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Graft-derived neurons and bystander effects are maintained for six months after human iPSC-derived NESC transplantation in mice’s cerebella – Scientific Reports

Ethics declarations

Human fibroblast isolation was performed under informed consent from all participants, was approved by the Ethics Committee of the Medical Faculty of the University of Coimbra, Portugal, and all research was performed in accordance with relevant guidelines/regulations.

All the experiments with animals were carried out in accordance with the European Community Council directive (86/609/ EEC) for the care and use of laboratory animals, were approved by the Responsible Organization for the Animals Welfare of the Faculty of Medicine and Center for Neuroscience and Cell Biology of the University of Coimbra and by the Portuguese Regulator (DGAV- 0421/000/000/2015) under ORBEA 66 (ORBEA_66_2015/22–06-2015), and all research was performed in accordance with relevant guidelines/regulations.

This study is reported in accordance with ARRIVE guidelines.

Lentivirus production

The lentiviral vectors encoding for green fluorescent protein (GFP, SIN-PGK-GFP-WHV) and the lentivirus encoding for the 4 reprogramming factors52 were produced in 293 T HEK cells using a four-plasmid system, as previously described53. The lentiviral particles were stored at -80ºC and their concentration was determined by measuring the HIV-1 p24 antigen (RETROtek, Gentaur).

Fibroblasts reprogramming into iPSC

Fibroblasts of one MJD patient (women, 31 years old, 79 CAG repetitions in the mutant ataxin-3 allele) and one Control (women, 52 years, no mutant allele) were previously established, and a normal diploid karyotype and genotyping indicating the number of CAG repetitions was also previously demonstrated54.

To reprogram human fibroblasts into iPSC, 10 000 fibroblasts growing in DMEM High-glucose (Sigma) medium supplemented with 10% FBS (Gibco), 1% Penicillin–Streptomycin (Gibco), 1% non-essential amino acids (Sigma), and 2 mM L-Glutamine (Gibco) were transduced with lentivirus (200 ng p24) encoding for the 4 reprogramming factors (c-Myc, Klf4, Sox2, and Oct4) and the dTomato fluorescent protein52. Fibroblasts were incubated with the virus for 12 h, then the culture medium was changed and cells were kept in this medium for 3 days. The lentivirus titration optimization in the fibroblasts used in this study has been performed by us55 according to a previously described study56. Transduction efficiency was evaluated in Control fibroblasts through the percentage of cells expressing dTomato (Supplementary Fig. S10). Afterward, fibroblasts were harvested via trypsinization, transferred onto mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeders, and subsequently cultured in human pluripotent cell culture medium (DMEM F-12 (ThermoFisher), 20% Knock-out serum replacement (Gibco), 2 mM L-glutamine (Gibco), 1% nonessential amino acids, 100 μM β-mercaptoethanol, 1% Penicillin–Streptomycin, and 10 ng/mL of bFGF (Peprotech)). The culture medium was replaced every 1–3 days depending on the cell density. iPSC single colonies were picked based on morphology and self-silenced reprogramming factors (absence of dTomato)52. IPSC were split (1:4) by manual dissection and expanded on MEF feeder cells. Stable iPSC clones with at least 5 passages were established.

iPSC-derived neuroepithelial stem cells (NESC) derivation

One Control iPSC clone and 3 MJD iPSC clones (from the same MJD patient) were then submitted to a neural induction protocol, as previously described12. Briefly, iPSC colonies were detached with 1 mg/mL dispase (STEMCELL Technologies), the cell clusters were collected through sedimentation and resuspended in human pluripotent cell culture medium (without bFGF) supplemented with 10 μM SB-431542 (Axon), 1 μM dorsomorphin (Sigma), 3.0 μM CHIR 99,021 (TargetMol), and 0.5 μM Purmorphamine (TargetMol) and cultured in non-treated MW6 plates. Then, the culture medium was replaced (50% on day 2, and 100% on day 3) by N2B27 medium (DMEM/F-12 no glutamine / Neurobasal (Gibco) 50:50, complemented with 1:200 N2 supplement (Gibco), 1:100 B27 supplement minus vitamin A (Gibco), 1% penicillin/streptomycin, and 1% L-glutamine) supplemented with 10 μM SB-431542, 1 μM dorsomorphin, 3.0 μM CHIR99021, and 0.5 μM Purmorphamine. On day 4, SB-431542 and dorsomorphin were withdrawn and 150 μM Ascorbic Acid (Sigma) was added to the medium. On day 6, the embryonic bodies showing neuroepithelial outgrowth were disrupted into smaller cell clusters and plated on Matrigel (hESC-qualified Matrix LDEV-Free, BD Matrigel, Corning) coated 12-well plates in NESC culture medium (N2B27 medium with 3.0 μM CHIR99021, 0.75 μM Purmorphamine, and 150 μM Ascorbic Acid). After a maximum of 5 splits, cultures were virtually free of contaminating non-NESC. One Control (CNT NESC) and three MJD (MJD CLA, MJD CLB, and MJD CLC NESC) cell lines were established and cultured in an incubator at 37ºC with 5% CO2. Frozen cell stocks were established in early passages (passage 5 to passage 10). Whenever needed cells were defrosted and cultured showing no change in morphology, proliferation, and differentiation capacity.

