*3.2. iPSC Generation and Characterization*

We generated human muscular PC-derived iPSCs and skin FB-derived iPSCs from the same donor (n = 3 donors) using a polycistronic vector harboring the four Yamanaka factors (OCT4, Sox2, Klf4, cMyc) [33,38]. Colonies were initially expanded on a feeder layer of inactivated mouse embryonic fibroblasts (iMEF) and then adapted to feeder free conditions replacing iMEF with geltrex matrix (Figure 2A, left panels). iPSC colonies expressed typical pluripotent markers, including OCT4, SSEA4, SOX2, and TRA-1-60, as assessed by immunofluorescence staining (Figure 2A, middle and right panels). These results were confirmed by quantitative real-time PCR (qRT-PCR) for the expression of OCT4, SOX2, NANOG, LIN28, and TERT genes. Certified Episomal iPSC lineage (EpiPSC, Thermo Fisher Scientific) was used as control (Figure 2B).

**Figure 2.** Characterization of human pericyte (PC)- and fibroblast (FB)-derived iPSCs. (**A**) Morphology of PC-derived iPSCs (left upper panel) and FB-derived iPSCs (left lower panel) cultured in feeder-free conditions.

Scale bar represents 100 μm. Immunofluorescence labeling for the expression of the pluripotent markers SSEA4 (yellow), OCT4 (magenta; middle panels), SOX2 (yellow), and TRA-1-60 (magenta; right panels) on PC- and FB-derived iPSCs (PC-iPSCs and FB-iPSCs, upper and lower panels respectively). Nuclei were stained with DAPI. Scale bar represents 100 μm and, for the lower right image, 200 μm. (**B**) Quantitative RT-PCR analysis for the expression of the embryonic genes, OCT4, SOX2, NANOG, LIN28, and TERT—in PC- and FB-derived iPSC lines, derived from three donors (named FB-/PC-derived iPSCs 1, 2, 3), compared to the certified Episomal iPSC lineage (EpiPSC), used as control. Results were normalized to GAPDH. (**C**) Representative fluorescence images demonstrating multilineage differentiation capacity of PC- and FB-derived iPSCs toward cardiomyocyte (alpha-sarcomeric actin–α-SA, Nkx2.5), neuronal (Nestin, TUJ1) and endothelial (CD31, VE-cadherin–VE-cad) lineages. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm (left and middle panels) and 100 μm (right panels).

Qualitative RT-PCR was conducted to verify the silencing of the exogenous transgenes in PC- and FB-derived iPSCs (Figure S2).

In addition, both derived iPSC lines retained the ability to differentiate toward multiple lineages, including cardiomyocytes, neuronal precursors, and endothelial cells (Figure 2C).

#### *3.3. MT-derived EV Characterization*

EVs represent an important vector of intercellular communication, acting as vehicles for the transfer of cytosolic factors, proteins, lipids and RNA [39]. EV cargo is cell-type specific, and the molecular composition reflects specific functions of the donor cells.

EV secretion and extracellular signaling occurs during muscle differentiation, repair and regeneration [25,40]. Both myoblasts and myotubes release EVs, but their contribution in muscle physiology and specific biological functions on recipient cells have not been fully elucidated.

Proteomic analysis of muscle-derived EVs revealed that, in addition to proteins involved in their biogenesis, EVs also contain functionally relevant proteins such as myogenic growth factors and contractile proteins [26,40,41].

On the basis of these premises, we have exploited the capacity of EVs, secreted during myotube formation of skeletal myoblasts (C2C12), to promote the differentiation of iPSCs into the myogenic lineage.

EVs were purified from conditioned media of C2C12-derived MTs cultured in 2% EV-depleted horse serum. EV isolation was performed by differential centrifugations according to well-established protocols [42]. EVs were further analyzed by FACS analysis for the expression of CD81 and CD63. We found that MT-derived EVs were enriched in membrane-bound tetraspanins CD63 and CD81, which are common markers for EV subsets released from most cell types (Figure 3A,B). The expression of CD81 is observed on vesicles of various sizes indicating that multivesicular endosomes in muscle cells contain intraluminal vesicles of heterogeneous sizes [27,41]. The expression of proteins associated with EVs, such as tumor susceptibility gene 101 (TSG101), α heat shock 70 kDa protein 4 (HSP70), CD81 and CD63 was further confirmed by Western blot analysis.

