Hepatic Oval Cells Research Articles

Fk506 Inhibit Liver Regeneration in HOC Model Rat

Studies have shown that lymphocytes support hepatic oval cell (HOC)-dependent liver regeneration and FK506（Tacrolimus） is known as an immunosuppressor. Therefore, we studied the role of FK506 in HOC activation and/or proliferation to guide the clinical use of FK506. Thirty male Lewis rats were randomly divided into 4 groups: (A) intervene in activation (n=8), (B) intervene in proliferation (n=8), (C) control HOC model (n=8), and (D) pure partial hepatectomy (PH) (n=6). The HOC model was established by 2AAF(2-acetylaminofluorene)/PH in groups A to C. FK506 (at a dose of 1 mg/kg/d) was given subcutaneously in group A except on operation day, and not until day 8 post-operation (PO) in group B. Half of the animals were euthanized on days 10 and 14 PO, respectively. The remnant liver was weighed and stained by hematoxylin and eosin and immunohistochemical staining of proliferating cell nuclear antigen and epithelial cell adhesion molecule enabled HOC proliferation analysis. FK506 intervention exacerbated liver damage and hindered the recovery of the HOC model rat. Weight gain was severely retarded or even negative. Liver weight and the liver body weight ratio were lower than control group. HE and immunohistochemistry showed pooer proliferation of hepatocytes and fewer HOC numbers in group A. FK506 inhibited HOC activation by affecting T and NK cells, ultimately blocking liver regeneration. Poor liver regeneration after auxiliary liver transplantation might be associated with the inhibition of HOC activation and proliferation caused by FK506 treatment.

Expression and significance of histone methyltransferase SET domain containing 2 with histone H3 lysine 36 trimethylation in mouse hepatic oval cells differentiated into bile duct epithelial cells invitro.

The present study aimed to identify the function and expression of trimethylated protein histone H3 lysine 36 (H3K36)me3 and the upstream specific enzyme histone methyltransferase SET domain containing 2 (SETD2), during the differentiation of hepatic oval cells (HOCs) into cholangiocytes in mice following partial liver resection and fed with 2‑acetamidofluorene. HOCs were isolated from Kunming male mice fed with 2‑acetamidofluorene for 10days. Their liver tissues were then isolated following partial liver resection and another week of 2‑acetamidofluorene treatment. HOCs were collected following a two‑step enzyme digestion procedure involving protease E and collagenase 4. The target cells were cultured in DMEM/F12 supplemented with 10µg/ml EGF, 5µg/ml stem cell growth factor and 5µg/ml leukemia inhibitory factor. Target cells using the markers OV‑6, CK‑19, SETD2, H3K36me3, were detected with flow cytometry and immunofluorescence microscopy; reverse transcription‑quantitative PCR and western blotting were used to quantify the protein levels of SETD2 and H3K36me3. The retrieved primary hepatocytes developed into cholangiocytes with increasing CK‑19 and decreasing OV‑6 expression in each subsequent passage, whereas the SETD2 and H3K36me3 levels gradually increased, suggesting the possible involvement of both of these factors in differentiation.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

Kangxianruangan granule‑containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt‑1/β‑catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)-containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt-1/β-catenin signaling pathway. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB-F344 into HCCs. The overexpression plasmid, Wnt-1-up, was utilized to increase Wnt-1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG-treated WB-F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin-19 (CK-19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt-1 and β-catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription-quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt-1, β-catenin, Cyclin D1, C-myc, matrix metalloproteinase-7 (MMP-7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK-19 protein, and mRNA and protein expression levels of Wnt-1, β-catenin, Cyclin D1, C-myc, MMP-7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt-1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt-1/β-catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

Dkk1 inhibits malignant transformation induced by Bmi1 via the β-catenin signaling axis in WB-F344 oval cells.

Dickkopf‐1 (Dkk1) is an inhibitor of Wnt signaling involved in cancer cell proliferation, apoptosis, and migration and angiogenesis. It was previously reported that B cell‐specific Moloney mouse leukemia virus integration site 1 (Bmi1) activates the Wnt pathway by inhibiting the expression of DKK1 in breast cancer cell lines and 293T cells. Bmi1 and DKK1 are highly expressed in liver samples taken by biopsy from patients with hepatitis B virus‐related hepatocellular carcinoma (HCC), but the effect of both Bmi1 and DKK1 on the carcinogenesis of adult hepatic stem cells (oval cells) has not previously been reported. In this study, we used WB‐F344 cells to explore the function and regulation of Dkk1 upon Bmi1 treatment. Overexpression of Dkk1 repressed differentiation, proliferation, and migration induced by Bmi1 but promoted the apoptosis of hepatic WB‐F344 oval cells. In addition, Dkk1 reduced the enhancement of β‐catenin levels induced by Bmi1. Finally, we used transcriptome sequencing to perform a comprehensive evaluation of the transcriptome‐related changes in WB‐F344 oval cells induced by Dkk1 and Bmi1. These results may provide evidence for future studies of the pathogenesis of HCC and the design of possible therapies.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche

