Animals 8-week-old C57Bl/6JR female mice were purchased from Janvier Labs (Rodent Research Models and Associated Services) and used for engraftment. SC were obtained from green fluorescent protein-tagged actin (GFP+) transgenic mice, Krox20Cre/+R26RYFP/+ mice, and TdTomato Krox20Cre/flox,R26mT/+ mice which were previously characterized [18, 44], and maintained at ICM and IBENS animal facilities. Animal experiments were performed according to European Community regulations, ICM and INSERM ethical committee (authorization 75-348; 20/04/2005) and were approved by the local Darwin ethical committee.
SC isolation and purification Sciatic nerves from GFP+ mice were isolated at postnatal day 15. Purification procedure was adapted from the previously described protocol . Briefly, enzymatic dissociation was performed by incubation with trypsin 0.025% and collagenase (420 U/ml) for 10 min at 37 °C, followed by mechanical dissociation through different needle gauges. After ending dissociation with fetal calf serum (FCS), SC were seeded in FN-coated flasks, and expanded in Dulbecco’s modified Eagle medium, containing 10% heat-inactivated FCS serum, penicillin (100 mg/ml), streptomycin (100 U/ml), human recombinant Neu-differentiation factor ß (hrNDFß) (125 ng/ml), insulin (10 µg/ml) and forskolin (2 µg/ml). SC were purified by differential adhesion , and used at passage P2 or P3. Purification was controlled by immunocytochemistry for p75 and GFAP as markers of non-myelinating Schwann cells , and exclusion of the Thy1-2 marker of mouse fibroblasts . For adhesion, migration and blocking receptor assays, SC were maintained in Sato serum-free medium  supplemented with hrNDFβ (125 ng/ml), and forskolin (2 µg/ml).
iDISCO whole-mount immunofluorescence and imaging Spinal cords were processed as described in the iDISCO protocol , including modifications described in the updated online protocol (https://idisco.info, Dec 2016). The primary antibody used was rabbit anti-RFP (1:1000, Rockland). Secondary antibodies used were donkey anti-rabbit Cy3 (1:800, Jackson Immunoresearch) and donkey anti-mouse IgG Cy5 (1:800, Jackson Immunoresearch) for intravascular staining. The cleared samples were imaged with a light sheet microscope (Ultramicroscope II; LaVision Biotec).
RNA transcriptome analysis For RNA preparations, SC from Krox20Cre/+,R26mT/+ (TdTomato) were freshly isolated from 2-week-old sciatic nerves. Nerves were cultured 4 days in vitro in the absence of growth factor to allow SC to de-differentiate and migrate out of the nerve. SC were then selected by FACS based on tdTomato expression. Library preparation and Illumina sequencing were performed at the IBENS genomic core facility. Briefly, (polyA +) mRNAs were purified from 250 ng of total RNA using oligo(dT). Libraries were prepared using the strand-specific RNA-Seq library preparation TruSeq Stranded mRNA kit (Illumina). Libraries were multiplexed by 6 in 1 high-output flow cells. A 75-bp read sequencing was performed on a NextSeq 500 device (Illumina). A mean of 94 ± 9.5 million reads passing Illumina quality filter was obtained for each of the six samples.
The analyses were performed using the Eoulsan pipeline , including read filtering, mapping, alignment filtering, read quantification, normalization and differential analysis. Before mapping, poly N read tails were trimmed, reads of ≤ 40 bases were removed, and reads with quality mean of ≤ 30 bases were discarded. Reads were then aligned against the Mus musculus genome from Ensembl version 84 using STAR . Alignments from reads matching more than once on the reference genome were removed using the Java version of SamTools . All overlapping regions between alignments and referenced gene were counted using HTSeq-count 0.5.3 . The sample counts were normalized using DESeq 1.8.3 . Statistical treatments and differential analyses were also performed using DESeq 1.8.3.
Data availability The RNASeq gene expression data and raw fastq files are available on the GEO repository (www.ncbi.nlm.nih.gov/geo/) under accession number: GSE107401 (accession password: mlkfkwoezxujvsn).
Myelin protein extract isolation Myelin was purified by sucrose gradient centrifugation . Cerebral hemispheres of adult mice (3 months old) were homogenized on ice in 0.35 M sucrose and 5 mM EGTA, and the suspension was overlaid onto an equivalent volume of 0.85 M sucrose and 5 mM EGTA, and centrifuged at 100,000×g at 4 °C for 20 min. The myelin-containing fraction at the interface was collected, diluted threefold in distilled water, and centrifuged at 100,000×g at 4 °C for 30 min. After washing with distilled water, the isolated myelin pellet was resuspended in 20 mM Tris–HCl, aliquoted, and stored at – 20 °C.
