Liver Injury Repair Research Articles

Regulation of hepatic differentiation of human embryonic stem cells by calcium silicate extracts for liver injury repairing

Directing embryonic stem cell (ESC)-derived hepatocytes is critical in understanding hepatic differentiation and applying cell-based treatment to severe liver diseases. While growth factor-based strategies are widely used, using chemical cues could present an alternative to optimize the strategies for stem cell differentiation. Here, for the first time, an inorganic calcium silicate (CS, CaSiO3)-based approach, together with a modified four-stage differentiating protocol, was proposed to quantify the effects of CS extracts on inducing hepatic differentiation of human ESCs (H9 cells). The roles of CS-activated H9 cells in liver injury repair were tested by cell tracking and immunohistochemical staining. Results indicated that high concentrations of CS extracts initially enhance definitive endodermal (DE) lineage, followed by gradual DE differentiation at low CS concentrations. The order of CS addition is also crucial, since the presence at stemness stage and the absence at DE stage could optimize hepatic differentiation capacity of H9 cells, resulting in optimized cells that differentiate into functional hepatocyte-like cells. The addition of CS extracts at precursor hepatocyte stage enhances their maturity, which favors the turnover of liver injury in CCl4-treated mice. These results provide an insight into applying bioactive inorganic biomaterials to foster hepatic differentiation of human ESCs for cell therapy.

Multidimensional imaging of liver injury repair in mice reveals fundamental role of the ductular reaction

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks were lost. The canalicular collapse was an incipient defect commonly associated with hepatocyte injury irrespective of cholestatic statuses, and could sufficiently provoke the ductular reaction when artificially induced. We propose a unifying model for the induction of the ductular reaction, where compensatory biliary epithelial tissue remodeling ensures bile-excreting network homeostasis.

Extraction of Silymarin Complex from Milk Thistle (Silybummarianaum) Seeds and Study its Effect on the Liver Functions and some Antioxidants in Rats

This study included extraction of silymarin complex from milk thistle seeds and study the effect of different concentrations of this complex on the liver functions and levels of some antioxidants in rats exposed to liver injury induced byethanol, then study the effect of these concentrations on the repair of liver injury induced by ethanol by evaluating the hepatic pathology of different animal groups. Sixty male rats with the age of 3-4 months were divided into 6 groups: Group (1) control which received drinking tap water, group (2) treated with 5% ethanol in drinking water, group (3) treated with 5% ethanol and 1 mg silymarin complex by oral dose once daily, group (4) treated with 5% ethanol and 2 mg silymarin complex by oral dose once daily, group (5) treated with 5% ethanol and 4 mg silymarin complex by oral dose once daily, group (6) treated with 5% ethanol and 8 mg silymarin complex by oral dose once daily, by the end of the experimental period of 3 weeks all animals groups were killed and collection of blood samples and liver tissues for the measuring of liver functions parameters and some antioxidants in blood, and processing the histological examination of liver. Treatment with ethanol revealed a significant prolongation of the coagulation time and a significant increase of Alanine transaminase (ALT) and Aspartate transaminase (AST) enzymes activity, total and direct bilirubin, malondialdehyde, peroxynitrite radical and xanthine oxidase activity and a significant decrease of albumin, Vit. C, Vit. E, glutathione and ceruloplasmin levels in serum comparing with the control group (P < 0.05). Different concentrations of silymarin complex have different effects on the protection from the liver injury induced by ethanol, and return these parameters to about normal ranges. Concentrations of 1 and 2 mg of silymarin complex can reduce the fatty changes induced by ethanol, while the concentrations of 4 and 8 mg led to reduce the necrosis and inflammation in addition to fatty changes.

Gas6/TAM Signaling Components as Novel Biomarkers of Liver Fibrosis.

Liver fibrosis consists in the accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells. This is commonly the result of chronic liver injury repair and represents an important health concern. As liver biopsy is burdened with many drawbacks, not surprisingly there is great interest to find new reliable noninvasive methods. Among the many are new potential fibrosis biomarkers under study, some of the most promising represented by the growth arrest-specific gene 6 (Gas6) serum protein and its family of tyrosine kinase receptors, namely, Tyro3, Axl, and MERTK (TAM). Gas6/TAM system (mainly, Axl and MERTK) has in fact recently emerged as an important player in the progression of liver fibrosis. This review is aimed at giving an overall perspective of the roles played by these molecules in major chronic liver diseases. The most promising findings up to date acknowledge that both Gas6 and its receptor serum levels (such as sAxl and, probably, sMERTK) have been shown to potentially allow for easy and accurate measurement of hepatic fibrosis progression, also providing indicative parameters of hepatic dysfunction. Although most of the current scientific evidence is still preliminary and there are no in vivo validation studies on large patient series, it still looks very promising to imagine a possible future prognostic role for these biomarkers in the multidimensional assessment of a liver patient. One may also speculate on a potential role for this system targeting (e.g., with small molecule inhibitors against Axl) as a therapeutic strategy for liver fibrosis management, always bearing in mind that any such therapeutic approach might face toxicity.

