**1. Introduction**

Skeletal muscle is a dynamic tissue with remarkable features for endogenous regeneration provided by muscle progenitors, such as satellite cells. However, in the presence of progressive muscle loss or degeneration, such as muscular dystrophies or aging, satellite cell function is largely affected due to an incorrect asymmetric division or aberrant transcriptional regulation [1–4]. Other adult progenitor cells with myogenic properties, including mesangioblasts [5], pericytes [6,7], muscle side cell population [8], interstitial cells PW1+/PAX7<sup>−</sup> [9], or stem cells derived from bone marrow [10] would be considered promising candidates for muscle repair therapy. Despite this, the reduction of proliferative capacity after isolation and the progressive loss of self-renewal potential strongly limit the use of adult progenitor cells for clinical application [11].

On the other hand, induced pluripotent stem cells (iPSCs) represent a valuable source of myogenic progenitors (MPs), essential for cell-based therapy. Indeed, iPSCs not only would allow autologous transplantation but they can also be produced in large quantities, with an unlimited replication ability in vitro. Furthermore, differentiated iPSCs can be used as individual-specific tissue modeling for the validation of innovative therapies, limiting the toxic effects for the patient and providing early indications on the efficacy [12,13].

Many efforts have been made to establish efficient methods for obtaining MPs from iPSCs, mostly relying on the transgenic expression of major myogenesis regulators, such as myoblast determination protein 1 (MyoD) and Pax7 (key myogenic transcription factors) [14–17]. The main disadvantage of these approaches is that forced expression of the MyoD protein leads to cell cycle arrest along with the consequent loss of the in vitro muscle progenitor generation. The risk of unwanted genetic recombination is a widespread limiting issue for future clinical application.

The use of chemical modulators to activate relevant myogenic pathways represents a promising approach to enhance the efficiency of myogenic iPSC differentiation [18–20]. In particular, a myogenic differentiation improvement of human ESC/iPSC through the treatment with a homologous wingless and Int-1 (Wnt) agonist, the glycogen synthase kinase-3 inhibitor (GSK-3, CHIR9902), has been reported [18,19]. Early inhibition of GSK3β is mandatory for the induction of paraxial mesoderm and activation of the myogenic program [21].

In this study we explored the possibility to exploit the content of extracellular vesicles (EVs), released from differentiated myotubes (MTs), in combination with GSK3 inhibitor, in order to synergistically enhance myogenic differentiation.

To date, the scientific interest regarding the role of EVs in cell-to-cell communication, both in physiological and pathological conditions, is rapidly increasing. EVs are similar in composition to their cell of origin, and their cargo can activate signaling pathways in target cells, thus modulating their activities. In particular, the content of EVs derived from skeletal muscle plays a fundamental role for skeletal muscle homeostasis and development [22,23]. Several studies have shown that skeletal muscle cells release protein/nucleic acid complexes within microvesicles, which promote myogenesis and muscle regeneration [24–27]. EVs derived from MTs (MT-derived EVs) were found to be able to promote the differentiation of myoblasts by altering the expression of cyclin-D1 and myogenin [27]. Another study reported that exosomes, a subclass of EVs measuring approximately 100 nm in diameter, secreted during myotube differentiation, contribute significantly to the myogenic differentiation of stem cells derived from human adipose tissue [28].

Previous researches have shown that iPSCs retain molecular characteristics of the cell from which they originate, named 'epigenetic memory', which is able to strengthen the propensity for re-differentiation in the same tissue [29,30]. On the basis of this, in order to enhance muscle differentiation and exploit myogenic predisposition, muscle-derived pericytes (PCs) and skin fibroblasts (FBs) derived from the same donor were employed as cell sources for iPSC generation. PCs surround the endothelial layer of small/medium vessels that reside beneath the microvascular basement membrane. Despite their role in regulating blood flow, angiogenesis, and maintenance of vascular tissue homeostasis [31], not much is known about pericytes as a source of muscle progenitor cells [6,7]. However, several studies have shown that pericytes are strongly predisposed to differentiate into myogenic lineage and repair muscle damage [6,7,32].

In this study, we established a defined transgene-free protocol, which allows iPSCs, derived either from muscular pericytes or skin fibroblasts, to differentiate into MT-like cells when exposed to GSK-3 inhibitor and EV cargo. This combination improved the differentiation yield into muscle cells up to 70% and the fusion index. After 30 days, evidence of an enhanced muscle differentiation was further revealed by an increased expression of myogenic markers. Furthermore, we found a propensity in pericyte-derived iPSCs to re-differentiate toward the skeletal muscular fate compared to fibroblast-derived counterpart.

Finally, in a pilot study, differentiated iPSCs were injected intramuscularly into anterior tibialis (TA) muscle of immunodeficient alpha-sarcoglycan knockout (KO) mice. The differentiated cells were able to integrate into the host regenerating myofibers, revealing a possible application of the proposed method in regenerative medicine.
