Isolation, culture of hAMSC and preparation of CM
Human term placentae were collected at the Department of Obstetrics and Gynecology of Fondazione Poliambulanza hospital in Brescia from healthy women after vaginal delivery or caesarean section at term. Placentae were collected after obtaining informed written consent according to the guidelines set by the local ethics committee (Comitato Etico Provinciale of Brescia, Italy: number NP 2243, approved 19.01.2016). hAMSC were isolated from the amniotic membrane using well-established techniques (30). Briefly, amniotic membrane was cut in fragments and enzymatically digested at 37°C with 2.5 U/mL dispase (VWR, Radnor, PA, USA). The digestion continued in the presence of collagenase and DNase I (both from Roche, Basel, Switzerland). The supernatant of cell suspension obtained by low centrifugation, was centrifuged at 300g for 10min to collect the cells. After isolation hAMSC (p0) were phenotypically characterized as previously reported (30). Cells with > 80% expression of mesenchymal markers CD13 and CD90 and < 10% expression of the hematopoietic marker CD45 and of the epithelial marker CD324 were used in this study. Freshly isolated cells referred to as hAMSC p0 were expanded until passage 1 (p1) by plating at a density of [104cells/cm2] in Chang medium C (Irvine Scientific, Santa Ana, CA, USA) supplemented with 2 mM L-glutamine (Sigma-Aldrich) at 37°C in 5% CO2. Conditioned medium from hAMSC was generated by culturing hAMSC p1 for 5 days in 24-well plates (Corning Inc, Corning, NY) (0.5×106 cells/well in a final volume of 0.5mL), in serum-free Gibco™ DMEM/F-12, HEPES complete medium compose of DMEM/F-12, HEPES supplemented with 2 mM L-glutamine (Sigma-Aldrich) and100 U/mL penicillin and 100 µg/mL streptomycin (herein referred to P/S, all from Merck, St. Louis, MO, USA). The control (CTR) was DMEM/F-12, HEPES complete medium that was left for 5 days at 37°C in 5% CO2. After 5 days the supernatants (or control media) were collected, centrifuged at 300×g for 10 min, filtered through a 0.2-µm sterile filter (Sartorius Stedim, Florence, Italy) and stored at − 80°C until use.
For the preparation of hAMSC conditioned media devoid of EVs, during cell expansion to p1 hAMSC were treated, for 30 minutes with the neutral sphingomyelinase inhibitor GW4869 (10µM) which selectively blocks exosome biogenesis or with respective vehicle (DMSO). Treated hAMSC were then washed, detached and replated to produce CM as described above.
EVs isolation and characterization
EVs were isolated from CM-hAMSC and in parallel from CM-CTR with Total Exosomes Isolation Reagent -TEIR- (#4478359, Invitrogen by Thermo Fisher Scientific) according to manufacturer instructions.
- Field Emission Scanning Electron Microscopy:
Ultrastructural analysis of EVs was performed by Field Emission Scanning Electron Microscopy (FESEM). EVs suspension was allowed to dry on 13-mm -diameter glass coverslip. EVs were fixed with 2.5% v/v glutaraldehyde in 0.1 mol l-1cacodylate buffer (pH 7.4) at room temperature for 1 h, washed in cacodylate buffer and dehydrated through graded ethanols (30, 50, 70, 85, 95, 100%- 10 mins each). After 100% ethanol, 1:1 ethanol:hexamethyldisilazane (HMDS) was added for 3–5 min at RT followed by a final step of 3–5 min incubation in HMDS and air drying. Glass coverslip was glued on SEM pin stub and gold coated (V150R quorum). Finally, EVs were examined by FESEM (Sigma-Zeiss).
- Nanosight:
Concentration and size of secreted vesicles was quantified by the nanoparticle tracking analysis (NTA) system (Nanosight NS300) and the number of particles isolated from 1 mL of CM was determined.
-Western Blot:
EVs isolated were lysed for protein extraction in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; protease inhibitors). The total extravesicular protein content was quantified using the micro bicinchonic acid protein assay (BCA) (#23235, Thermo Fisher scientific).
Western blot was performed on 12 µg of EV proteins using rabbit anti-CD81 (1/500, Thermo Fisher scientific #PA513582) and mouse anti-Alix (3A9) (1/500, Cell Signaling Technologies #2171).
