Interferon-ß Overexpression in Adipose Tissue-Derived Stem Cells Induces HepG2 and Macrophage Cell Death in Liver Tumor Organoids via Induction of TNF-Related Apoptosis-Inducing Ligand Expression.

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-ß 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-ß 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-ß, but cell death was only observed in HepG2 cells and macrophages. Further, MSCs overexpressing IFN-ß (ASC-IFN-ß) also expressed TRAIL, contributing to the reduced viability of liver tumor organoids. In summary, IFN-ß or ASC-IFN-ß 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.

Role of TGF-ß and p38 MAPK in TSG-6 Expression in Adipose Tissue-Derived Stem Cells In Vitro and In Vivo.

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-ß (TGF-ß) regulates TSG-6 expression in adipose tissue-derived stem cells (ASCs) and whether effective immunosuppression can be achieved using ASCs and TGF-ß signaling inhibitor A83-01. TGF-ß 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-ß or p38 inhibitors is not adequate to modulate inflammation.

Skeletal muscle satellite cell-derived mesenchymal stem cells ameliorate acute alcohol-induced liver injury.

Cultured human skeletal-muscle satellite cells have properties of mesenchymal stem cells (skeletal muscle satellite cell-derived mesenchymal stem cells, SkMSCs) and play anti-inflammatory roles by secreting prostaglandin E2 and hepatocyte growth factor (HGF). To evaluate the utility of SkMSCs in treating liver diseases, we determined whether SkMSCs could ameliorate acute liver and gut inflammation induced by binge ethanol administration. Binge drinking of ethanol led to weight loss in the body and spleen, liver inflammation and steatosis, and increased serum ALT and AST levels (markers of liver injury), along with increased IL-1ß, TNF-α, and iNOS expression levels in mice. However, levels of these binge-drinking-induced indicators were reduced by a single intraperitoneal treatment of SkMSCs. Furthermore, levels of bacteria-derived lipopolysaccharide decreased in the livers and sera of ethanol-exposed mice after SkMSC administration. SkMSCs decreased the extent of tissue inflammation and reduced villus and crypt lengths in the small intestine after alcohol binge drinking. SkMSCs also reduced the leakage of blood albumin, an indicator of leaky gut, in the stool of ethanol-exposed mice. Alcohol-induced damage to human colonic Caco-2/tc7 cells was also alleviated by HGF. Therefore, a single treatment with SkMSCs can attenuate alcoholic liver damage by reducing inflammatory responses in the liver and gut, suggesting that SkMSCs could be used in cell therapy to treat alcoholic liver diseases.

Antifibrotic TSG-6 Expression Is Synergistically Increased in Both Cells during Coculture of Mesenchymal Stem Cells and Macrophages via the JAK/STAT Signaling Pathway.

The pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß 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-1ß, 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-1ß. STAT1/3 activation synergistically increased TNF-α/IL-1ß-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-ß-induced Smad3 phosphorylation, resulting in decreased α-smooth muscle actin (SMA) expression. Moreover, fibrotic activities of LX-2 cells induced by TGF-ß 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 cell therapy for liver disease: current status and future perspectives.

PURPOSE OF REVIEW: Liver transplantation is the gold standard for the treatment of end-stage liver disease. However, a shortage of donor organs, high cost, and surgical complications limit the use of this treatment. Cellular therapies using hepatocytes, hematopoietic stem cells, bone marrow mononuclear cells, and mesenchymal stem cells (MSCs) are being investigated as alternative treatments to liver transplantation. The purpose of this review is to describe studies using MSC transplantation for liver diseases based on the reported literature and to discuss prospective research designed to improve the efficacy of MSC therapy. RECENT FINDINGS: MSCs have several properties that show potential to regenerate injured tissues or organs, such as homing, transdifferentiation, immunosuppression, and cellular protective capacity. Additionally, MSCs can be noninvasively isolated from various tissues and expanded ex vivo in sufficient numbers for clinical evaluation. SUMMARY: Currently, there is no approved MSC therapy for the treatment of liver disease. However, MSC therapy is considered a promising alternative treatment for end-stage liver diseases and is reported to improve liver function safely with no side effects. Further robust preclinical and clinical studies will be needed to improve the therapeutic efficacy of MSC transplantation.

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors.

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1ß secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1ß expression, it suppressed the secretion of cleaved IL-1ß. In contrast, HGF decreased active IL-1ß secretion without affecting pro-IL-1ß expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1ß expression level and active IL-1ß secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.

Adipose Tissue-Derived Mesenchymal Stem Cells Suppress Growth of Huh7 Hepatocellular Carcinoma Cells via Interferon (IFN)-ß-Mediated JAK/STAT1 Pathway in vitro.

