These regulatory effects on HSCs were concentration-dependent. In addition, hAMSC-CM upregulated the expression of ECM degradation-related genes, such as metalloproteinase- ( Mmp-) 2, Mmp-9, Mmp-13, and tissue inhibitor of metalloproteinase- ( Timp-) 1 however, it did not affect the expression of collagen type 1α1 ( Col1a1). We observed that in routine culture, hAMSC-CM in HSCs significantly inhibited the expression of alpha-smooth muscle actin (α-SMA), an activation marker of HSCs, and the production of collagen type 1 (COL1), a dominant component of the extracellular matrix (ECM) in the culture medium. In this study, we investigated the effects, and underlying mechanisms, of a conditioned medium obtained from hAMSC cultures (hAMSC-CM) on a primary culture of rat hepatic stellate cells (HSCs). However, the mechanism for the prevention of liver fibrosis is poorly understood. Human amnion-derived mesenchymal stem cell (hAMSC) transplantation has been reported to ameliorate liver fibrosis in animal models. Mesenchymal stem cells (MSCs), or multipotent mesenchymal stromal cells, are present in almost all organs and tissues, including the amnion. Supplementary Table 1: sequences of primers. Data are expressed as mean ± SD ( n = 3). Supplementary Figure 4: gene expressions of primary HSCs cultured in SM or skin fibroblast-CM. (B) Desmin (HSC marker), CD31 (endothelial cell marker), CD163 (Kupffer cell marker) expressions of sorted HSCs were analyzed by flow cytometry. ∗ P < 0.05 and ∗∗ P < 0.01 versus sorted HSCs. Beta-type platelet-derived growth factor receptor (Pdgfb) is a marker of HSCs, C-type lectin domain family 4f (Clec4f) is a marker for Kupffer cells, and albumin is a marker for hepatocytes. (A) Cell type-specific gene expression analysis of sorted HSCs and remaining cells. Supplementary Figure 3: characterization of sorted HSCs. (B) Sorted HSCs proliferated during culture and presented a fibroblast-like phenotype. Arrows indicate lipid droplets, and the inset at the upper right corner shows the lipid droplets close-up. (A) Most of the seeded HSCs present an asteroid phenotype with remaining a great quantity of lipid droplets after 1 day of culture. Supplementary Figure 2: morphology of sorted HSCs in vitro. Gene expression was normalized to 18s rRNA.
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