Karyotype analysis

The karyotype analysis was performed by a certificated Genetic Laboratory (Centro de Medicina Laboratorial Germano de Sousa, Lisboa, Portugal) using standard G-banding techniques. Cells growing in a T25 flask covered with Matrigel were treated with 10 μg/ml Colcemid (0.05 μg/ml final) for up to 1 h, followed by dissociation with StemPro Accutase Cell Dissociation Reagent (Gibco). The cells were pelleted via centrifugation, re-suspended in pre-warmed 0.075 M KCl hypotonic solution, and incubated for 5 min at 37ºC. Following centrifugation, the cells were re-suspended in Carnoy’s fixative (3:1 ratio of methanol: glacial acetic acid). Metaphase spreads were prepared on glass microscope slides and GTG-banded by brief exposure to trypsin and stained with Leishman. Fifty metaphases were analyzed for each cell line and karyotypes were established according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016.

iPSC-derived NESC culture and differentiation

Cells growing in monolayers in Matrigel-coated cell culture flasks cultured in NESC culture medium, as previously described, were detached with pre-warmed StemPro Accutase Cell Dissociation Reagent for 3–5 min at 37ºC. Then, cells were diluted in KnockOut DMEM (Gibco) and precipitated through centrifugation at 1100 RPM for 5 min. For cell differentiation in neural cultures composed of neurons and glia, 250 000 cells/well (MW12 plates) were plated on Matrigel-coated coverslips in N2B27 medium supplemented with 250 μM Dibutyryl cyclic-AMP sodium salt (dbcAMP) (BIOLOG), 5 μM Forskolin (TargetMol), and 2 μM retinoic acid (Sigma) as previously reported5,46. Cells were maintained for up to 2 months and the culture medium was changed every five days.

Human brain organoids establishment

Brain organoids were obtained as previously described19. Briefly, Control and MJD iPSC-derived NESC cultured in N2B27 medium complemented with 3.0 μM CHIR99021, 0.75 μM Purmorphamine, and 150 μM Ascorbic Acid were plated at 9000 cells/well/100 µL in an ultra-low attachment 96-well flat-bottom plate (Corning Costar). This culture medium was added every other day for 6 days. Then, spheroids were transferred to a 24-well ultra-low attachment flat-bottom plate (Corning Costar). Two days later (day 8), the spheroids were transferred to cold droplets of Matrigel and cultured in the same culture medium for 2 days. On day 10, N2B27 medium supplemented with 10 ng/mL human BDNF, 10 ng/mL human GDNF, 1 ng/mL human TGFβ3, 500 μM dbcAMP, 200 μM ascorbic acid, and 1 μM purmorphamine was used to induce differentiation. On day 14, organoids were placed on an orbital shaker (65 rpm), in an incubator at 37ºC with 5% CO2. From day 16, the culture medium was changed every 3–4 days using N2B27 medium supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 1 ng/mL TGFβ-3, 500 μM dbcAMP, and 200 μM ascorbic acid. Organoids were kept in these culture conditions until the end of the experiment.

GFP expression and maturation of iPSC-derived NESC prior to cerebellar transplantation

NESC plated in T75 flasks covered with Matrigel were infected with lentivirus encoding for GFP as previously reported5. Briefly, 3 × 106 cells were infected with lentivirus (1.33 × 103 ng of p24 antigen) and 24 h later half of the culture medium was replaced to dilute the virus. On the second day, the lentiviral vectors were completely removed by changing the medium. Five days before transplantation, cells were submitted to a maturation protocol with the N2B27 medium supplemented with 10 ng/ml human BDNF (Peprotech), 10 ng/ml human GDNF (Peprotech), 1 ng/ml human TGFβ-3 (Peprotech), 200 μM Ascorbic Acid, and 250 μM dbcAMP. For transplantation, cells were prepared in Hank’s Balanced Salt Solution HBSS (Sigma) at 150 000 cells/2 μl.