Interferon (IFN)-ß and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) secreted by adipose tissue-derived mesenchymal stem cells (ASCs) have been proposed as key mechanistic factors in anti-cancer efficacy in lung cancer and breast cancer cells, where they act through paracrine signaling. We hypothesized that IFN-ß and TRAIL produced by ASCs suppress proliferation of hepatocellular carcinoma cells (HCCs). The present study evaluated the anti-cancer effects of ASCs on HCCs in vitro. We found that indirect co-culture with ASCs diminished growth of Huh7 hepatocellular carcinoma cells with increased protein levels of p53/p21 and phosphorylated STAT1 (pSTAT1), without apoptosis. Treatment with ASC-conditioned medium (ASC-CM) also decreased growth of Huh7 cells through elevated p53/p21 and pSTAT1 signaling. ASC-CM-mediated inhibition of cell growth was neutralized in Huh7 cells treated with anti-IFN-ß antibody compared to that in ASC-CM-treated Huh7 cells incubated with an anti-TRAIL antibody. Treatment with JAK1/JAK2 inhibitors recovered inhibition of growth in Huh7 cells incubated in ASC-CM or IFN-ß via down-regulation of pSTAT1/p53/p21. However, treatment of IFN-ß resulted in no alterations in resistance of Huh7 cells to TRAIL. Our findings suggest that ASCs decrease growth through activated STAT1-mediated p53/p21 by IFN-ß, but not TRAIL, in Huh7 cells.

M1 Macrophages Promote TRAIL Expression in Adipose Tissue-Derived Stem Cells, Which Suppresses Colitis-Associated Colon Cancer by Increasing Apoptosis of CD133+ Cancer Stem Cells and Decreasing M2 Macrophage Population.

We have previously reported that adipose tissue-derived stem cells (ASCs) cultured at high cell density can induce cancer cell death through the expression of type I interferons and tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAIL). Here, we investigated whether TRAIL-expressing ASCs induced by M1 macrophages can alleviate colitis-associated cancer in an azoxymethane (AOM)/dextran sodium sulfate (DSS) animal model. M1 macrophages significantly increased the TRAIL expression in ASCs, which induced the apoptosis of LoVo cells in a TRAIL-dependent manner. However, CD133knockout LoVo cells, generated using the CRISPR-Cas9 gene-editing system, were resistant to TRAIL. In the AOM/DSS-induced colitis-associated cancer model, the intraperitoneal transplantation of TRAIL-expressing ASCs significantly suppressed colon cancer development. Moreover, immunohistochemical staining revealed a low CD133 expression in tumors from the AOM/DSS + ASCs group when compared with tumors from the untreated group. Additionally, the ASC treatment selectively reduced the number of M2 macrophages in tumoral (45.7 ± 4.2) and non-tumoral mucosa (30.3 ± 1.5) in AOM/DSS + ASCs-treated animals relative to those in the untreated group (tumor 71.7 ± 11.2, non-tumor 94.3 ± 12.5; p < 0.001). Thus, TRAIL-expressing ASCs are promising agents for anti-tumor therapy, particularly to alleviate colon cancer by inducing the apoptosis of CD133+ cancer stem cells and decreasing the M2 macrophage population.

Co-treatment with interferon-γ and 1-methyl tryptophan ameliorates cardiac fibrosis through cardiac myofibroblasts apoptosis.

Cardiac remodeling characterized by cardiac fibrosis is a pathologic process occurring after acute myocardial infarction. Fibrosis can be ameliorated by interferon-gamma (IFN-γ), which is a soluble cytokine showing various effects such as anti-fibrosis, apoptosis, anti-proliferation, immunomodulation, and anti-viral activities. However, the role of IFN-γ in cardiac myofibroblasts is not well established. Therefore, we investigated the anti-fibrotic effects of IFN-γ in human cardiac myofibroblasts (hCMs) in vitro and whether indoleamine 2,3-dioxygenase (IDO), induced by IFN-γ and resulting in cell cycle arrest, plays an important role in regulating the biological activity of hCMs. After IFN-γ treatment, cell signaling pathways and DNA contents were analyzed to assess the biological activity of IFN-γ in hCMs. In addition, an IDO inhibitor (1-methyl tryptophan; 1-MT) was used to assess whether IDO plays a key role in regulating hCMs. IFN-γ significantly inhibited hCM proliferation, and IFN-γ-induced IDO expression caused cell cycle arrest in G0/G1 through tryptophan depletion. Moreover, IFN-γ treatment gradually suppressed the expression of α-smooth muscle actin. When IDO activity was inhibited by 1-MT, marked apoptosis was observed in hCMs through the induction of interferon regulatory factor, Fas, and Fas ligand. Our results suggest that IFN-γ plays key roles in anti-proliferative and anti-fibrotic activities in hCMs and further induces apoptosis via IDO inhibition. In conclusion, co-treatment with IFN-γ and 1-MT can ameliorate fibrosis in cardiac myofibroblasts through apoptosis.

