Study of the effects of stem cell derived EVs on an in vitro model of hepatic fibrosis

Progressive liver fibrosis occurs in response to chronic liver injury and, if uncontrolled, can lead to cirrhosis, cancer and end-stage liver disease. For these reasons, there is increasing interest in finding new anti-fibrotic treatments. Extracellular vesicles (EVs) are lipid bilayer membrane-bound vesicles involved in cell-to-cell communication both in physiological and pathophysiological processes, as inflammation and fibrosis. Mesenchymal stromal cell (MSC)-derived EVs were reported to exert anti-inflammatory and anti-fibrotic functions. Moreover, previous studies have demonstrated that human liver stem cells (HLSCs), a hepatic multipotent population of adult stem cells with MSC-like phenotype, release EVs with pro-regenerative activity. The aim of this work is to set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) and then to study the potential anti-fibrotic effects of stem cell-EVs on LX-2. The stimulation of LX-2 with 10 ng/mL of TGF-β1 increases the expression of the pro-fibrotic markers α-SMA, COL1α1, TGF-B1 and matrix metalloproteinase 2 (MMP2). There is also an increase of pro-collagen I α1 released in LX-2 cell supernatant. The incubation of activated LX-2 with stem cell-EVs shows a dose-dependent downregulation of α-SMA. However, only in the presence of HLSC-EVs a slight decrease in the expression of pro-fibrotic genes COL1A1, TGF-β1 and MMP2 has been observed. In conclusion, this study demonstrates the TGF-β1-dependent in vitro activation of LX-2. Moreover, this study shows that bone marrow (BM)-MSC-derived EVs can partially revert the LX-2 activated phenotype, while HLSC-EVs have a more potent in vitro anti-fibrotic effect on the same cells.