Pre-clustering of recombinant EphrinB3–Fc Mouse EphrinB3–Fc fragments and human Fc were purchased from R&D Systems. The soluble forms of EphrinB3–Fc and its control Fc have low effect on receptor activation ; therefore, they were mixed with anti-mouse Fc–IgG and anti-human Fc–IgG (Alexa 555), respectively (ratio = 1:5), and incubated for 1 h at 37 °C prior to addition to SC .
Adhesion and spreading assays Adhesion and spreading in vitro assays were performed in 24-well dishes. Silicon strips on coverslips were used to separate two coated areas of each coverslip . Surfaces were coated overnight at 37 °C with recombinant EphrinB3–Fc fusion at 10 µg/mL and Fc equimolar (as control) on each half, or myelin extract (100 µg/mL) and PBS buffer (as control). Before cell seeding, strips were removed and coverslips were washed carefully with PBS. 105 SC were seeded in serum-free Sato medium to avoid proliferation, and allowed to adhere for 3 h. Data were always expressed as ratio in respect to the intra-coverslip control .
Survival assay GFP+SC were seeded on uncoated glass coverslips in normal medium. After overnight adhesion, medium was changed, adding Sato serum-free medium supplemented with clustered EphrinB3 at 10 µg/mL or Fc equimolar (as control), or with myelin extract (100 µg/mL) or PBS (as control). SC were incubated for 3 h or 24 h as specified in each experiment. After fixation in 4% paraformaldehyde (5 min), SC were immuno-stained for caspase 3 adding Hoechst dye to visualize all nuclei, and coverslips were mounted with fluoromount.
Migration assay SC were resuspended at 3 × 106 cells/ml in Sato medium containing 0.8% low-melting point agarose (Sigma). One drop (1.5 μL) of this suspension was applied to the center of FN +EphrinB3, or FN +Fc-coated glass coverslips, which were placed at 4 °C for 1 min to allow the agarose to solidify. The cooled drop was covered with Sato medium with hrNDFß (125 ng/ml) and forskolin (2 µg/ml), and placed up to 6 h at 37 °C in the incubating chamber of a video-microscope (ZEISS).
SC receptor blocking assay EphA4 and EphB6 receptors or Integrinβ1 were neutralized in SC by incubation with anti-EphA4 (1.2 µg/10.000 cells, R&D, AF641), anti-EphB6 (1.2 µg/10.000 cells, Santa Cruz Biotechnology, sc-7282), anti-integrinβ1 (0.6 µg/10.000 cells, MA2910, Thermo Fisher Scientific) antibodies or IgG (as control) in Sato medium for 1 h at 37 °C prior to cell seeding or transplantation.
Immuno-staining Cultured SC were fixed for 5 min in 4% paraformaldehyde prior to immuno-staining and mice were killed by trans-cardiac perfusion of PBS followed by cold 4% paraformaldehyde, and post-fixed in the same fixative for 1 h. Spinal cords were cryo-protected by immersion in 20% sucrose solution overnight, embedded in cryomatrix (Thermo Scientific), and frozen in cold isopentane at − 60 °C. Finally, they were sectioned with a cryostat at 12 µm (Leica Microsystems). Both cells and sections were washed, blocked in 5% BSA for 40 min and incubated with the primary antibodies. While cells were incubated 1 h at room temperature, slides were incubated overnight at 4 °C. For MOG staining, sections were incubated with absolute ethanol for 10 min followed by primary antibody, and then washed profusely. Primary antibodies were as follows: anti-EphA4 (1:50, AF641, R&D Systems); anti-EphA4-Tyr(602) (1:50, EP2731, ECM Biosciences); anti-EphB6 (1:50, SAB4503476, Sigma); anti-EphB1 (1:50, SAB4500776, Sigma; anti-Eph receptor B1 + Eph receptor B2 (phospho Y594) (1:50, ab61791, Abcam); anti-Ki67 (1:100, 556003, BD Biosciences); anti-cleaved caspase3 (1:500, 9661S, Cell Signalling); anti-GFP (1:400, GFP-1020, Aves); anti-MOG (1:20, mouse IgG1 hybridoma, clone C18C5; provided by C. Linnington, University of Glasgow, Glasgow, United Kingdom); anti-MBP (1:50, ab7349, Sigma); anti-Glut1 (1:100, 07-1401, Merck Millipore; and 1:400, MABS132, Sigma); anti-Fibronectin (1:600, F6140, Sigma); anti-CD31 (1:200, 553370, BD Pharmigen); anti-NF200 (1:200, N4142,Sigma), anti-p75 (1:100, 8238S, Ozyme), anti-CD13 (1:50, MCA2183, BioRad), anti-CD68 (1:400, MCA1957, BioRad), anti-CD11b (1:400, MCA74G, BioRad), anti-F8/40 (1:100, MCA497R, BioRad), anti-Collagen 4(1:400, ab19808, Abcam), anti-Olig2 (1:300, MABN50, Millipore); anti-sox10 (1:50, AF2864, R&D Systems); and anti-CD13 (1:200, 553370, BD Pharmingen). Next, cells or sections were washed and incubated with secondary antibodies and Hoechst dye for 1 h at room temperature. The excess secondary antibody was removed by several PBS washes, and coverslips/slides were mounted using fluoromount.
