Primary cell extractions
Six deltoid muscle biopsies were obtained from ALS patients (50.0 ± 6.5 years old) who attended the Motor Neuron Diseases Center (Pitié Salpétrière, Paris), and 17 muscle biopsies from healthy subjects (51.4 ± 18.2 years old) from the BTR (Bank of Tissues for Research, a partner in the EU network EuroBioBank) in accordance with European recommendations and French legislation. The protocol (NCT01984957) was approved by the local Ethical Committee. Written informed consent was obtained from all patients. All biopsies were isolated from deltoid muscle.
Cell culture Proliferation and Differentiation
Primary human myoblasts were extracted from fresh muscle biopsies as described previously [31]. Briefly, myoblasts were sorted using CD56 magnetic beads (Milteny®) and expanded in 0.22mm filtered proliferating medium containing DMEM/M199 medium supplemented with 20% FBS, 25µg/ml Fetuin, 0.5ng/ml bFGF, 5ng/ml EGF, 5µg/ml insulin and incubated at 5%CO2, 37 °C. The number of cell divisions was calculated using the formula below. The myogenicity of the culture was determined by counting the number of nuclei positive for desmin against the total number of nuclei using the primary antibody anti-desmin (D33, 1 :100, Dako). Secondary antibody was goat anti-mouse IgG1 AlexaFluor 594 (1:400, Invitrogen™), and counterstaining was performed with 1mg.ml-1 DAPI as described below. After CD56 MACS sorting, 91.78±8.32% of the cells were myogenic.
See formula 1 in the supplementary files.
For differentiation into myotubes, 7.5 x 106 myoblasts were plated in 225cm2 flask (Falcom™) and let adhere overnight. Seeded myoblasts were then washed six times with supplement free DMEM and differentiated in DMEM for 72 hours. Conditioned medium was then collected and used for exosome extraction.
Beta-galactosidase staining
The senescence level was assessed using a Senescence β-Galactosidase Staining Kit (Cell Signaling Technology®).
Cell immunostaining
The cells were fixed with 3.6% formaldehyde, permeabilised, blocked and stained as described previously [32]. Primary antibody anti-myosin heavy chain (MF20, 1:50, DSHB) and secondary antibody goat anti-mouse IgG2b AlexaFluor 594 (1:400, Invitrogen™) were used to determine the formation of myotubes. The slides were washed and counter-stained with 1mg.ml-1 DAPI for 2 min and then rinsed twice with PBS before being mounted with ibidi mounting medium (ibidi®).
Protein extraction from cells
Myoblasts were scraped into 50 ml of chilled RIPA lysis buffer (Invitrogen™) supplemented with 1x Halt™ protease inhibitor cocktail (Thermo Scientific™) and, incubated on ice for 10min. Cell lysates were then centrifuged at 14,000 g for 10min at 4 °C and protein supernatants were collected and stored at -80ºC for downstream SDS-PAGE and immunoblotting.
Condition culture media clearance
At the time of collection, the conditioned medium is centrifuged at 200g for 10min. The subsequent supernatant was then centrifuged at 4,000g for 20min. The resulting supernatant was centrifuged for 70min at 4°C at 20,000g and then filtered through a 0.22µm filter. The cleared medium was then stored at -80°C prior to exosome extraction.
Muscle exosome extraction using ultracentrifugation
Cleared media were centrifuged at 100,000g for 70min at 4°C following a method described previously [24]. The subsequent pellet was resuspended in PBS and washed three times by centrifugation at 100,000g for 70min at 4°C. The clean pellet was then resuspended in 100ul of PBS or in NuPAGE™ LDS sample buffer for Western blot experiments.
Exosome Extraction using polymer precipitation
Cleared culture media was mixed with the Total Exosome Isolation kit (LifeTechnologies™) at a 2:1 volume ratio and incubated at 4°C overnight. The mixture was then centrifuged at 10,000g for 60min at 4°C. The subsequent pellet was resuspended in 500 µl of PBS and washed three times using 100kDa Amicon® filter column. The exosomes were then resuspended in 100ml of PBS or in NuPAGE™ LDS sample buffer for Western blot experiments.