Immunocytochemistry

Cells in coverslips were washed two times with PBS, fixed with 4% paraformaldehyde (PFA) (Sigma) for 20 min at room temperature, washed with PBS, and stored at 4ºC until further processing. Then, cells were permeabilized for 5 min with 1% Triton X-100 (Sigma), washed three times with PBS, and blocked in 3% bovine serum albumin (BSA) (Sigma) in PBS for 1 h at room temperature. Cells were then incubated overnight at 4ºC with primary antibodies (MAP2, GFAP, β3 Tubulin, S100B, and O4; Supplementary Table S1) diluted in 3% BSA in PBS. Afterward, coverslips were washed two times with PBS and incubated for 2 h at room temperature with secondary antibodies Alexa Fluor-594 anti-mouse and Alexa Fluor-488 anti-rabbit (1:200, Invitrogen) diluted in 1% BSA in PBS. Subsequently, cells were washed two times with PBS; nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (1:5000, Applichem) for 5 min, and then washed 3 times with PBS. Finally, coverslips were mounted with a fluorescence mounting medium (Dako). Images were acquired at room temperature with an Axio Imager Z2 widefield microscope (CCD monochromatic digital camera Axiocam HRm) using EC Plan-Apochromat 10x/0.3NA or Plan-Apochromat 20x/0.8NA air objectives.

Total neurite length measurement and synapses quantification in cell cultures

Cells plated in coverslips, as previously described, were washed two times with PBS, fixed with 4% PFA in PBS for 20 min at room temperature, washed again with PBS, and stored at 4ºC until further processing. Then, cells were permeabilized for 5 min in 0.25% Triton X-100 at room temperature, washed three times with PBS, and blocked in 10% BSA in PBS for 1 h. Primary antibodies (VGluT1, PSD95, MAP2, β3 Tubulin, and Gephyrin; Supplementary Table S2) were prepared in 3% BSA in PBS and centrifuged for 10 min at 13,000 rpm, 4ºC, to remove antibody aggregates. Cells were incubated overnight with the primary antibodies. Afterward, cells were washed two times with PBS, followed by a 45 min incubation at 37ºC with Alexa Fluor conjugated secondary antibodies, diluted in 1% BSA in PBS. Coverslips were then washed two times with PBS, nuclei were stained with DAPI (1:5000) for 5 min, washed 3 times with PBS, dried, and mounted with fluorescence mounting medium (Dako). Images were acquired with an Axio Imager Z2 widefield microscope (CCD monochromatic digital camera Axiocam HRm). For neurite length measurement it was used EC Plan-Apochromat 10x/0.3NA or Plan-Apochromat 20x/0.8NA air objectives after randomly selecting MAP2-positive cellular tracts. For synapse quantification it was used a Plan-Apochromat 63x/1.4NA oil objective after randomly selecting MAP2/ β3 Tubulin-positive cellular tracts. At least five pictures were acquired and analyzed from each condition. Images were analyzed blindly to condition using Fiji software. The number of colocalized puncta or the total number of inhibitory postsynaptic terminals were normalized per dendritic section length.

Western blot

Cells were disrupted using a lysis buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS added freshly with protease inhibitor (cOmplete Mini, Roche), phosphatase inhibitor (PhosStop, Roche), 1 mM Phenylmethane Sulfonyl Fluoride (PMSF) (Sigma-Aldrich), and 10 μg/mL Dithiothreitol (DTT) (Sigma-Aldrich)) and a strong vortex followed by 3 sonication cycles of 10 s (40 kHz). The amount of protein in each sample was quantified using Pierce BCA Protein Assay Kit (Thermo Fischer Scientific) and 50 μg of protein per sample was loaded. Samples were prepared with sample buffer (0.5 M Tris–HCl, pH 6.8, 30% glycerol, 10% SDS, 0.6 M DTT, and 0.1 mg/mL blue bromophenol), denatured at 95ºC for 5 min, and stored at -20ºC until use. Proteins were separated by SDS–polyacrylamide gel electrophoresis, in 10% resolving acrylamide gels at 70 V for 10–15 min and 100 V for 45—60 min. Afterward, proteins were transferred into polyvinylidene difluoride (PVDF) membranes (Immobilon-P Membrane, Millipore). The transference was done at 0.75 A and 4ºC for 2 h and 30 min. Then, membranes were blocked with 5% milk in Tris-Buffered Saline (TBS) with 0.1% Tween 20 (TBS-T) for 1 h. Incubation with primary antibodies (Tra-1–60, Pax6, ataxin-3, and Actin; Supplementary Table S2), prepared in 5% milk in TBS-T, was done overnight at 4ºC. Subsequently, membranes were washed three times with TBS-T and incubated with alkaline phosphatase-linked secondary antibodies for 2 h at room temperature. Membranes were washed with TBS-T three times and proteins were detected using Enhanced Chemifluorescence substrate (ECF, Amersham Biosciences) in the Chemidoc (Bio-Rad) and analyzed in Image Lab software (Bio-Rad).