Electron microscopy For electron microscopy, mice were perfused with PBS followed by 4% paraformaldehyde/2.5% glutaraldehyde (Electron Microscopy Science) in PBS for 45 min. Dissected spinal cords were post-fixed with the same solution for 2 h, then sectioned into 60-µm slices with a vibratome and washed twice with PBS before enzyme immunolabeling. For DAB revelation, endogenous peroxidase was inhibited with a methanol/oxygen peroxide incubation, and washed and blocked by 5% BSA for 1 h. Sections were incubated with anti-GFP overnight at 4 °C, then washed and incubated with a secondary biotinylated antibody for 2 h at room temperature. After several washes with PB 0.1 M, sections were incubated with the ABC kit (VECTASTAIN® ABC-HRP Kit, Vector Lab) containing peroxidase–anti-peroxidase for 40 min followed by a DAB/oxygen peroxide mix before stopping the reaction with distilled water. Samples were fixed in 2% osmium tetroxide (Sigma-Aldrich) 30 min, washed gently and incubated with 5% uranyl acetate for 30 min in the dark. After dehydration, samples were embedded in Epon resin 812. Ultra-thin sections (80 nm) were examined with a HITACHI 120 kV HT-7700 electron microscope.
Demyelinating lesions and grafts Wild-type mice were anaesthetized with a ketamine/xylazine mixture. Demyelination was induced by stereotaxic injection of lysolecithin (LPC) (1%, 0.5 µl) in PBS. LPC or PBS (in control animals) was injected into the dorsal funiculus of the spinal cord at the level of T8–T9 in the dorsal column of white matter using a glass micropipette. SC (105/2 µL) were injected the same day, two vertebrates caudally (4 mm) in the same tract.
Western blotting SC (3 × 105 cells/well) were lysed in RIPA buffer with Complete® and Phosphostop® inhibitor, and analyzed by electrophoresis in an SDS 4–20% MINI PROTEAN TGX gel. After electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride membranes and probed with the following antibodies:anti-EphB6 (1:500, SAB4503476, Sigma), anti-EphB1 (1:500, SAB4500776, Sigma), anti-EphA4 (4 µg/mL, 37-1600, ThermoFisher), anti-p-EphB1 + 2 (1:300, ab61791, Abcam), anti-p-EphA4 (1:300, EP2731, ECM Biosciences), anti-Integrinβ1 (1:500, 550531, BD Pharmingen), anti-EphrinB3 (1:250, 1 µg/mL, AF395 R&D Systems), anti-MBP (1:1000, ab980, Millipore), anti-GAPDH (1:5000, MAB374, Millipore) and anti-Actin (1:50,000, A2228, Sigma). Peroxidase-conjugated anti-rabbit, anti-goat or anti-mouse IgG secondary antibodies (Jackson Immuno Research) were used at a dilution of 1:5000, 1:10,000 and 1:20,000, respectively, and anti-Rat biotinylated (1:100, Vector Labs) followed by peroxidase-conjugated streptavidin. Protein bands were visualized by chemoluminescence (ECL BioRad). Intensity of the bands was quantified with FIJI.
Neutralization of EphrinB3 epitopes in myelin extracts EphrinB3 epitopes in myelin extract proteins were neutralized by incubation with anti-EphrinB3 antibodies (AF395, R&D system and sc-271328, Santa Cruz Biotechnology, ratio: 1:1) for 2 h at room temperature prior to the addition to the cells .
