*2.11. Immunohistochemistry and Immunocytochemistry*

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed twice in PBS and blocked with 10% donkey serum (Sigma-Aldrich) for 30 min at room temperature. For intracellular immunostaining, we permeabilized cells for 10 min in 0.1% Triton X-100 (Sigma-Aldrich). Immunofluorescence assays were carried out with the following primary antibodies as follows: pericytes were stained with anti-PDGFRβ (1:100 Abcam), anti-α-smooth muscle actin (1:400, SMA; Dako, Santa Clara, CA, USA) and anti-NG2 (1:200, Millipore, Burlington, MA, USA); fibroblasts were labelled with anti-vimentin (1:50, Sigma-Aldrich); HUVEC were incubated with anti-von Willebrand factor (1:100, Abcam); iPSC pluripotency was verified by anti-OCT4, anti-SSEA4, anti-SOX2 and anti-TRA-1-60 (1:100, all from Invitrogen); iPSC-derived MT were identified by anti-myosin heavy chain (MHC; 1:100; R&D).Incubations with the secondary antibodies (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were performed for 1 hour at 37 ◦C. Cells were then counterstained using Vectashield Mounting Medium with DAPI (4 ,6-diamidino-2-phenylindole). Fluorescence was detected by microscope (Axio Observer A1, Zeiss, Oberkochen, Germany).

Differentiation evaluation was assessed by immunofluorescence for MHC and fusion index scoring, defined as the ratio between the number of myosin heavy chain expressing myotubes with greater than 2 nuclei with respect to the total number of nuclei.

#### *2.12. Western Blotting Analysis*

EVs were also characterized by Western blotting for the expression of specific markers. EVs were lysed in radioimmunoprecipitation assay buffer (RIPA buffer), and supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Proteins (30 μg) were separated on the Nupage Novex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a Polyvinylidene fluoride (PVDF) membrane Amersham Hybond (GE Healthcare Biosciences, Piscataway, NJ, USA). Membranes were incubated overnight at 4 ◦C with primary antibodies anti-CD81, anti-CD63, anti-αHSP70 (ExoAb Antibody Kit, System Biosciences, Palo Alto, CA, USA), anti-TSG-101 (Thermo Fisher Scientific), anti-MyoD1 (Abcam), anti-MHC (R&D) followed by peroxidase-linked anti-rabbit IgG or anti-mouse IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).
