## 4 Discussion  
Use of MuSCs as therapeutic agents has been widely investigated [2; 7; 57]. The therapeutic application of MuSCs is not completely understood but is largely attributed to their ability to differentiate into myoblasts and fuse into damaged fibers to support regeneration [6]. However, MuSC-mediated therapy is hindered by several major limitations, including the number of MuSCs  
Figure 5: Proteomic Analysis of MuSC-EVs. Significantly enriched (\(p<0.05\)) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (LEGG) terms identified using DAVID enrichment analysis following UPLC MS/MS of MuSC-EVs. (**A**) Biological processes (**B**) Molecular function (**C**) Cellular component (**D**) KEGG Pathway results. needed to treat systemic muscle diseases and the loss of myogenic potential during ex vivo expansion [2; 14; 15; 16]. Interestingly, mesenchymal stem cells (MSCs) release EVs that confer many of the same regenerative benefits as the cells themselves [58]. Further, typically cells continually release EVs and thus from a therapeutic standpoint EVs may provide a more efficient and scalable production method than cell-based therapeutics. Additionally, EVs have been shown to efficiently deliver functional molecular cargo into cells [59; 60; 61]. Therefore, MuSC-EVs could feasibly overcome the major limitations of MuSC-based therapeutic approaches. The purpose of this investigation was to characterize MuSC-EVs and evaluate the extent to which MuSC-EVs can aid in reversing hydrogen peroxide-induced muscle damage. Calculations in the present study show approximately \(2.35\times 10^{5}\) EVs were released per MuSC per 24 h of culture. To our knowledge, this is the first study quantifying the number of EVs released per MuSC per 24 h. However, most studies quantifying number of EVs present the data as EVs per milliliter of media or bodily fluid. This is likely due to nanoparticle tracking analysis platforms quantifying data as particle per milliliter. Reporting the data this way makes it difficult to compare the quantity of EVs released from a specific cell line under differing conditions. Back calculations can be performed on studies that report cell number, amount of time, and concentration of EVs. However, even when back calculating for number of EVs released per cell per 24 h, comparing number of EVs released both by other stem cell types and other cell lines presents several challenges regarding EV isolation and cell culture methods [62]. For example, studies reporting the number of EVs released from MSCs vary widely when back calculating to number of EVs released per cell per 24 h of culture [63; 64]. This is also the case when examining the number of EVs released from other cell types, such as human umbilical vein endothelial cells and human embryonic kidney cells [65; 66]. Therefore, it is evident the use of varying isolation and cell culture methodologies as well as varying cell types result in different numbers of EVs obtained during cell culture. To accurately make a comparison of EVs released based on cell type, one must control for isolation and culture methods and implement a standardized approach of reporting EV release data. In addition to variations in the number of EVs obtained, the isolation method utilized may also impact the identity and cargo of the EVs. In the current study, MuSC-EVs were isolated using a precipitation based method (ExoQuick TC from System Bioscience, Palo Alto, CA, USA). Compared to ultracentrifugation, precipitation methods and kits using precipitation methods have been shown to result in both increased number of EVs as well as higher extraction efficiency and purity of small RNA in EVs isolated from cell culture media [67]. Precipitation-based methods have also been shown to yield an overall greater number of EVs and higher protein concentration than ultracentrifugation [68]. Our data suggest MuSCs produce an abundance of EVs that deliver large amounts of cargo into myotubes, some of which colocalizes to intracellular mitochondria.