Liver organoids generated with single or multiple cell types have been used to investigate liver fibrosis development, toxicity, pathogenesis, and drug screening. However, organoid generation is limited by the availability of cells isolated from primary tissues or differentiated from various stem cells. To ensure cell availability for organoid formation, we investigated whether liver organoids could be generated with cell-line-based Huh-7 hepatocellular carcinoma cells, macrophages differentiated from THP-1 monocytes, and LX-2 hepatic stellate cells (HSCs) and primary liver sinusoidal endothelial cells (LSECs). In liver organoids, hepatocyte-, LSEC-, macrophage-, and HSC-related gene expression increased relative to that in two-dimensional (2D)-cultured Huh-7/LSEC/THP-1/LX-2 cells without Matrigel. Thioacetamide (TAA) increased α-smooth muscle actin expression in liver organoids but not in 2D-cultured cells, whereas in TAA-treated organoids, the expression of hepatic and LSEC markers decreased and that of macrophage and HSC markers increased. TAA-induced fibrosis was suppressed by treatment with N-acetyl-L-cysteine or tumor-necrosis-factor-stimulated gene 6 protein. The results showed that liver toxicants could induce fibrotic and inflammatory responses in liver organoids comprising Huh-7/LSEC/macrophages/LX-2 cells, resulting in fibrotic liver organoids. We propose that cell-line-based organoids can be used for disease modeling and drug screening to improve liver fibrosis treatment.

The pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β upregulate TNF-α-stimulated gene 6 (TSG-6); however, current knowledge about the optimal conditions for TSG-6 expression in mesenchymal stem cells (MSCs) is limited. Here, we investigated whether TSG-6 expression varies depending on the polarization state of macrophages co-cultured with adipose tissue-derived stem cells (ASCs) and analyzed the optimal conditions for TSG-6 expression in ASCs. TSG-6 expression increased in ASCs co-cultured with M0, M1, and M2 macrophages indirectly; among them, M1 macrophages resulted in the highest increase in TSG-6 expression in ASCs. TSG-6 expression in ASCs dramatically increased by combination (but not single) treatment of TNF-α, IL-1β, interferon-gamma (IFN-γ), and lipopolysaccharide (LPS). In addition, phosphorylation of signal transducer and activator of transcription (STAT) 1/3 was observed in response to IFN-γ and LPS treatment but not TNF-α and/or IL-1β. STAT1/3 activation synergistically increased TNF-α/IL-1β-dependent TSG-6 expression, and JAK inhibitors suppressed TSG-6 expression both in ASCs and macrophages. In LX-2 hepatic stellate cells, TSG-6 inhibited TGF-β-induced Smad3 phosphorylation, resulting in decreased α-smooth muscle actin (SMA) expression. Moreover, fibrotic activities of LX-2 cells induced by TGF-β were dramatically decreased after indirect co-culture with ASCs and M1 macrophages. These results suggest that a comprehensive inflammatory microenvironment may play an important role in determining the therapeutic properties of ASCs by increasing TSG-6 expression through STAT1/3 activation.

Mesenchymal stem cells (MSCs) regulate immune cell activity by expressing tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) in inflammatory environments; however, whether anti-inflammatory responses affect TSG-6 expression in MSCs is not well understood. Therefore, we investigated whether transforming growth factor-β (TGF-β) regulates TSG-6 expression in adipose tissue-derived stem cells (ASCs) and whether effective immunosuppression can be achieved using ASCs and TGF-β signaling inhibitor A83-01. TGF-β significantly decreased TSG-6 expression in ASCs, but A83-01 and the p38 inhibitor SB202190 significantly increased it. However, in septic C57BL/6 mice, A83-01 further reduced the survival rate of the lipopolysaccharide (LPS)-treated group and ASC transplantation did not improve the severity induced by LPS. ASC transplantation alleviated the severity of sepsis induced by LPS+A83-01. In co-culture of macrophages and ASCs, A83-01 decreased TSG-6 expression whereas A83-01 and SB202190 reduced Cox-2 and IDO-2 expression in ASCs. These results suggest that TSG-6 expression in ASCs can be regulated by high concentrations of pro-inflammatory cytokines in vitro and in vivo, and that A83-01 and SB202190 can reduce the expression of immunomodulators in ASCs. Therefore, our data suggest that co-treatment of ASCs with TGF-β or p38 inhibitors is not adequate to modulate inflammation.