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis.

Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells.

Mesenchymal stromal cell therapy for liver diseases.

The therapeutic potential of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis is predominantly based on their immunosuppressive properties, and their ability to secrete various trophic factors. This potential has been investigated in clinical and preclinical studies. Although the therapeutic mechanisms of MSC transplantation are still not fully characterised, accumulating evidence has revealed that various trophic factors secreted by MSCs play key therapeutic roles in regeneration by alleviating inflammation, apoptosis, and fibrosis as well as stimulating angiogenesis and tissue regeneration in damaged liver. In this review, we summarise the safety, efficacy, potential transplantation routes and therapeutic effects of MSCs in patients with liver fibrosis. We also discuss some of the key strategies to enhance the functionality of MSCs, which include sorting and/or priming with factors such as cytokines, as well as genetic engineering.

Adipose-derived stem cells ameliorate colitis by suppression of inflammasome formation and regulation of M1-macrophage population through prostaglandin E2.

Inflammatory bowel disease (IBD) is an idiopathic disease caused by a dysregulated immune response to intestinal microbes in an individual with a genetic predisposition. Therefore, alleviation of inflammation is very important to treat IBD. Mesenchymal stem cells (MSCs) have been highlighted as new candidates for treating autoimmune disease based on their immunomodulatory properties. In this study, we investigated the anti-inflammatory mechanism and therapeutic effects of adipose tissue-derived MSCs (ASCs) using THP-1 macrophages and dextran sodium sulfate (DSS)-induced mice with chronic colitis. LPS-treated THP-1 cells expressed mRNA of CD11b, an M1 macrophage marker, at day 2. However, THP-1 co-cultured with ASCs expressed mRNA of CD206, CD68, CCL18, legumain, and IL-10, markers of M2 macrophages. In THP-1 cells co-cultured with ASCs, precursor (pro)-IL-1ß, Cox-2, and NLRP3 increased dramatically compared to LPS-treated THP-1 cells. Secretion of IL-1ß and IL-18 was significantly inhibited by ASCs, but PGE2 production was highly increased in co-culture conditions of THP-1 and ASCs. IL-18 secretion was inhibited by PGE2 treatment, and PGE2 inhibited inflammasome complex (ASC/Cas-1/NLRP3) formation in THP-1 cells. In the DSS-induced chronic colitis model, ASCs ameliorated colitis by decreasing the total number of macrophages and the M1 macrophage population. Our results suggest that ASCs can suppress the inflammatory response by controlling the macrophage population, and ASCs may be therapeutically useful for the treatment of IBD.

L-ascorbic acid 2-phosphate and fibroblast growth factor-2 treatment maintains differentiation potential in bone marrow-derived mesenchymal stem cells through expression of hepatocyte growth factor.

l-ascorbic acid 2-phosphate (Asc-2P) acts as an antioxidant and a stimulator of hepatocyte growth factor (HGF) production. Previously, we reported that depletion of growth factors such as fibroblast growth factor (FGF)-2, epidermal growth factor (EGF), FGF-4 and HGF during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF). In this study, we investigated the proliferation and differentiation potential of BMSCs by FGF-2 and Asc-2P. Co-treatment with FGF-2 and Asc-2P induced optimal proliferation of BMSCs and increased the accumulation rate of BMSC numbers during a 2-month culture period. Moreover, differentiation potential was maintained by co-treatment with FGF-2 and Asc-2P via HGF expression. Adipogenic differentiation potential by FGF-2 and Asc-2P was dramatically suppressed by c-Met inhibitors (SU11274). These data suggest that co-treatment with FGF-2 and Asc-2P would be beneficial in obtaining BMSCs that possess "stemness" during long-term culture.

Therapeutic Effects of Mesenchymal Stem Cells for Patients with Chronic Liver Diseases: Systematic Review and Meta-analysis.

