br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments This work was supported by grants from the Ministerio de Ciencia e Innovación (Grant SAF2012-38914), Plan Nacional de I + D + I, Spain and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (Grant 2014 SGR229), Departament d’Economia i Coneixement, Catalonia, Spain to J.A.M. T.A. acknowledges financial support from the Ministerio de Ciencia e Innovación (MICINN) under grant MTM2011-29342 and Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) under grant 2014SGR1307. We are thankful to José M.A. Vaquero (Center of Regenerative Medicine, CMR[B] Core Facility-Cytometry Unit, Barcelona, Spain) for excellent assistance with flow cytometry.
Introduction Human bone marrow mesenchymal stem ion channels (hBMSCs) are attractive candidates for advanced cell therapies, including bone regeneration (Costa-Pinto et al., 2012). Satisfactory treatments are hampered by the difficulty in obtaining a well-defined population of terminally differentiated cells. This causes heterogeneity of hBMSCs, enhances the possibility of spontaneous differentiation into other lineages (Nombela-Arrieta et al., 2011), and may shorten the cells’ engraftment-activation time (Tsubota et al., 1999). Therefore, approaches beyond the standard chemical cocktails have been investigated (Heng et al., 2004) such as genetic modulation through the overexpression of genes (e.g., runt-related transcription factor 2, RUNX2) (Karsenty et al., 2009; Monteiro et al., 2014), and the use of synthetic/recombinant factors such as bone morphogenetic protein 4 or cell-derived conditioned medium (CM). Indeed, CM contains an array of growth factors, cytokines, proteins (Makridakis et al., 2013), and the recently highlighted extracellular vesicles (EVs) (Collino et al., 2010). EVs are nanosized particles (exosomes, 30–100 nm; microvesicles, 50–2000 nm) carrying lipids, proteins, and nucleic acids (Akers et al., 2013). It has been suggested that hBMSC-secreted EVs include differentiation cues (miRNA, Collino et al., 2010; Baglio et al., 2015; tRNA, Baglio et al., 2015; and proteins, Kim et al., 2012), even upon osteogenic induction (Xu et al., 2014). The regenerative potential of MSC-EVs is supported by preclinical studies showing the improvement of at least one clinical outcome associated with acute kidney/liver/lung injury, myocardial infarction, or hindlimb ischemia (Akyurekli et al., 2015). Furthermore, the literature shows that hBMSCs undergo osteogenic differentiation induced by EVs derived from monocytes (Ekstrom et al., 2013) or platelet lysate (Torreggiani et al., 2014). These data enable us to hypothesize that EVs may be vehicles of communication toward tissue regeneration. The knowledge on the bone regenerative potential of hBMSC-EVs is scarce. Therefore, the purpose of this study is to validate the functionality of hBMSC-EVs in the osteoinduction of hBMSCs. We hypothesized that if EVs mirror the content and fate of parent cells, then EVs derived from osteogenically committed hBMSCs will induce the osteogenic commitment of homotypic cells without further supplementation.
Results
Discussion Bone regenerative therapies have been challenged by the limited understanding of the induction of hBMSC fate. This hinders the clinical translation of the most promising strategies. For example, genetically modified cells raise safety, efficacy, and fate issues (Kumar et al., 2008); synthetic/recombinant factors, used at supra-physiological doses, are associated with severe side effects and high costs (Jakob et al., 2012); the CM concentration of biotherapeutics is low, at least for some applications, and contains medium contaminants, such as phenol red (Tran and Damaser, 2015). Our study demonstrates that hBMSCs secrete vesicles with features of EVs that have osteoinductive potential. We showed that hBMSCs secrete a population of EVs heterogeneous in size, as noted earlier (Sokolova et al., 2011; Lobb et al., 2015; Van Deun et al., 2014). Although this can be attributed to the method of EV isolation (Lobb et al., 2015; Van Deun et al., 2014) or to sample aggregation, the sizes obtained (Figures 1A–1C) roughly fall within the range reported for hBMSC-EVs, 47–180 nm (Bian et al., 2014; Bruno et al., 2013). Consistent with MSCs expressing CD9 (Chen et al., 2010), CD63 (Stewart et al., 2003), and CD81 (Lee et al., 2009), EVs also showed these surface markers (Figures 1D and 1E), suggesting that they originated at the parent cell plasma membrane lipid rafts (Tan et al., 2013). Therefore, it is reasonable to accept that the parent cell source affects the EVs’ surface proteins, their glycosylation or lipid composition, and consequently their charge. We found that the surface charge of hBMSC-EVs was relatively less negative (Figure 1B) than that reported for umbilical cord MSC-EVs, −52 mV (Sokolova et al., 2011). Furthermore, our data showed that the environmental stimuli affect the yield of EVs (Figure 1E) probably in parallel with cell activation (Figures 2A and 2B). This is in line with the oxygen-dependent placental secretion of MSC-EVs (Salomon et al., 2013). Due to their biophysical and biochemical properties, namely the size (Figures 1A–1C), EVs may interact differentially with cells. In general, the uptake of EVs is dose, time (Franzen et al., 2014), and recipient-cell (Feng et al., 2010) dependent, while the fate of the EV cargo varies with the particle subtype (Kanada et al., 2015). We indirectly showed that EVs added in concentrations comparable with those produced in vitro undergo uptake by hBMSCs, although the subtype and percentage of EVs being internalized was not determined.