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)

NESC cultures and organoids (a 3–5 organoids pool depending on the time point/organoids’ size) were washed 3 times with PBS and kept at -80ºC until processing. RNA was extracted using the NucleoSpin RNA kit (Macherey–Nagel) according to the manufacturer’s instructions. The RNA purity and concentration were measured with NanoDrop 2000 (Thermo Scientific). Then, 1000 ng of total RNA was converted into cDNA with iScript cDNA Synthesis Kit, according to the manufacturer’s instructions (Bio-Rad). Quantitative real-time PCR was performed with the SsoAdvanced SYBR Green Supermix Kit (Bio-Rad) using cDNA diluted 10 times with DNase-free water (Sigma) and the protocol: a single cycle of 95ºC for 30 s followed by 45 cycles of two steps, one step of 5 s at 95ºC followed by a step of 15 s at a temperature depending on each primer set (Supplementary Table S3). StepOne Software (Applied Biosystems) generated automatically a threshold cycle (CT) for each gene. In each experiment, and for all genes, a standard curve was done, and no template and no reverse transcriptase controls were performed. Subsequently, the software determined the PCR amplification efficiency and R2. The mRNA relative quantification with respect to control samples was determined by the Pfaffl method taking into consideration the different amplification efficiencies of all genes.

Metabolic activity

Metabolic activity was assessed by the resazurin reduction assay. For this, a single organoid/well (in 96-well plates) was incubated with 0.1 mg/mL resazurin, prepared in the organoids culture medium previously described, for 4 h at 37ºC and 5% CO2. The absorbance for the reduced and oxidized species of resazurin was read at 570 nm and 600 nm, respectively, using a spectrophotometer (SpectraMax Plus 384, Molecular Devices). Metabolic activity was evaluated through absorbance at 570 nm subtracted from 600 nm absorbance.

Single-cell calcium imaging

For cells plated in coverslips, the intracellular calcium concentration changes were evaluated upon potassium and histamine depolarization, markers of functional differentiated neurons and undifferentiated neural progenitors, respectively5,17. For brain organoids, cultured as previously described, the intracellular calcium concentration changes were evaluated upon potassium depolarization. Cells and organoids were washed twice in 0.1% BSA/Krebs buffer (142 mM NaCl, 1 mM KCl, 1 mM MgCl2, 10 mM Glucose, 10 mM HEPES, 10 mM NaHCO3, and 1 mM CaCl2, pH 7.4) and incubated with 5 μM Fluo-4/AM (a calcium indicator, increasing its fluorescence excitation at 488 nm upon Ca2+ binding, thus increasing the fluorescence signal) (Invitrogen) and 0.02% pluronic acid in 0.1% BSA/Krebs buffer for 45 min at 37ºC. Afterward, cells and organoids were transferred to 0.1% BSA/Krebs buffer and kept at 37ºC during microscopy observation. For observation, the basal fluorescent levels were measured for 4 min. Then, the depolarization stimulus, 50 mM KCl/Krebs (96 mM NaCl, 50 mM KCl, 1 mM MgCl2, 10 mM Glucose, 10 mM HEPES, 10 mM NaHCO3, and 1 mM CaCl2, pH 7.4) for cells, and 50 mM KCl in 0.1% BSA/Krebs buffer for organoids, was added for 2 min, followed by a 4-min repolarization period in 0.1% BSA/Krebs. Subsequently, for the cell cultures, the second depolarization stimulus (100 μM histamine in 0.1% BSA/Krebs buffer) was added for 2 min, followed again by a 4-min repolarization period in 0.1% BSA/Krebs buffer. Fluorescence measurement at 488 nm was performed at 37ºC and continuously for all the experiments with a Carl Zeiss Cell Observer Spinning Disk microscope (EM-CCD Evolve Delta camera) using a Plan-Apochromat 20x/0.8NA air objective. Image analysis was performed in Fiji, where cell bodies were drawn and the shift in signal intensity was measured. Cells responding to potassium were identified as functional neurons, while cells responding to histamine were identified as neural progenitors. Cells were considered to respond to the potassium stimulus when a 15% increase in signal (Ca2+ concentration) was observed, while cells presenting a histamine/potassium ratio equal to or above 1 were considered to respond to histamine and consequently considered as neural progenitors as previously reported5. The response amplitude for each neuron was determined by the maximum fluorescence value normalized by the basal fluorescence.