Preparation of high Fischer’s ratio corn oligopeptides using directed enzymatic hydrolysis combined with adsorption of aromatic amino acids for efficient liver injury repair

Directed hydrolysis of corn crude peptides was performed by sequential digestion with α-chymotrypsin and carboxypeptidase A. With the additional step of adsorption of aromatic amino acids by activated carbon, corn oligopeptides with a Fischer’s ratio of 41.87 and low bitterness were prepared. The fraction with molecular weight of 180–1000 Da was found to constitute 71.37% of these oligopeptides, where the proportion of Phe and Tyr among all amino acid residues was only 0.73%. In addition, a model of liver injury induced by carbon tetrachloride was established. Through the analysis of the body–mass index, liver index, liver histopathological features, and various serum biochemical indicators of liver function, high Fischer’s ratio corn oligopeptides were found to be safe and slightly superior in terms of liver injury repair when compared with a commercial high Fischer’s ratio amino acid product. Crude corn peptides (raw material) and skimmed milk powder had no effect on repairing liver injury.

Effect of pregnant rat adipose-derived stem cells on repair of acute liver injury

To assess the effect of pregnant rat adipose-derived stem cells (ADSCs) on repair of acute liver injury. ADSCs were isolated from 18-week pregnant Sprague Dawley rats and were identified by flow cytometry. Twenty Sprague Dawley rats were randomly divided into groups A, B, C, and D ( n=5); rats in group A were not treated as normal controls; rats in groups B, C, and D were injected intraperitoneally with CCl 4 to establish the acute liver injury model. At 2 hours after modeling, DPBS, 0.1 mL normal rat ADSCs (2×10 6cells/mL), and pregnant rat ADSCs (2×10 6cells/mL) were injected into the spleen in groups A, C, and D respectively; rats in group B was not treated. After 7 days, total bilirubin (TBIL), alanine aminotransferase (ALT), aspartic acid transaminase (AST), albumin (ALB), and total protein (TP) in serum were measured. The liver tissue sections were stained with HE. The expressions of Ki67, alpha-fetoprotein (AFP), and ALB were measured by immunohistochemistry. The serum levels of TBIL, ALT, and AST in group B were significantly higher than those in groups A, C, and D ( P<0.05), but ALB and TP were significantly lower than those in groups A, C, and D ( P<0.05). The levels of TBIL, ALT, and AST were significantly higher in groups C and D than group A, and in group C than group D ( P<0.05). There was no significant difference in serum levels of ALB among groups A, C, and D ( P>0.05). The serum level of TP in groups C and D was significantly lower than that in group A ( P<0.05), but no significant difference was found between group C and group D ( P>0.05). HE staining showed that the liver tissue of group A had clear structure; the cells arranged neatly with uniform size. The hepatocytes in group B showed obvious edema, disorderly arrangement, dot necrosis in liver lobules, and diffuse infiltration of inflammatory cells. In groups C and D, the inflammation and hepatocellular necrosis were obviously reduced when compared with group B, and the number of vacuoles caused by dilation of mitochondria and rough endoplasmic reticulum was decreased; especially in group D, improvement of liver injury was more effective. The Ki67 positive cell rate was significantly higher in groups C and D than groups A and B ( P<0.05), in group B than group A ( P<0.05), and in group D than group C ( P<0.05). There was no expression of AFP in groups A and B, but positive expression was observed in groups C and D, and AFP positive cell rate of group D was significantly higher than that of group C ( t=3.006, P=0.017). ALB expression was significantly higher in groups C and D than groups A and B ( P<0.05), and in group D than group C ( P<0.05). Pregnant rat ADSCs could promote repair of liver injury induced by CCl 4.

