*2.3. Pericyte and C2C12 Myogenic Di*ff*erentiation*

Spontaneous skeletal myogenic differentiation of human pericytes and C2C12 cells was induced by plating 104cells/cm2 in αMEM, 20% FBS and penicillin/streptomycin. After the cells reached confluence, we replaced the medium with low-serum medium (2% horse serum, HS, Thermo Fisher Scientific) for about 10 (pericyte differentiation) and 5 (C2C12 differentiation) days.

#### *2.4. Lentiviral Vector Generation*

The lentiviral vector employed for the induction of reprogramming was composed of a single excisable polycistronic lentiviral stem cell cassette (STEMCCA), encoding the Yamanaka factors [33]. Low passage 293T cells (Cell Biolabs, San Diego, CA, USA) were used to produce lentiviruses, employing the psPAX2 and vesicular stomatitis virus G protein (VSV-G) packaging constructs and a calcium phosphate transfection protocol. Supernatants containing STEMCCA lentiviruses were collected 48 h later, filtered, and used immediately right after preparation. The lentiviral vector used to introduce a Green Fluorescent Protein (GFP) transgene for the isolation of GFP+-EVs was produced employing the calcium phosphate method into 293FT packaging cells.

#### *2.5. iPSC Generation*

To induce reprogramming, we exposed pericytes and fibroblasts, at early passages, to fresh lentiviral medium 3 times at 12 h intervals. Lentiviral medium was then replaced with fresh medium. After a further 5 days, 1 <sup>×</sup> 103 transduced cells/cm2 were plated on a feeder layer constituted of inactivated mouse embryonic fibroblast (iMEF). Cells were then cultured in iPSC medium composed of knockout DMEM (Life Technologies, Carlsbad, CA, USA), supplemented with 20% knockout Serum Replacement (Life Technologies), 20 ng/mL of basic fibroblast growth factor (bFGF; Life Technologies), 1% N-2 (Life Technologies), 2% B27 (Life Technologies), 2 mM Glutamax (Life Technologies), 100 μM Eagle s minimum essential medium non-essential amino acid solution (MEM-NEAA, Life Technologies), 100 μM β-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. After iPSC line expansion and characterization was carried out, cells were adapted to feeder-free condition, by seeding them on Geltrex matrix (Thermo Fisher Scientific) in Essential 8 medium (Life Technologies).

#### *2.6. iPSC Multilineage Di*ff*erentiation*

Cardiomyocyte differentiation was performed using STEMdiff Cardiomyocyte Differentiation Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. Briefly, uniform undifferentiated iPSC colonies were harvested and seeded as single cells at 3.5 <sup>×</sup> 105 cells per well in a 12-well format. After 48 h, the iPSC medium was replaced with Medium A to induce the cells toward a cardiomyocyte fate. On day 2, a full medium change was performed with fresh Medium B. On days 4 and 6, medium B was replaced with fresh Medium C. On day 8, medium was switched to cardiomyocyte Maintenance Medium with full medium changes on days 10, 12 and 14, to promote further differentiation into cardiomyocyte cells.

Neural differentiation was promoted plating 1 <sup>×</sup> 106 cells/mL in Neural Induction Medium (Thermo Fisher Scientific) for 7 days. On day 8, iPSC-derived neural stem cells were harvested and expanded in Neural Expansion Medium (Thermo Fisher Scientific).

For endothelial differentiation, human iPSC cells were cultured in Roswell Park Memorial Institute medium (RPMI; Sigma-Aldrich, St. Louis, MO, USA) plus B27 medium with 6 μM CHIR99021 (CHIR; Sigma-Aldrich). On day 2, we replaced the medium with fresh RPMI supplemented with B27 and 2 μM CHIR. After 48 h, the medium was changed with EGM-2 medium supplied with vascular endothelial growth factor (VEGF; PeproTech, London, UK), bFGF, and SB431542 (Merck, Darmstadt, Germany). Every other day, the medium was changed with fresh EGM-2 medium supplied with VEGF, bFGF, and SB431542.

#### *2.7. Isolation of MT-derived EVs*

EVs were isolated from conditioned medium of C2C12 myoblasts, differentiated into myotubes, using HS, previously centrifuged at 100,000× *g* for 16 h at 4 ◦C for EV depletion. After 48 h of incubation in fresh medium, EVs were harvested and purified by differential centrifugation—cell debris and organelles were eliminated at 500× *g* for 20 min followed by another centrifugation at 3500× *g* for 15 min at 4 ◦C. EVs were pelleted by ultracentrifugation at 100,000× *g* for 70 min at 4 ◦C by L-80-XP ultracentrifuge (Beckman-Coulter, Brea, CA, USA). Finally, the pellet was washed with cold PBS (Phosphate Buffered Saline) in order to minimize sticking and trapping of non-vesicular materials. Purified EVs were used immediately after isolation.