Based on their ability to differentiate into multiple cell types including hepatocytes, the transplantation of mesenchymal stem cells (MSCs) has been suggested as an effective therapy for chronic liver diseases. The aim of this study was to evaluate the safety, efficacy and therapeutic effects of MSCs in patients with chronic liver disease through a literature-based examination. We performed a systematic review (SR) and meta-analysis (MA) of the literature using the Ovid-MEDLINE, EMBASE and Cochrane Library databases (up to November 2014) to identify clinical studies in which patients with liver diseases were treated with MSC therapy. Of the 568 studies identified by the initial literature search, we analyzed 14 studies and 448 patients based on our selection criteria. None of the studies reported the occurrence of statistically significant adverse events, side effects or complications. The majority of the analyzed studies showed improvements in liver function, ascites and encephalopathy. In particular, an MA showed that MSC therapy improved the total bilirubin level, the serum albumin level and the Model for End-stage Liver Disease (MELD) score after MSC treatment. Based on these results, MSC transplantation is considered to be safe for the treatment of chronic liver disease. However, although MSCs are potential therapeutic agents that may improve liver function, in order to obtain meaningful insights into their clinical efficacy, further robust clinical studies must be conducted to evaluate the clinical outcomes, such as histological improvement, increased survival and reduced liver-related complications, in patients with chronic liver disease.

Mesenchymal Stem Cell-Mediated Effects of Tumor Support or Suppression.

Mesenchymal stem cells (MSCs) can exhibit a marked tropism towards site of tumors. Many studies have reported that tumor progression and metastasis increase by MSCs. In contrast, other studies have shown that MSCs suppress growth of tumors. MSCs contribute to tumor growth promotion by several mechanisms: (1) transition to tumor-associated fibroblasts; (2) suppression of immune response; (3) promotion of angiogenesis; (4) stimulation of epithelial-mesenchymal transition (EMT); (5) contribution to the tumor microenvironment; (6) inhibition of tumor cell apoptosis; and (7) promotion of tumor metastasis. In contrast to the tumor-promoting properties, MSCs inhibit tumor growth by increasing inflammatory infiltration, inhibiting angiogenesis, suppressing Wnt signaling and AKT signaling, and inducing cell cycle arrest and apoptosis. In this review, we will discuss potential mechanisms by which MSC mediates tumor support or suppression and then the possible tumor-specific therapeutic strategies using MSCs as delivery vehicles, based on their homing potential to tumors.

The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.

Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study.

BACKGROUND: In experimental models, bone marrow-derived mesenchymal stem cells (BM-MSCs) have the capacity to differentiate into hepatocytes and exhibit antifibrotic effects. However, there have been no studies in humans with alcoholic cirrhosis. AIM: The aim of this study was to elucidate the antifibrotic effect of BM-MSCs in patients with alcoholic cirrhosis, as a phase II clinical trial. METHODS: Twelve patients (11 males, 1 female) with baseline biopsy-proven alcoholic cirrhosis who had been alcohol free for at least 6 months were enrolled. BM-MSCs were isolated from each patient's BM and amplified for 1 month, and 5 × 10(7) cells were then injected twice, at weeks 4 and 8, through the hepatic artery. One patient was withdrawn because of ingestion of alcohol. Finally, 11 patients completed the follow-up biopsy and laboratory tests at 12 weeks after the second injection. The primary outcome was improvement in the patients' histological features. RESULTS: According to the Laennec fibrosis system, histological improvement was observed in 6 of 11 patients (54.5%). The Child-Pugh score improved in ten patients (90.9%) and the levels of transforming growth factor-ß1, type 1 collagen and α-smooth muscle actin significantly decreased (as assessed by real-time reverse transcriptase polymerase chain reaction) after BM-MSCs therapy (P < 0.05). No significant complications or side effects were observed during this study. CONCLUSIONS: Bone marrow-derived mesenchymal stem cells therapy in alcoholic cirrhosis induces a histological and quantitative improvement of hepatic fibrosis.

Generation of Fibrotic Liver Organoids Using Hepatocytes, Primary Liver Sinusoidal Endothelial Cells, Hepatic Stellate Cells, and Macrophages.

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.

Synthesis of substituted benzimidazolyl curcumin mimics and their anticancer activity.

A novel curcumin mimic library (14a-14h and 15a-15h) possessing variously substituted benzimidazole groups was synthesized through the aldol reaction of (E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one (7) or (E)-4-(3-hydroxy-4-methoxyphenyl)but-3-en-2-one (13) with diversely substituted benzimidazolyl-2-carbaldehyde (12a-12h). The MTT assay of the cancer cells MCF-7, SH-SY5Y, HEP-G2, and H460 showed that compound 14c with IC(50) of 1.0 and 1.9µM has a strong inhibitory effect on the growth of SH-SY5Y and Hep-G2 cells, respectively, and that compound 15h with IC(50) of 1.9µM has a strong inhibitory effect on the growth of MCF-7 cancer cells.

12-O-tetradecanoylphorbol-13-acetate Reduces Activation of Hepatic Stellate Cells by Inhibiting the Hippo Pathway Transcriptional Coactivator YAP.

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.