Lipopolysaccharide promotes tumorigenicity of hepatic progenitor cells by promoting proliferation and blocking normal differentiation

Hepatic progenitor cells (HPCs) are bipotential stem cells that can differentiate into mature hepatocytes or biliary epithelial cells (BECs). They are thought to be involved in repair of liver injury and the incidence of hepatic carcinoma. Their physiology is closely associated with the microenvironment where they reside. Lipopolysaccharide (LPS), an important component of the hepatic pathological microenvironment, is stored in the liver and affects many types of cells in various hepatosis. HPCs may also be influenced by LPS. In this paper, mouse ED13.5 E-cadherin+ foetal liver cells were isolated as mouse hepatic progenitor cells (mHPCs). Proliferation of mHPCs was promoted under LPS conditions both in vivo and in vitro. Moreover, LPS enhanced colony formation ability of mHPCs, and blocked them differentiation into mature hepatocytes and formation of a bile duct-liked structure. More importantly, long-term treatment with LPS promoted tumorigenesis of mHPCs in nude mice. Thus, we conclude that LPS may promote aberrant proliferation of mHPCs and restrict their normal differentiation. Long-term exposure of mHPCs to LPS increased the risk of tumour formation. These data provide insight into the links between LPS, HPCs fate, and tumorigenesis, and present novel insight into the relationship between HPCs and their microenvironment.

Manipulating senescence in health and disease: emerging tools

Manipulating senescence in health and disease: emerging tools

Liver-inherent immune system: its role in blood-stage malaria.

The liver is well known as that organ which is obligately required for the intrahepatocyte development of the pre-erythrocytic stages of the malaria-causative agent Plasmodium. However, largely neglected is the fact that the liver is also a central player of the host defense against the morbidity- and mortality-causing blood stages of the malaria parasites. Indeed, the liver is equipped with a unique immune system that acts locally, however, with systemic impact. Its main “antipodal” functions are to recognize and to generate effective immunoreactivity against pathogens on the one hand, and to generate tolerance to avoid immunoreactivity with “self” and harmless substances as dietary compounds on the other hand. This review provides an introductory survey of the liver-inherent immune system: its pathogen recognition receptors including Toll-like receptors (TLRs) and its major cell constituents with their different facilities to fight and eliminate pathogens. Then, evidence is presented that the liver is also an essential organ to overcome blood-stage malaria. Finally, we discuss effector responses of the liver-inherent immune system directed against blood-stage malaria: activation of TLRs, acute phase response, phagocytic activity, cytokine-mediated pro- and anti-inflammatory responses, generation of “protective” autoimmunity by extrathymic T cells and B-1 cells, and T cell-mediated repair of liver injuries mainly produced by malaria-induced overreactions of the liver-inherent immune system.

Chronic restraint stress decreases the repair potential from mesenchymal stem cells on liver injury by inhibiting TGF-β1 generation.

Chronic psychological stress has been demonstrated to play an important role in several severe diseases, but whether it affects disease therapy or not remains unclear. Mesenchymal stem cells (MSCs) have been demonstrated to have therapeutic potentials in treating tissue injury based on their multidifferentiation potential toward various cell types. We investigated the effect of chronic restraint stress on therapeutic potential of MSCs on carbon tetrachloride (CCl4)-induced liver injury in mice. CCl4-induced mice were injected with enhanced green fluorescent protein–MSCs, which was followed by chronic restraint stress administration. Corticosterone and RU486, a glucocorticoid receptor (GR) antagonist, were employed in vivo and in vitro, too. In the present study, we illustrated that MSCs could repair liver injury by differentiating into myofibroblasts (MFs) which contribute to fibrosis, whereas stress repressed differentiation of MSCs into MFs displayed by reducing α-smooth muscle actin (α-SMA, a solid marker of MFs) expression. Whereas RU486 could maintain the liver injury reduction and liver fibrosis increases induced by MSCs in stressed mice and block the decrease of α-SMA expression induced by stress. Furthermore, chronic stress inhibited MFs differentiation from MSCs by inhibiting transforming growth factor-β1 (TGF-β1)/Smads signaling pathway which is essential for MFs differentiation. Chronic stress reduced autocrine TGF-β1 of MSCs, but not blunted activation of Smads. All these data suggested that corticosterone triggered by chronic stress impaired liver injury repair by MSCs through inhibiting TGF-β1 expression which results in reduced MFs differentiation of MSCs.

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

Antifibrotic effects of CCN1/CYR61 in primary portal myofibroblasts through induction of reactive oxygen species and resulting apoptosis

Cysteine-rich protein 61 (CCN1/CYR61) is a matricellular protein of the CCN family that comprises six secreted CCN proteins in mammals [1, 2]. In adults, CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies showed that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transform into an extracellular matrix-degrading phenotype [3]. We here investigated CCN1/CYR61 expression in primary profibrogenic liver cells (i.e. hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels in primary parenchymal liver cells (i.e. hepatocytes) were only marginal compared to that found in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly found during early periods of insult, while the expression declined during prolonged phases of fibrogenesis. We further generated an adenovirus type 5 encoding CCN1/CYR61 (i.e. Ad5-CMV-CCN1/CYR61) and transduced it into primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 inhibits production of collagen type I at both mRNA and protein levels in these cells as evidenced by quantitative real time PCR, Western blot and immunocytochemistry. Interestingly, CCN1/CYR61 further induces production or reactive oxygen species (ROS) leading to apoptosis as shown by ROS staining and TUNEL assay, while β-galactosidase staining of senescence phenotype showed no difference from respective controls. Therefore, we conclude that in line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in the process of liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells therefore might be beneficial in liver fibrotic therapy.

