*3.2. Physico-Chemical and Biological Properties of EVs from mHSC or hHSC*

Serial low speed centrifugation and ultracentrifugation of serum-free medium from each cell type resulted in a significantly greater yield (4.5-fold) of EVs from P1-P2 mHSC than D4 mHSC showing that EV production was positively correlated with mHSC activation (Figure 2A). EVs from D4 mHSC or P1 mHSC were very similar in terms of mean size (144.3 ± 3.2 nm and 133.5 ± 20.61 nm, respectively) and size range (approx. 50–500 nm) (Figure 2B). Western blot revealed that EVs from either D4 or P1 mHSC were positive for proteins such as CD9, Tsg101 and flotillin-1 that are commonly associated with or enriched in EVs from other systems (Figure 2C). Thus, EVs from D4 or P1 mHSC exhibited shared physical properties and expression of key EV proteins. Additional characterization of mHSC EVs including conventional or cryogenic transmission electron microscopy and dynamic light scattering have been reported by us previously [25].

We have previously demonstrated and characterized the binding interactions between target HSC and EVs from mHSC or hHSC, including functional delivery of specific EV payload components [24,25,27,28,34]. When added to P1 mHSC, EVs from D4 mHSC caused expression of CCN2 or Col1a1 to be suppressed in the cells (Figure 2D), an observation that is consistent with our earlier reports that EVs from quiescent mHSC suppress fibrogenic gene expression as well as levels of proteins such as αSMA [25,27,28]. We extended these findings by showing that EVs from P1 mHSC did not alter expression of these genes in P1 mHSC (Figure 2D), yet they stimulated expression of CCN2, Col1a1 or αSMA in D4 mHSC (Figure 2E), the latter of which is consistent with our prior demonstration that αSMA mRNA or protein in HSC is stimulated by EVs from activated HSC or by EVs carrying an enriched CCN2 payload [24]. By contrast, EVs from D4 mHSC had no significant effect on gene expression in D4 mHSC (Figure 2E). We previously conducted transmission electron microscopy and cell binding assays of EVs purified from LX-2 hHSC [24,25] and expanded those data in this study by using NTA to show that they were of similar size as those from mHSC (151.9 ± 5.8 nm; size range 50–500 nm), contained EV-associated proteins such as flotillin-1 and CD63 (Figure 2F), and, like EVs from P1 mHSC, were able to stimulate gene expression in D4 mHSC (Figure 2E).

**Figure 2.** Characterization of EVs from mouse HSC. NTA was performed on EVs that had been purified by differential centrifugation of 2-day serum-free conditioned medium from D4 HSC or P1-P2 HSC, with particle number expressed as a function of (**A**) cell number or (**B**) particle size (mean ± S.E.M. for particle diameter (nm) is indicated). (**C**) Western blot analysis of D4 or P1 EVs (25 μg EV protein per lane) showing the presence of common EV proteins. (**D**) P1 HSC or (**E**) D4 HSC were treated for 48 h with 8 μg/mL EVs from D4 mHSC, P1 mHSC or LX-2 hHSC after which expression for the indicated transcripts was determined by qRT-PCR. (**F**) EVs purified from LX-2 hHSC under serum-free conditions were analyzed by NTA or Western blot (inset). \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.005; n.s., not significant.
