*3.1. Characterization of Primary mHSC*

Autofluorescence for vitamin A, which is stored in cytoplasmic oil droplets in HSC and is useful for identifying or characterizing HSC, was detected in 91.3 ± 3.4% of D4 mouse cells and 85.4 ± 8.0% of P1 mouse cells (Figure 1A). Staining for αSMA, a marker of HSC activation, was detectable in only 3.3 ± 0.7% of D4 cells but in 93.3 ± 5.0% of P1 cells demonstrating the high degree of activation of P1 cells as compared to D4 cells (Figure 1B). Staining for reelin, which is preferentially expressed in quiescent HSC but not in other hepatic cell types [33], was detected in 98.4 ± 1.6% of D4 mHSC and 13.1 ± 1.2% of P1 mHSC (Figure 1C). This is consistent with previous findings showing that reelin expression declines during HSC activation [33] and also argues that our early (and hence later) primary HSC cultures were essentially devoid of contaminating cells that might otherwise confound analysis. Since absolute quiescence is not feasible because HSC start to autonomously activate after isolation, the above staining data nonetheless led us to conclude that D4 (the earliest time point at which we could reproducibly harvest sufficient numbers of EVs for detailed analysis) represented a relatively quiescent state as compared to P1 cells which were highly activated, thus validating our subsequent comparative EV analysis.

**Figure 1.** Characterization of primary mouse HSC. (**A**) Primary D4 or P1 mHSC were evaluated for vitamin A autofluorescence (left) or examined by phase contrast (right), and imaged by confocal microscopy. Based on six fields, Vitamin A was positive in 91.3 ± 3.4% of D4 mHSC and 85.4 ± 8.0% of P1 mHSC. (**B**) D4 or P1 mHSC were stained with anti-αSMA (left) which was then merged with DAPI (right). Based on three fields, α SMA was positive in 3.3 ± 0.7% of D4 mHSC and 93.3 ± 5.0% of P1 mHSC. (**C**) D4 or P1 mHSC were stained with anti-reelin (left) which was then merged with DAP1 (right). Based on two fields, the frequency of reelin-positive cells was 98.4 ± 1.6% for D4 mHSC and 13.1 ± 1.2% for P1 mHSC. Bars: 50 μm for A and C; 100 μm for B.