Exosome Protein extraction
Exosomes were lysed in 8M urea supplemented with 1x Halt™ Protease Inhibitor cocktail (Thermo Scientifc™) and 2% SDS. Samples were incubated at 40C for 15min, and exosome lysates were centrifuged at 14,000g for 10min at 40C. Supernatants containing soluble proteins were stored at -800C.
SDS-PAGE and Western Blotting
SDS-PAGE was performed as follows. For cell lysates, protein concentrations were measured at 562 nm using the bicinchoninic acid assay kit (Pierce™) and 20 mg of protein was mixed with 4x NuPAGE™ LDS sample buffer. For exosome extracts, proteins were also mixed with 4x NuPAGE™ LDS sample buffer. For reducing conditions, samples were supplemented with 10x NuPAGE™ reducing agent. For immunoblotting of tetraspanins, samples were prepared similarly but for the omission of reducing agents. All samples were then denatured at 70 °C for 10 min before being added to a 4-12 % polyacrylamide Bis-Tris gel (Life Technologies™) and electrophoresed at 200 v for 70 min in MOPS SDS Running buffer (LifeTechnologies™). Following electrophoresis, the gel was incubated in 20 % ethanol for 10 min and proteins were transferred onto polyvinylidene fluoride membrane using the iBlot™ 2 Dry Blotting system (LifeTechnologies™) according to manufacturer’s instructions.
Immunoblotting was performed using the iBind™ Flex western system following the manufacturer’s instructions (Life Technologies™). PVDF membrane was probed with primary antibodies forPARP-1 (9542, Cell Signaling, rabbit IgG, 1:1000), or CD63 TS63 (10628D, Life Technologies™, mouse, 2µg/ml), or CD81 (MA5-13548, Life Technologies™, mouse IgG, 1:100, v:v dilution), Flotillin (PA5-18053, Life Technologies™, 0.3µg/ml) or HSPA8 (MABE1120, Millipore, mouse IgG, 1:1000 ) or Alix (SC-53540, Santa Cruz, 1:1000) and Goat anti-mouse or Goat anti-rabbit secondaries conjugated with HRP (LifeTechnologies™, 1:400, and 1:10,000 respectively). The membrane was then incubated with Amersham ECL Prime Western Blotting Detection Reagent for 5 minutes at room temperature and images were subsequently acquired using the UVP ChemiDoc-It™2 Imager and UVP software.
Electron microscopy and immunogold
Extracted and further whole-mounted vesicles were processed as described in [24]. Observations were made using a CM120 transmission electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV and images recorded with a Morada digital camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany).
Determination of the exosome density
Exosomes extracted from cell culture medium using either ultracentrifugation or polymer-based precipitation were resuspended in 100ml of PBS and loaded on the top of the sucrose gradient as previously described [5,32]. Samples were then centrifuged at 100,000g for 17 hr at 40C. Twelve fractions were sequentially collected, diluted in 12 ml PBS and centrifuged at 100,000g for 70min at 40C. Each pellet was then resuspended in non-reducing NuPAGE™ LDS sample buffer and used for western blot analyses as described above. The density gradient of each fraction was determined using the method described by [33] by measuring the absorbance at 244 nm:
See formula 2 in the supplementary files.
Nanoparticle tracking analysis (NTA)
Exosome pellets were resuspended in 100ml of filtered PBS. The exosome suspension was then diluted 10x in PBS. Size and distribution of exosomes secreted by primary muscle cells were evaluated by a NanoSight LM10 instrument (NanoSight) equipped with NTA analytic software (version 2.3 build 2.3.5.0033.7-Beta7). Three videos of 30 s were as previously described [34,35] at temperature set to 22.5C. The minimum particle size, track length and blur were set to “automatic”.