Experimental Model And Animal Procedure
Animal procedures performed according to European Guidelines (2010/63/EU) and Italian law requirements (D.L. 26/2014) were approved by Animal Welfare Office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care, and Veterinary Drugs of Italian Ministry of Health and were therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. C57BL6/mdx mice were bred, handled, and maintained under housing and feeding conditions according to the standard animal facility procedures and the ethics committee of the Fondazione Santa Lucia. Mice were euthanized by cervical dislocation. Efforts were made to minimize animal suffering and the number of animals necessary to produce reliable results.
hAMSC-derived EVs at a final concentration of 3.2x10^9 particles in 20µl of PBS1x were injected 3 times in the left TA of 1.5 months old C57BL6/mdx mice: every 7 days for 21 days. The rights TA were injected with vehicle (20µL PBS1x) following the same timing described for EV injection.
Human Myoblast Culture
Human DMD myoblasts were kindly provided by Dr. Pier Lorenzo Puri, Rome in collaboration with Telethon Biobank. Informed consent to use Telethon Network of Genetic Biobanks biological samples for diagnosis and research was obtained from the patients in accordance with protocols approved by the Institutional Review Board of the C. Besta Neurological Institute (Milan). Primary cultures were derived by enzymatic digestion of excised human skeletal obtain single cell Cells were cultured in DMEM + Glucose supplemented with: 20% FBS (Corning Fetal Bovine Pen/Strep; 500 ul Insulin (Sigma Aldrich #I9278); 10ng/mL Epidermal growth factor (EGF) (Thermo #PHG0311); 1ng/mL Fibroblast growth factor (FGF) (PreproTech Corning Fetal Bovine #100-18B).
Isolation and culture of MuSCs.
Murine MuSCs were isolated from 1.5 month old C56BL6/mdx tibialis anterior, gastrocnemius and Extensor Digitorum Longus muscles following Mozzetta’s protocol (31). Briefly, tissues were digested by Dispase II/ Collagenase A and cells were isolated based on their size, granulosity, and fluorophore levels using a FACS MoFlo Astrios EQ High Speed Cell Sorter (Beckman Coulter) and analyzed using FlowJo. In detail, MuSCs were isolated as TER119neg/CD45neg/CD31neg/alpha7INTEGRINpos/SCA-1neg cells. MuSCs were cultured after sorting directly in growth culture media GM2: 20% FBS (#16000044, Gibco), 10% HS (#26050-070, Gibco), 1% penicillin– streptomycin (#15140, Gibco), 1% Chicken Embryo Extract (CEE, #CE-650-F, Seralab) in DMEM + Pyruvate (#41966, Gibco). MuSCs were plated at low density on regular cell culture dishes coated with gelatin 0.1% (#07903, Stemcell) and then treated with CM -hAMSC diluted 1:5 in the culture medium or with 6.5x10^8 EVs-hAMSC isolated from the same amount of diluted CM-hAMSC used for treatment.
Single Fiber Isolation And Culture
Single fibers were isolated from gastrocnemius, soleus and Extensor Digitorum Longus muscles of 1.5 month old C57BL6J- WT mice. The muscles were digested at 37°C under gentle agitation for 45 minutes in digestion solution (DMEM + pyruvate + 4.5 g/l glucose + glutamate, 10 units/ml penicillin and 10 µg/ml streptomycin, 0.35% Collagenase I (#C0130, SIGMA). After serial washes to remove debris, the single fibers were cultured floating into a 100 mm petri dish pre-coated with pre-warm proliferating medium (GM1: DMEM + pyruvate + 4.5 g/l glucose + glutamate, 10% Horse Serum, 0.5% Chicken Embryo Extract) for 24 hours. Then the single fibers were gently and carefully moved into a low-adherence 6-well plate and exposed for the next 48hrs to CM-hAMSC or CM-CTR diluted 1:5 in GM1. Subsequently, single fibers were fixed with 4% PFA for immunofluorescence analysis (32).
