*3.1. Identification of Candidate HSC-Linked miRNAs*

As hepatic stellate cell (HSC) activation is an early event of liver fibrosis initiation and progression, we hypothesized that HSC-derived circulating miRNAs could be suitable markers for early stage liver fibrosis. In order to identify candidate miRNAs, NanoString analysis was performed on extracellular vesicles (EVs), both microvesicles and small extracellular vesicles (sEV), obtained from the conditioned medium of in vitro activating primary murine HSCs. To this end, primary mouse HSCs were plated on plastic tissue culture dishes for 10 days. The activation of cultured HSCs was verified on a protein level by the up-regulation of HSC-activation markers Desmin, α-SMA, and Vimentin (Figure 1A), and on an mRNA level by *Acta2*, *Col1a1*, and *Lox* (Figure 1B). Although the obtained miRNA counts by NanoString analysis were insufficient to compare between the quiescent and activated conditions, several miRNAs were found to be highly enriched in such EVs, as compared to the average expression level of all tested miRNAs. This list of highly shed miRNAs, and thus potential fibrosis markers, was further restricted to the miRNAs that are conserved among mouse and human, to ensure translational value, ending up with a list of nine candidate miRNAs (Supplementary Figure S1). The expression levels of these miRNAs were analyzed in the in vitro activated primary mouse HSC cultures. Expression analysis of all nine candidate miRNAs was performed by using qPCR on the cell lysate of activated HSCs, as compared to freshly isolated HSCs (Figure 1C), and identified the significant dysregulation of four miRNAs: miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p (Figure 1D).