Spinal cord live imaging LPC lesion followed by GFP + SC engraftment was performed in 60-day-old mice and terminally anesthetized for imaging 36 h later. Rhodamine-labeled BSL I (Vector Labs RL-1102) was injected at 2 mg/ml in the beating heart to label BV. After 5 min allowing dye circulation, spinal cords were dissected in ice-cold HBSS solution supplemented with 6.4 mg/mL D-(+)-glucose and bubbled for 30 min with bubbled with 95% O2/5% CO2. Spinal cord segments including lesion and graft sites were laid onto Millicell-CM slice culture inserts (Millipore) over culture medium (50% DMEM + Glutamax, 25% HBSS, 25% heat-inactivated horse serum, 5 mg/mL D-(+)-glucose, 20 mM Hepes, penicillin (100 mg/ml), streptomycin (100 U/ml), hrNDFß (125 ng/ml), and forskolin (2 µg/ml) in glass bottom plates, and then placed in an inverted Leica SP8X confocal microscope with an on-stage incubator, while streaming 95% O2 and 5% CO2 into the chamber. Spinal cords were imaged using a 25 × immersion objective at intervals of 15 min during 12 h with intermittent repositioning of the focal planes. Maximum intensity projections of the collected stacks (~ 60 μm at 2 μm step size) were compiled in FIJI.
Lesions The lesion area was identified by immune detection of GFAP combined with Hoechst+-labeled nuclei to reveal astrocyte reactivity and hyper-cellularity, respectively.
SC adhesion and spreading In vitro SC adhesion on different surfaces was quantified as the ratio of the number of adhered GFP+ on myelin- or EphrinB3-coated areas, over those adhered to uncoated or Fc-coated area within the same coverslip. All coated areas were of equal size. SC spreading was evaluated by quantifying the ratio of “round” GFP+SC (lacking processes) out of the total number of cells on myelin- or EphrinB3-coated areas over those on non-coated or Fc coated areas. “Round cells” were defined as cells with no processes at all, thus with a perfectly circular shape.
In vitroSC extent of migration SC migration was quantified by measuring the number of SC outside the 1.5-µL agarose drop and the maximum extent of their migration from the edge drop.
In vitroSC velocity SC speed of migration was quantified by manual cell tracking plugging in FIJI, calibrating pixel size and duration of time lapse of each frame.
Size of lesion and graft area Lesion and grafted cells within the dorsal funiculus were quantified by delimiting Hoechst + nuclei hyper-density and GFAP-positive area on 12-µm section. Lesion and graft areas were quantified by ImageJ 1.49 s. For each animal, at least three serial sections with 60-µm intervals were quantified.
In vivoextent of SC migration SC migration within the dorsal funiculus was quantified on longitudinal sections evaluating the distance between the graft injection site and the most proximal GFP+ cell to the lesion (LPC injection site) in each animal from the different groups.
SC–BV association SC–BV association was quantified in the intermediate zone, at 1 mm from the graft edge in the direction of the lesion or in the lesion site. GFP+/Hoechst+ SC, with the whole or more than half of the cell area in contact with Glut1+ endothelial cells, were counted as “closely associated cells” while those with only “tip” contacts or no contacts were considered as “not associated cells”. Data are expressed as the percentage of total counted cells in both groups.
SC–axon alignment in the lesion. GFP+/Hoechst+ SC, with the whole or more than half of the cell in parallel orientation and aligned closely to NF200+ axons (excluding alignment to BV), were counted as “closely associated cells” while those with only “tip” contacts or no contacts were considered as “not associated cells”. Data are expressed as the percentage of total counted cells in both groups.
The sample size calculation was performed by the resource equation method to minimize the sample size, following the ARRIVE guidelines for reporting animal research. Each n represents one animal or SC sample in the experiment. The grafting experiments were repeated at least three times with a different set of animals each. For the in vitro analysis, experiments were performed at least three times with SC obtained from different dissections and dissociations. Statistical analysis was carried out using GraphPad Prism 6 software. All values were expressed as mean ± SD. Normality in the variable distributions was assessed by the D’Agostino–Pearson omnibus test and Grubbs’ test was used to detect and exclude possible outliers. When normality test was passed, means were compared by two-tailed Student’s t test. When one or both groups did not follow a normal distribution, means were compared by two-tailed Mann–Whitney U test. When different independent groups were compared, we performed a one-way ANOVA plus Tukey’s multiple comparison tests. One-sample t test was used to compare values to the hypothetical mean: 1 for ratios and 100 for percentages. Repeated measure ANOVA was used to analyze the difference along time of a certain parameter. P-values lower than 0.05 were used as a cut-off for statistical significance.