Role of STAT3 signaling pathway in repair of liver injury

Role of STAT3 signaling pathway in repair of liver injury

Microarray Data Reveal Relationship between Jag1 and Ddr1 in Mouse Liver

Alagille syndrome is an autosomal dominant disorder involving bile duct paucity and cholestasis in addition to cardiac, skeletal, ophthalmologic, renal and vascular manifestations. Mutations in JAG1, encoding a ligand in the Notch signaling pathway, are found in 95% of patients meeting clinical criteria for Alagille syndrome. In order to define the role of Jag1 in the bile duct developmental abnormalities seen in ALGS, we previously created a Jag1 conditional knockout mouse model. Mice heterozygous for the Jag1 conditional and null alleles demonstrate abnormalities in postnatal bile duct growth and remodeling, with portal expansion and increased numbers of malformed bile ducts. In this study we report the results of microarray analysis and identify genes and pathways differentially expressed in the Jag1 conditional/null livers as compared with littermate controls. In the initial microarray analysis, we found that many of the genes up-regulated in the Jag1 conditional/null mutant livers were related to extracellular matrix (ECM) interactions, cell adhesion and cell migration. One of the most highly up-regulated genes was Ddr1, encoding a receptor tyrosine kinase (RTK) belonging to a large RTK family. We have found extensive co-localization of Jag1 and Ddr1 in bile ducts and blood vessels in postnatal liver. In addition, co-immunoprecipitation data provide evidence for a novel protein interaction between Jag1 and Ddr1. Further studies will be required to define the nature of this interaction and its functional consequences, which may have significant implications for bile duct remodeling and repair of liver injury.

Liver renewal: detecting misrepair and optimizing regeneration.

Cirrhosis and liver cancer, the main causes of liver-related morbidity and mortality, result from defective repair of liver injury. This article summarizes rapidly evolving knowledge about liver myofibroblasts and progenitors, the 2 key cell types that interact to orchestrate effective repair, because deregulation of these cells is likely to be central to the pathogenesis of both cirrhosis and liver cancer. We focus on cirrhosis pathogenesis because cirrhosis is the main risk factor for primary liver cancer. Emerging evidence suggests that the defective repair process has certain characteristics that might be exploited for biomarker development. Recent findings in preclinical models also indicate that the newly identified cellular and molecular targets are amenable to therapeutic manipulation. Thus, recent advances in our understanding about key cell types and fundamental mechanisms that regulate liver regeneration have opened new avenues to improve the outcomes of liver injury. clinicaltrials.gov Identifier: NCT01899859.

Alcohol Exposure Impairs Human Liver Stem Cell Self-Renewal and Differentiation

The proliferative capacity of liver parenchymal cells, particularly hepatocytes is restricted in diverse pathological conditions, including exposure to hepatotoxic agents and infection with hepatitis viruses. Activation of liver stem/progenitor cells (LSPCs) may consequently play an important role in the repair of liver injury. However, little is known about LSPC function in liver injury repair or hepatic regeneration after resection of diseased liver tissue. In particular, alcohol abuse injures the liver, causing hepatitis, liver steatosis, fibrosis, and cirrhosis.

Role of stem cells in repair of liver injury: Experimental and clinical benefit of transferred stem cells on liver failure

Although the liver has a high regenerative capacity, as a result of massive hepatocyte death, liver failure occurs. In addition to liver failure, for acute, chronic and hereditary diseases of the liver, cell transplantation therapies can stimulate regeneration or at least ensure sufficient function until liver transplantation can be performed. The lack of donor organs and the risks of rejection have prompted extensive experimental and clinical research in the field of cellular transplantation. Transplantation of cell lineages involved in liver regeneration, including mature hepatocytes, fetal hepatocytes, fetal liver progenitor cells, fetal stem cells, hepatic progenitor cells, hepatic stem cells, mesenchymal stem cells, hematopoietic stem cells, and peripheral blood and umbilical cord blood stem cells, have been found to be beneficial in the treatment of liver failure. In this article, the results of experimental and clinical cell transplantation trials for liver failure are reviewed, with an emphasis on regeneration.

