi-quantitative immunohistochemistry
Three age and weight matched Macaca fascicularis served as controls. We evaluated the extent and amplitude of the nigrostriatal lesion through semi-quantitative immunohistochemistry of striatal structures, as previously described19,55. The mean optical density (O.D.) of TH and DAT in immunopositive regions were computed on at least 5 sections per animal (5-20) from 8bit images, normalized and compared with two-sample t-test separately for each ROI (ttest2, Matlab). In Fig.2a, composite CTR and MPTP images has been enhanced for brightness and contrast, for illustration purpose.
Statistics
We segmented data for MPTP, post-MPTP and post-Graft periods into five equal epochs and grouped variables into each of these epochs. This method of segmentation, described in18,19, thus reveals normalized stages of the progression of processes and allows for comparison of different parameters across an equivalent number of epochs for all subjects, called Quantiles (Fig.1, 27 ± 3 days for the CTR period; duration per quantile for premotor 10 ± 5 days , motor 18 ± 8 days and post-Graft 42 ± 12 days). Results are presented as means ± standard errors.
Parameters were compared between groups by a treatment-contrast test using one group as the first level and Bonferroni corrected for multiple comparisons and, within groups across periods by a treatment-contrast test using control measures as the first level (estimated standard errors and z-ratio were computed using GLM fit and contrast result was given by two-tailed p-values corresponding to z-ratio based on a Student t-test). Significance was considered at p<0.05. Statistical analyzes were computed using R software (R Foundation for Statistical Computing, Vienna, Austrian http://www.R-project.org) and the summary function (Chambers, J. M. and Hastie, T. J. (1992) Statistical Models in S. Wadsworth & Brooks/Cole).
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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