Human umbilical cord mesenchymal stem cells attenuate liver fibrosis in mice and inhibit hepatic stellate cell activation by secreting soluble factors

Abstract Background Mesenchymal stem cells (MSCs) have emerged as promising candidates to treat clinical liver fibrosis. However, the key factors and mechanisms underlying their antifibrotic effects remain largely unclear. Our aims were to assess the therapeutic efficacy of human umbilical cord (UC) MSCs in a murine liver fibrosis model, and to dissect the influence of their secretome in vitro. Methods Liver fibrosis was induced in mice using carbon tetrachloride (CCl₄). Fluorescent labeling was used to track UC-MSC distribution post-injection, comparing intraperitoneal (IP) and intravenous (IV) delivery. UC-MSCs were administered intraperitoneally weekly, starting at either fibrosis induction (prevention) or after six weeks (therapeutic). Fibrosis severity was evaluated by hematoxylin and eosin (H&E) staining, Masson’s trichrome staining and immunohistochemical (IHC) staining for alpha smooth muscle actin (α-SMA). Human hepatic stellate LX-2 cells were treated with complete UC-MSC-conditioned medium (CM UC−MSC ), its soluble factors (SF UC−MSC ) or extracellular vesicle fraction of the secretome (EV UC−MSC ). Fibrosis-related gene and protein expression were analyzed by Q-PCR, immunofluorescence, and Western blotting. Results IP-injected UC-MSCs showed increased liver-specific accumulation compared to IV-injected UC-MSC that distributed mostly to the lungs. In vivo, IP-injected UC-MSC significantly reduced fibrosis, as evidenced by decreased histological damage, collagen deposition, and α-SMA expression. Both preventive and therapeutic UC-MSC IP-treatments were effective. In vitro, CM UC−MSC suppressed TGF-β-induced ACTA2 /α-SMA expression and proliferation of human LX-2 cells, but not COL1A1 /collagen type I expression. These effects persisted in EV-depleted SF UC−MSC . EV UC−MSC inhibited proliferation, but did not suppress ACTA2 /α-SMA or COL1A1 /collagen type I levels in LX-2 cells. Conclusions IP-administered therapeutic UC-MSCs preferentially accumulate in the liver and effectively prevent and reverse liver fibrosis in mice, while inhibiting hepatic stellate cell activation in vitro. The antifibrotic effects in vivo are likely primarily mediated by excreted soluble factors rather than extracellular vesicles.