Organoids sectioning and immunolabeling

Brain organoids were collected on day 40, washed three times in PBS, and fixed with 4% PFA for 20 min at 4ºC. Then, organoids were washed three times with PBS and kept in 30% sucrose in PBS for 24 h at 4ºC. Organoids were subsequently frozen at -80ºC in Tissue-Tek O.C.T. Compound (Sakura) and kept at -80ºC until further sectioning. Organoids were sliced at 35 μm using a cryostat (Thermo Fisher Scientific) and collected in Superfrost Plus slides (Thermo Scientific). Slides were dried at 37ºC for 25 min and kept at 4ºC until immunohistochemistry processing. Afterward, sections were washed three times with PBS and blocked and permeabilized with 0.1% Triton X-100 and 10% normal goat serum (NGS) in PBS, for 2 h at room temperature. Then, sections were incubated overnight at 4ºC with the primary antibodies (Nestin, GFAP, MAP2, PCP4, Ataxin-3, Msi1, and NeuroD1; Supplementary Table S2) prepared in 10% NGS in PBS. Subsequently, sections were washed three times with PBS and incubated with the Alexa Fluor conjugated secondary antibodies, prepared in 2% NGS in PBS, for 2 h at room temperature. The nuclei were stained with DAPI (1:5000) for 10 min at room temperature. Sections were then washed three times with PBS, dried, and mounted with Mowiol reagent (Sigma). Fluorescence images were acquired with a Plan-Apochromat 20x/0.8NA air objective on an Axio Imager Z2 widefield microscope.

Organoids’ size

Sections from organoids collected as previously described were washed three times with PBS before incubation with the nuclear DNA staining reagent DAPI for 10 min. The sections were then washed three times with PBS, air dried, and mounted with Mowiol reagent (Sigma). Widefield fluorescence images were acquired with an EC Plan-Apochromat 20x/0.8NA air objective. A tile image was acquired and stitched together to reconstitute the entire organoid. ImageJ/Fiji software was used to determine the organoids’ area in the DAPI channel.

Stereotaxic injection of iPSC-derived NESC

For in vivo experiments, immunodeficient NOD.CB17-Prkdc scid/J female mice (NOD.SCID) (Charles River) were used. In each experiment, littermates with similar weights were caged randomly and submitted to 7 days of acclimatization before the stereotaxic surgery. The NOD.SCID (postnatal days 42–46) were submitted to deep anesthesia (ketamine/medetomidine) and received a single stereotaxic injection of 150 000 cells prepared in 2 μl HBSS (Sigma) injected in lobule 5 of the cerebellum at a rate 0.25 μl/min with an automatic injector (Stoelting Co., Wood Dale, USA) at the following coordinates: anteroposterior: − 2.3 mm, lateral: 0 mm and ventral: − 3.0 mm, relative to lambda. For the 2-month-time point the following number of animals were transplanted: CNT NESC n = 4, MJD CLA NESC n = 3, MJD CLB n = 5, and MJD CLC NESC n = 4 mice. For the 6-month-time point CNT NESC n = 5, MJD CLA NESC n = 3, and HBSS-injected n = 4 mice were transplanted. Mice were housed in sterile conditions (Ventiracks) and food and water were provided ad libitum.