Immunofluorescence
For immunofluorescence analysis, cryo-sections and cells were fixed in 4% PFA for 10 min and permeabilized with 100% cold acetone (#32201, Sigma) for 6 min at -20°C or 100% cold Methanol (#32213, Sigma) for 6 min at -20° or with 0.25% Triton for 15 min at RT. Muscle sections were blocked for 1h with a solution containing 4% BSA (#A7030, Sigma) in PBS. The primary antibodies incubation was performed O.N. at 4°C and then the antibody binding specificity was revealed using secondary antibodies coupled to Alexa Fluor 488, 594, or 647 (Invitrogen). Sections were incubated with DAPI in PBS for 5 minutes for nuclear staining, washed in PBS, and mounted with glycerol 3:1 in PBS. The primary antibodies used for immunofluorescences are: rabbit anti-Laminin (1/400, #L9393, Sigma); mouse anti-eMyHC (1:20, #F1.652, Developmental Studies Hybridoma Bank, DSHB, http://dshb.biology.uiowa.edu/F1-652); mouse anti-MF20 (1:20, Developmental Studies Hybridoma Bank, DSHB, http://dshb.biology.uiowa.edu/MF-20), mouse anti-PAX7 (1:10, Developmental Studies Hybridoma Bank, DSHB, http://dshb.biology.uiowa.edu/PAX7), rabbit anti-MyoD-318 (1:50 #SC760, Santa Cruz Biotechnology), EDU (#C10350, Invitrogen), rabbit anti-Ki67 (1:1000, #15580 Abcam). Myoblast fusion, essential for muscle regeneration, was evaluated by the fusion index analysis. Specifically, MuSCs were stained with anti-MF20 to detect MyHC and the percentage of nuclei were measured, and more specifically the nuclei that were i) in MyHC- or MyHC + mononucleated myotubes (n < 2), ii) inside myotubes containing between two and five nuclei (2 < n < 5), ii) inside myotubes containing more than five nuclei (n > 5). Collagen deposition was evaluated through Sirius Red staining. Muscle cryosections were fixed for 1 h at 56°C in Bouin’s solution and then stained in Picro-Sirius Red (0.1%) solution for 1 h (Direct Red 80; #365548-5G Sigma and picric acid solution; #P6744-1GA Sigma). Then sections were washed in acidified water 0.5% vol/vol and fixed in 100% ethanol. The final dehydration was performed in xylene 100% and sections were mounted with Eukitt (#03989 Sigma).
Muscle cryosections were fixed for 5 min at room temperature with 2% PFA and washed three times with PBS. Picro-sirius red staining (Direct Red 80 (Sigma #365548-5G)) in picric acid solution (Sigma #P6744-1GA)). Tissue area positive for Picro-sirius red staining was quantified by image analysis (Image J) (33).
RNA preparation and RT-qPCR.
TRIzol Reagent (#T9424, Sigma) was used to extract total RNA including small RNA according to the manufacturer’s recommendations and 0.5–1ug was retro-transcribed using the TaqMan reverse transcription kit (Applied Biosystems). Real-time qPCR was performed using TB Green Premix Ex Taq II (Tli RNase H Plus, Takara) that includes TB Green, a reagent designed for intercalator-based qPCR and the following primers:
MmCol3a1 Fw: 5’ CCCAACCCAGAGATCCCATT 3’; MmCol3a1 Rev: 5’ GGTCACCATTTCTCCCAGGA 3’; MmCol1a1 Fw: 5’ CCTCAGGGTATTGCTGGACA 3’; MmCol1a1 Rev: 5’ GAAGGACCTTGTTTGCCAGG 3’; MmPax7 FW: 5’ AGGACGACGAGGAAGGAGA 3’; MmPax7Rev: 5’TCATCCAGACGGTTCCCTTT 3’; MmKi67 FW: 5’ TCACCTGGTCACCATCAAGC 3’; MmKi67 Rev: 5’ TCAATACTCCTTCCAAACA 3’; MmMyoG FW 5’ GTCCCAACCCAGGAGATCA; MmMyoG Rev: 5’ CAGACATATCCTCCACCGT 3’.
Statistical Analysis
The number of independent experiments, replicates and precision measures are reported in the Figure legends (n, mean ± SEM). Statistical analysis was performed using Prism 8.2.1 software (Graph Pad Prism Software). Unpaired t-test was used for comparing two different groups; one-way analysis of variance (one-way ANOVA) was used to analyze differences between more than two different group (i.e count of the mean of nuclei), while two-way analysis of variance (two-way ANOVA) was used for comparing different groups to examine the influence of different independent variables on dependent variables (i.e Fusion index or Pax7 and Myod quantification). When data met the assumptions of the tests (e.g., normal distribution) and the ANOVA was significant, post hoc testing of differences between groups were performed using Tukey's honestly significant difference (HSD). When data did not meet the assumptions of the tests (e.g., normal distribution) but the ANOVA was significant, post hoc testing of differences between groups was performed using Kruskal-Wallis test. A p < 0.05 was considered statistically significant. No statistical method was used to predetermine sample size for animal studies. The animal experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. No exclusion criteria were applied to exclude samples or animals from analysis.