Mechanism and Efficacy of Mobilization of Granulocyte Colony-Stimulating Factor in the Treatment of Chronic Hepatic Failure

To explore the efficacy of G-CSF mobilization in the treatment of chronic liver failure (CLF) and the mechanism of its action. The proportions of cluster-of-differentiation (CD)-34+ cells and their receptor-CXCR4 were detected by flow cytometry in patients with different types of chronic HBV infection. The levels of chemokines and cytokines were measured by enzyme-linked immunosorbent assay. The proportion of CD34+ cells in patients with cirrhosis was significantly increased compared with the healthy controls (p<0.05) and was increased obviously after treatment by G-CSF mobilization (p<0.01). The expression levels of SDF-1, SCF and MMP-9 were significantly elevated in patients with chronic hepatitis B and liver cirrhosis (p<0.01). The expression levels of SCF and MMP-9 were significantly elevated after treatment with G-CSF (p<0.05). No significant differences were found in the levels of total bilirubin, albumin and prothrombin time between the treated and control groups; furthermore, no significant differences were observed in the cure and improvement rates between the two groups. The basal levels of stem cell mobilization in patients with liver cirrhosis might be associated with the repair of liver injury. G-CSF could promote hematopoietic stem cell mobilization through regulation of the expression levels of stem-cell-mobilization-related factors in patients with liver cirrhosis. No apparent effects of G-CSF therapy on both liver function and short-term prognosis in patients with liver cirrhosis were confirmed.

Expression and Function of Fibroblast Growth Factor (FGF) 7 during Liver Regeneration

Background/Aim: Previous studies have shown that fibroblast growth factors (FGFs) are involved in the process of liver injury repair. Liver regeneration after partial hepatectomy (PH) is impaired in transgenic mice expressing dominant-negative FGFR2b in hepatocytes. Although FGF7, a ligand specifically bound to FGFR2b, is expressed by activated hepatic stellate cells (HSCs) in fibrotic livers, the expressions and functions of FGF7 and FGFR2b after PH remain unexplored. Therefore, this study sought to examine the potential role of FGF7 signaling during liver regeneration. Methods: We examined the expression of FGF7 and FGFR2b in normal and regenerating livers. Effects of FGF7 on hepatocytes were examined in vitro using primary hepatocyte culture with FGF7 recombinant protein and in vivo by hydrodynamic-based gene transfer method. Results: We found that FGF7 expression was increased according to the activation status of HSCs after PH. The receptor, FGFR2b, was also increased in hepatocytes during liver regeneration. In vitro treatment with FGF7 protein activated ERK1/2 and promoted proliferation of hepatocytes isolated from regenerating livers. In vivo overexpression of exogenous FGF7 could notably promote hepatic proliferation and activate MAPKs after PH. Conclusion: This study suggests a role for activated HSC-expressed FGF7 in stimulating FGF signaling pathways in hepatocytes and regulating liver regeneration.

Hepatocytic precursor (stem-like) WB-F344 cells reduce tumorigenicity of hepatoma CBRH-7919 cells via TGF-β/Smad pathway

Hepatic stem cells (HSCs) are involved in repair of liver injury. Stem cells may have inhibitory effects on tumor cell growth and apoptosis. However, it is unknown whether HSCs regulate the biological functions of hepatocarcinoma cells, especially tumor cell growth and apoptosis. The present study was designed to determine the effects of hepatocytic precursor (stem-like) WB-F344 cells on the growth and apoptosis of hepatoma CBRH-7919 cells. Using a Transwell chamber culture system, we co-cultured WB-F344 cells and CBRH-7919 cells in serum-free conditioned medium at 3 different ratios: 1:1 (2 x 10(5): 2 x 10(5) cells/well), 1:5 (4 x 10(4): 2 x 10(5) cells/well), and 5:1 (2 x 10(5): 4 x 10(4) cells/well). We determined the effects of stem cells on tumor cells using in vivo xenograft assay in nude mice and determining gene expression by RT-PCR and Western blot analyses. With the increment proportion of the WB-F344 cells in the co-culture system, tumor formation was inhibited in nude mice. Moreover, down-regulation of bone morphogenetic protein 4 (BMP4), Bcl-2, and c-Myc and upregulation of PTEN also occurred along with the inhibitory effects. Western blotting showed that the TGF-beta/Smad pathway played a prominent role in tumor inhibition, which may have been mediated by the cytokines released from the stem cells. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibit the tumorigenicity of hepatoma CBRH-7919 cells, and the effect is mediated by TGF-beta/Smad signaling pathway.