Mice brain tissue preparation and immunohistochemistry

After induction of deep anesthesia (ketamine/medetomidine) mice were intracardiacally perfused with 4% cold PFA, brains were removed, post-fixed in 4% PFA for 24 h at 4ºC, and cryoprotected in 25% sucrose in PBS for 48 h at 4ºC. Then, brains were frozen at -80ºC and 25 μm-sagittal sections were cut using a cryostat (Thermo Fisher Scientific). Brain slices were collected in 48-well plates as free-floating sections in PBS/0.05 mM sodium azide and were stored at 4ºC until processing. Brain sections (two sections per animal) were washed three times with PBS and blocked and permeabilized with 10% NGS and 0.1% Triton X-100 in PBS for two hours. Primary antibodies (GFAP, MAP2, Iba1, cleaved Caspase-3, Ki67, PCP4, Calbindin, Parvalbumin, GABA, Synaptophysin, Gephyrin, PSD95, VGluT1, NGF, GDNF, IL10, IL1B, IL6, Msi1, and NeuroD1; Supplementary Table S2) were diluted in 10% NGS and 0.05% Triton X-100 in PBS and incubated overnight at 4ºC. For synapses quantification the diluted antibodies were centrifuged at 4ºC for 10 min at 13,000 rpm prior to incubation, to remove antibody aggregates. Then, after three washes in PBS, brain sections were incubated for two hours with Alexa Fluor conjugated secondary antibodies (1:200, Invitrogen) diluted in 2% NGS in PBS. Sections were nuclei stained with DAPI (1:5000) for 10 min and washed three times in PBS. Brain sections were mounted in glass slides covered with gelatin, left to dry for 15 min at 37ºC, and mounted with Mowiol mounting medium. Widefield fluorescence images were acquired with an Axio Imager Z2 microscope (CCD monochromatic digital camera Axiocam HRm) using EC Plan-Apochromat 10x/0.3NA or Plan-Apochromat 20x/0.8NA air objectives. Confocal fluorescence images were obtained with an LSM 710 Axio Observer using Plan-Apochromat 20x/0.8NA air objective and Plan-Apochromat 63x/1.4NA oil objective. For synapse quantification in graft-derived neurons, Z-stack confocal microscopy pictures were acquired with the 63x/1.4NA Plan-ApoChromat oil objective with exposure times conserved in the experiments. IMARIS software (Oxford Instruments) was used to obtain the 3D representation of the confocal microscopy pictures and to quantify synapses as pre/post-synaptic spots adjacent to each other. For immunofluorescence quantification, two sections (immediately next to the cerebellum sections where the cell grafts were detected but not containing human cells) were analyzed. Exposure times were conserved in the experiments, tiff images (obtained from microscopy pictures) were processed using ImageJ/FIJI (National Institutes of Health), and mean intensity was obtained for each lobule(s) quantified (lobule 4 and 5 (Lobules 4–5), lobule 6, 7 and 8 (lobules 6–8), Lobule 9, and Lobule 10).

Quantification of undifferentiated and differentiated human iPSC-derived NESC

For in vivo cell differentiation, immunofluorescence Z-stack pictures of the whole human graft in cerebellar sections were acquired with a Plan-Apochromat 40 × /1.40 Oil DIC M27 objective on a Zeiss LSM 710 confocal microscope. Zeiss Zen software was used to obtain orthogonal reconstructions of the confocal z-stack pictures. Then, ImageJ software was used for the quantification of positive cells for each specific cell marker. The threshold of each channel was adjusted so that only positive staining remained. Following this, regions of interest (ROI) corresponding to the human nuclei (HuNu-positive cells) were selected, and these ROIs were placed on the channel of the cell marker of interest (Pax6, β3 Tubulin, NeuN, S100B). The colocalization percentage between both channels was then measured, and cells exhibiting more than 10% colocalization were considered positive for the marker. For in vitro cell differentiation, four immunofluorescence pictures were randomly acquired for each condition of each of the four independent experiments with a Zeiss LSM 710 confocal microscope using a Plan-Apochromat 20x/0.8NA air objective. The quantification method was equal to the previously described for in vivo cell differentiation, except that the regions of interest (ROI) corresponding to the human nuclei were established on the DAPI channel, which was then placed on the channel of the cell marker of interest (MAP2 and GFAP). The colocalization percentage between both channels was then measured, and cells exhibiting more than 8% colocalization were considered positive for the marker.

Cresyl violet staining and quantification of cerebellum total area and cellular layers area

Sections were pre-mounted and stained as previously described5. Quantification of molecular and granular cerebellar layers area as well as cerebellum total area was made over six sections per animal. Brightfield images were acquired with an Axio Scan.Z1 microscope using a Plan-Apochromat 20x/0.8 M27 air objective. For quantification, the ImageJ software (NIH, USA) was used.

Statistical analysis

All data are presented as mean ± SEM. Graphs and statistical analysis were performed in Graph Pad Prism 6 software. Statistical significance was assessed by unpaired t-test and One-way ANOVA; values of p < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 as indicated in the legend of the figures).

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