#### *2.8. Myogenic Di*ff*erentiation by MT-Derived EVs*

Human iPSCs with no differentiated colonies, expressing pluripotency markers were used for the differentiation process. The iPSCs were cultured under feeder-free conditions using Essential 8 medium on Geltrex matrix. A critical variable for the generation of robust myotube culture was the relative confluence at the onset of differentiation that it should be approximately 30%. After they were seeded for about 48 h, iPSCs were induced toward mesodermal commitment in Essential 6

medium (Life Technologies) and 1% ITS (insulin-transferrin-selenium) supplemented with 10 uM GSK3 inhibitor CHIR (Sigma-Aldrich). After 2 days, we withdrew CHIR from the culture medium. The mesodermal induction medium was replaced with fresh expansion medium composed of Essential 6 medium enriched with 1% ITS, 5 mM LiCl, 10 ng bFGF, 10 ng insulin-like growth factor 1 (IGF-1; Thermo Fisher Scientific) and 50 ug/mL MT-derived EVs. After further 4 days, LiCl was removed from the medium. During this period, cells underwent enhanced proliferation. Between days 8–10, cells reached confluence and were expanded using TryplE (Thermo Fisher Scientific) and Collagen Type I matrix coating (BD Biosciences). The final differentiation and maturation phase into myotubes took additional 2 weeks: by day 20, muscular progenitors were seeded on Collagen type I dishes; after cells reached confluence, growth factors and MT-derived EVs were removed from the medium, and cells were cultured only in Essential 6 medium supplemented with 1% ITS.

#### *2.9. Flow Cytometry and Cell Sorting*

Fluorescence-activated cell sorting (FACS) analysis on physical parameters (forward and side light scatter, FSC and SSC, respectively), was first performed in order to exclude small debris, while the LIVE/DEAD Fixable Dead Cell Stain (Invitrogen, Carlsbad, CA, USA) allowed for the discrimination between live and dead cells. Muscle pericytes were labelled with the following conjugated antibodies: anti-alkaline phosphatase-Cy5 (BD Pharmingen), anti-CD45-FITC/CD14-PE (BD Biosciences, San Jose, CA, USA), anti-NG2-PE (BD Pharmingen), anti-CD56-APC (NCAM; BD Biosciences), anti-CD146-Cy5 (MCAM; R&D Systems, Minneapolis, MN, USA), anti-PDGF-R-beta-FITC (R&D Systems), and anti-CD44-APC (BD Pharmingen). Skin fibroblasts were characterized by staining with anti-CD90-FITC (BD Pharmingen). iPSC-derived skeletal muscle progenitor cells were stained with primary antibodies: PAX3 (Thermo Fisher Scientific), MyoD1 (Abcam, Cambridge, UK), PAX7 (DHSB), MyoG (Clone F5D, eBioscience, San Diego, CA, USA), and myosin heavy chain (Clone MF20; R&D Systems) (Abcam), followed by staining with the FITC-conjugated secondary antibody (R&D System). All antibodies were diluted in accordance with the manufacturers' instructions. Fluorescence intensity for surface antigens and intracellular cytokines was detected by flow cytometry using a BD FACS Canto II analyzer. Flow data were analyzed with the FACSDiva 6.1.2 software (Becton Dickinson, Franklin Lakes, NJ, USA) and the FlowLogic software (Miltenyi Biotec, Bergisch Gladbach, Germany).

The ALP+/CD56<sup>−</sup> subpopulation was sorted by FACSAria II Cell Sorter (Becton Dickinson) and subsequently characterized by FACS analysis for the expression of pericyte markers (as listed above) following 2 passages in vitro.

To detect and analyze surface EVs markers by FACS analysis, we bound them to 4 μm aldehyde sulphate latex beads (Thermo Fisher Scientific) overnight at 4 ◦C in rotation. EV-coated beads were then incubated with fluorochrome-conjugated antibodies CD63-APC (eBioscience) and CD81-PE (Invitrogen), and diluted in accordance with the manufacturers' instructions. A "beads only" control sample was used to set gating parameters.

For EV internalization, we labelled the purified vesicles isolated from C2C12 with 5 μg/mL CellMask Deep Red plasma membrane stain (Molecular Probes, Eugene, OR, USA) and 5 mM CellTrace Violet (Invitrogen) at 37 ◦C for 30 min. The labeled EVs were washed in PBS and ultra-centrifuged at 100,000× *g* at 4 ◦C for 90 min, suspended in differentiating medium and used to treat the cells. After 48 h, we detected fluorescence on differentiating cells by flow cytometry.

#### *2.10. Gene Expression Analysis*

Total RNA was extracted using small RNA miRNeasy Mini Kit (Qiagen, Hilden, Germany). A total of 1 μg of total RNA was reverse-transcribed to cDNA using SuperScript VILO cDNA synthesis kit (Life Technologies). qRT-PCR were performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Each sample was analyzed in triplicate using QuantStudio 6 Flex Real-Time PCR System Software (Applied Biosystems). The relative gene expression for pluripotency markers was expressed relative to a certified Episomal iPSC lineage (EpiPSC, Thermo Fisher Scientific), and normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Table S1).

The expression of the lentiviral vector was assessed by qualitative RT-PCR according to standard procedure. Amplified products were separated by electrophoresis on a 1% agarose gel. Primers were designed to identify cMyc (one of the four human transcription factors included in the polycistronic lentiviral backbone—forward oligonucleotide) and WPRE (woodchuck hepatitis virus post-transcriptional regulatory element, a lentiviral-specific transgene—reverse oligonucleotide) (Table S1).