Although protein kinase C (PKC) regulates various biological activities, including cell proliferation, differentiation, migration, tissue remodeling, gene expression, and cell death, the antifibrotic effect of PKC in myofibroblasts is not fully understood. We investigated whether 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, reduced the activation of hepatic stellate cells (HSCs) and explored the involvement of the Hippo pathway transcriptional coactivator YAP. We analyzed the effect of TPA on the proliferation and expression of α-smooth muscle actin (SMA) in the LX-2 HSC line. We also analyzed the phosphorylation of the Hippo pathway molecules YAP and LATS1 and investigated YAP nuclear translocation. We examined whether Gö 6983, a pan-PKC inhibitor, restored the TPA-inhibited activities of HSCs. Administration of TPA decreased the growth rate of LX-2 cells and inhibited the expression of α-SMA and collagen type I alpha 1 (COL1A1). In addition, TPA induced phosphorylation of PKCδ, LATS1, and YAP and inhibited the nuclear translocation of YAP compared with the control. These TPA-induced phenomena were mostly ameliorated by Gö 6983. Our results indicate that PKCδ exerts an antifibrotic effect by inhibiting the Hippo pathway in HSCs. Therefore, PKCδ and YAP can be used as therapeutic targets for the treatment of fibrotic diseases.

Liver tumor organoids derived from liver tumor tissues and pluripotent stem cells are used for liver tumor research but have several challenges in primary cell isolation and stem cell differentiation. Here, we investigated the potential of HepG2-based liver tumor organoids for screening anticancer drugs by evaluating their responsiveness to IFN-β produced by mesenchymal stem cells (MSCs). Liver tumor organoids were prepared in three days on Matrigel using HepG2, primary liver sinusoidal epithelial cells (LSECs), LX-2 human hepatic stellate cells, and THP-1-derived macrophages at a ratio of 4:4:1:1, with 10⁵ total cells. Hepatocyte-related and M2 macrophage-associated genes increased in liver tumor organoids. IFN-β treatment decreased the viability of liver tumor organoids and increased M1 macrophage marker expression (i.e., TNF-α and iNOS) and TRAIL. TRAIL expression was increased in all four cell types exposed to IFN-β, but cell death was only observed in HepG2 cells and macrophages. Further, MSCs overexpressing IFN-β (ASC-IFN-β) also expressed TRAIL, contributing to the reduced viability of liver tumor organoids. In summary, IFN-β or ASC-IFN-β can induce TRAIL-dependent HepG2 and macrophage cell death in HepG2-based liver tumor organoids, highlighting these liver tumor organoids as suitable for anticancer drug screening and mechanistic studies.

Analysis of pulmonary vascular dysfunction in various lung pathologies remains challenging due to the lack of functional ex vivo models. Paracrine signaling in the lung plays a critical role in regulating endothelial maturation and vascular homeostasis. Previously, we employed single-cell RNA-sequencing (scRNAseq) to systematically map ligand–receptor (L/R) interactions within the lung vascular niche. However, the functional impact of these ligands on endothelial biology remained unknown. Here, we systematically evaluated selected ligands in vitro to assess their effects on endothelial barrier integrity, anti-inflammatory responses, and phenotypic maturation. Among the top soluble ligands, we found that adrenomedulin (ADM) exhibited superior barrier enhancing effect on human pulmonary endothelial cell monolayers, as evidenced by electrical cell impedance sensing (ECIS) and XperT assays. ADM also exhibited anti-inflammatory properties, decreasing ICAM1 and increasing IkBa expression in a dose-dependent manner. Perfusion is commonly used in bioengineered vascular model systems. Shear stress (15 dynes/cm²) alone increased endothelial characteristics, including homeostatic markers such as CDH5, NOS3, TEK, and S1PR1. ADM treatment maintained the enhanced level of these markers under shear stress and further improved anti-coagulation by increasing THBD and decreasing F3 expression and synergistically enhanced the expression of the native lung aerocyte capillary endothelial marker EDNRB. This effect was completely attenuated by a blockade of ADM receptor, RAMP2. Together, these findings identify ADM/RAMP2 signaling as a key paracrine pathway that enhances vascular barrier integrity, anti-inflammatory phenotype, and endothelial homeostasis, providing a framework for improving the physiological relevance of engineered vascular models.