**Figure 3.** Extracellular vesicle (EV) characterization and uptake. (**A**) Sample gating strategy indicating the percentage of CD63<sup>+</sup> and CD81<sup>+</sup> in myotube (MT)-derived EVs coated with beads (n = 3). In the upper plots, singlets and subsequently EVs were selected according to physical parameters (FSC-A vs. FSC-H and SSC-A vs. FSC-A, respectively) are shown. Fluorescent intensity signal for CD63 and CD81 was detected on gated EVs (SSC-A vs. APC-A and SSC-A vs. PE-A, respectively). Beads alone were used as control (lower four scatter plots). (**B**) Representative histograms displaying the percentage of EV specific markers, such as CD81 and CD63, determined by flow cytometry in purified MT-derived EVs. Matched isotypes were used as negative controls (grey peaks) (**C**) Western blot for specific the expression of EV markers, such as TSG101, CD81, CD63, aHSP70, and skeletal muscle markers, such as CAV-3, MYOD, MHC, in C2C12-derived myotubes and C2C12 myotube-derived EV lysates. (**D**) Size distribution profile of MT-derived EVs (n = 5). (**E**) Representative histograms, determined by flow cytometry, displaying the percentage of positive cells (black) after the treatment with EVs stained for cell trace violet or cell mask deep red. Non-treated cells (light grey peaks) and cells treated with unstained EVs (dark grey peaks) were used as controls. (**F**) Green spots indicate the presence of fluorescent (GFP+) EVs, derived from GFP transduced myotubes, in the cytoplasm of the recipient differentiating iPSC after 48 h of exposure (upper panels; scale bars represent 20 μm); GFP<sup>+</sup> cells 10 days after GFP<sup>+</sup> MT-derived EV exposure (lower panels; scale bars represent 100 μm), demonstrating that GFP was transferred through the EVs into the recipient cells.

We found that MT-derived EVs exhibited specific membrane proteins associated with mature muscle tissue, such as caveolin 3 (Cav3), expressed only during the late stage of differentiation, and MHC, a differentiated myotubes marker (Figure 3C). Interestingly, MyoD, a transcription factor implicated in myogenesis, was also detected within the EV cargo.

Finally, the purity, size and concentration of the MT-derived EVs were determined by nanoparticle tracking analysis (NTA), using instrument-optimized analysis settings in NTA 3.1 build 54 software. The results showed a mean size distribution, consistent with what is expected from a sample enriched in microvesicles and exosomes (130.3 ± 0.3 nm), and free from contamination by apoptotic bodies (>1 μm) (Figure 3D).

#### *3.4. Detection of the MT-derived EV Uptake*

To exert their functional influence, EV cargo must be internalized within the cell in adequate concentrations. As first step, in order to verify if EVs can transfer their contents to differentiating iPSCs, we performed EV uptake assays. For this experiment, we employed two different methods: FACS analysis and immunofluorescence microscopy. Upon isolation, MT-derived EVs were marked either with cell mask deep red or with cell trace violet. As controls we used untreated cells and cells exposed to unstained EVs. After 48 h, FACS analysis revealed an increased fluorescence of both tracers in recipient cells, indicating a successful uptake (Figure 3E).

In order to demonstrate the EV cargo delivery, we transduced C2C12 cells with a lentivirus expressing the GFP protein, and subsequently differentiated into myotubes. The EVs released by GFP<sup>+</sup> myotubes were collected and used to treat the differentiating iPSCs. After 48 h, a fluorescent signal was detected in the cytoplasm of cells undergoing differentiation (Figure 3F, upper panels).

Cells were treated with EVs released by GFP<sup>+</sup> myotubes every other day and, after 10 days, approximately 40% of cells expressed GFP in the cytoplasm (Figure 3F, lower panels) indicating a high functional cargo delivery to the recipient cells. EVs may enter into a cell via more than one route, depending on proteins and glycoproteins found on the surface of both the vesicle and the target cell [43]. The mechanisms responsible for MT-derived EVs delivery have not yet been determined highlighting the need for further research [44].

Considering these results, we have shown that EVs target iPSCs via delivery of effector molecules directly affecting their phenotype and functions.