Proteomic analysis
- The exosome pellets were re-suspended in 25μl 8M Urea, 50 mM ammonium bicarbonate, pH 8.5, and reduced with DTT for 1 h at 4˚C. Protein concentrations were then quantified using Pierce BCA Protein Assay kit (ThermoFisher®). Exosomal proteins were kept at -80°C.
- Proteome profile determined by Mass spectrometry - 20 mg of exosome protein were trypsin digested using a SmartDigest column (Thermo) for 2h at 70°C and centrifugated at 1400rpm. Peptides were then fractionated into 8 fractions using a high pH reverse phase spin column (Thermo). Fractioned peptides were vacuum dried, resuspended and analyzed by data-dependent mass spectrometry on a Q Exactive HF (Thermo) with the following parameters: Positive Polarity, m/z 400-2000 MS Resolution 70,000, AGC 3e6, 100ms IT, MS/MS Resolution 17,500, AGC 5e5, 50ms IT, Isolation width 3 m/z, and NCE 30, cycle count 15.
- Database Search and Quantification - The MS raw data sets were searched for protein identification for semi tryptic peptides against the Uniprot human database for semi tryptic peptides including common contaminants, using MaxQuant software (version 1.6.2.1) (https://wSww.biochem.mpg.de/5111795/maxquant). We used default parameters for the searches: mass tolerances were set at +/- 20 ppm for first peptide search and +/- 4.5 ppm for main peptide search, maximum two missed cleavage; and the peptide and resulting protein assignments were filtered based on a 1% protein false discovery rate (thus 99% confidence level). 1254 proteins were detected in at least 1 sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015736 [Accessible to reviewers under Username: [email protected]; Password: 10f1iUAq].
- To test for overlap with known exosome proteins from previous studies, all proteins detected in at least 1 proteomic sample were entered into the Funrich tool for vesicle functional analysis [36–39], and a Venn diagram generated against the subset of the Vesiclepedia database comprising previously observed exosomal proteins detected by mass spectrometry in human samples.
mRNA extraction from polymer precipitatedexosomes
Exosomes were first dissolved in 900 µl TRIzol® (Invitrogen™), then 200 µl of chloroform was added. After 5min of incubation at RT, samples were centrifuged at 12,000 g for 15min at 4°C. The aqueous phase containing the RNA was transferred into a collection and mixed with 75% ethanol (1:1, v:v). mRNA was then purified using PureLink® RNA Mini Kit (LifeTechologies™) following manufacturer’s instructions. RNA eluates were stored at -80°C until use. Concentration of each RNA sample was determined by NanoDrop® spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE). The quality of RNA samples was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA).
Immunoprecipitation of muscle exosome subpopulation
Polymer precipitated exosomes were immunoprecipitated using anti-CD63 magnetic beads (Invitrogen™) overnight according to manufacturer’s instructions. Magnetically captured beads were then washed 3 times in PBS and CD63 positive exosomes were eluted in 4x NuPAGE™ LDS sample buffer. Samples were then used for western blot analyses as described above.
Exosome functionality assessment
The exosomes were labelled with the PKH26 kit (Sigma-Aldrich®). Briefly, 100 µl of Diluent C was added to the exosome suspension and labelled with 100 µl of 4 µM PKH26 solution. After 5 min of incubation, samples were washed 3 times in PBS using a 100kDa Amicon® filter column and centrifuged at 12,000xg at 4°C for 15 min. Muscle exosomes extracted from 3,000 differentiated myoblasts were either added to 3,000 human iPSC derived motor neurons or to 3,000 differentiated human myoblasts. Human iPSC derived motor neurons were differentiated from human neuron progenitors as described in [40]. Uptake of muscle exosomes by recipient cells was observed after 24h incubation in living cells using an Olympus IX170 inverted microscope, with a 40x/0.60 Ph2 objective equipped with an AxiocamMR camera.
Statistical analysis
All values are presented as means ± SD. ANOVA 1 Factor followed by Tukey post-hoc test was used to compare differences between the different cell densities conditions. Differences were considered to be statistically different at P < 0.05.