BackgroundAs a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated.MethodsTo generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit−CD29+CD49f+/lowCD45−Ter-119− cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs.ResultsFollowing liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies.ConclusionsOur findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Matrine promotes hepatic oval cells differentiation into hepatocytes and alleviates liver injury by suppression of Notch signalling pathway

AimsRecent studies have shown that the hyperactive Notch pathway is involved in cirrhosis and hepatocellular carcinoma (HCC) development by regulating differentiation of hepatic oval cells (HOCs) into cancer cells. The aim of this study was to investigate whether matrine can alleviate liver injury and promote HOC differentiation into hepatocytes by suppression of Notch pathway. Main methodsWe evaluated the expression of Notch-1, Jagged-1, and Hes-1 in HCC tissue by immunohistochemistry. Stem cell characteristics of HOCs were evaluated by CCK-8, cell cycle, and apoptosis. The expression of Notch pathway, HOC markers and albumin (ALB) was detected by immunohistochemistry, QRT-PCR and western blotting. The effects of matrine in protecting liver in vivo were investigated in a rat Solt-Farber precancerous model. Key findingsWe found an abnormal activated Notch pathway in HCC tissue, and the hyperactive Notch pathway was strongly associated with poor liver function in patients with cirrhosis with HCC. Using siNotch-1 to inhibit Notch pathway confirmed that Notch pathway could maintain stem cell characteristics of HOCs. Matrine inhibited stem cell characteristics of HOCs, the expression of Notch pathway and HOC markers but upregulated ALB. Matrine in combined with siNotch-1 RNA decreased the more potently inhibited HOC markers and Notch pathway. In rat Solt-Farber precancerous model, prophylactic application of matrine alleviated liver injury, downregulated Notch pathway and HOC markers, and upregulated ALB in a dose-dependent manner. SignificanceMatrine could promote the differentiation of HOCs into hepatocytes by inhibiting the Notch signalling pathway and alleviate liver injury.

Knockout of LATS1 induces neoplastic phenotype in hepatic oval cells.

BackgroundPrimary liver cancer (PLC) is the second leading cause of cancer-related death worldwide. It has been reported that PLC can be originated from malignant transformed adult hepatic progenitor cells. Mammalian large tumor suppressor kinase 1 (LATS1) is one of the core components of the Hippo pathway and it has been implicated in regulating invasion and metastasis of different cancer cell. However, the underlying connections between hepatic progenitor cells and LATS1 in the pathogenesis of PLC are still elusive.MethodsLATS1 gene knockout (LATS1-KO) hepatic oval cells (HOCs) were constructed by the CRISPR/Cas9 system. Cell viability was evaluated by the CCK-8 assay. Cell migration was measured by scrape assay. Cell invasion was examined by Transwell assay. Cell apoptosis was evaluated by flow cytometry. The expression of LATS1 and Yes-associated protein (YAP) in HOCs was determined by Q-PCR and Western blot analysis.ResultsHere, we found that knockout of LATS1 significantly induced the migration and invasion of WB-F344 cells. Knockdown of YAP suppressed the neoplastic phenotype of LATS1-KO WB-F344 cells. Furthermore, overexpression of YAP promoted the migration and invasion of LATS1-KO WB-F344 cells.ConclusionsIn summary, the current study demonstrated that LATS1 is required for inhibiting the neoplastic phenotype of normal hepatic progenitor cell via downregulating YAP.

The Expression of Yes-Associated Protein (YAP) Maintains Putative Cancer Stemness and Is Associated with Poor Prognosis in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is resistant to most chemotherapeutic agents. Yes-associated protein (YAP) is related to tumor progression; however, its role in ICC remains unknown. We investigated the mechanism underlying YAP-mediated cancer progression by focusing on the property of cancer stem cells (CSCs) in ICC. Immunohistochemistry results revealed the positive YAP expression in 37 of 52 resected ICC cases. Those with positive YAP expression showed poor prognosis in Kaplan-Meier analysis (P=0.023). YAP expression was associated with vimentin and the putative CSC marker, hepatic oval cell marker 6 (OV-6). The knockdown of YAP expression using specific siRNAs in ICC cells decreased octamer-binding transcription factor 4 (OCT4) expression in Western blot analyses and OV-6 and CD133 expression in flow cytometry analysis. Verteporfin, a YAP inhibitor, decreased N-cadherin and OCT4 expression in Western blot analyses. Invitro sphere formation and anoikis resistance assays revealed the impairment in CSC property and anoikis resistance in response to the decrease in YAP expression. Verteporfin treatment activated the protein kinase B/mechanistic target of rapamycin signaling pathway and dramatically impaired IL-6-stimulated STAT3 phosphorylation in ICC cells. The combination of verteporfin and rapamycin, an inhibitor of mechanistic target of rapamycin phosphorylation, inhibited cell proliferation and tumor growth. In conclusion, verteporfin regulates multiple signaling pathways and, in combination with rapamycin, might be a promising therapeutic strategy for ICC treatment.

Histological and Immuno-Histochemical Study on the Possible Effect of Vitamin K2 on the Hepatic Oval Cells in Diabetic and Non-Diabetic Albino Rats

Histological and Immuno-Histochemical Study on the Possible Effect of Vitamin K2 on the Hepatic Oval Cells in Diabetic and Non-Diabetic Albino Rats

Intrasplenic Transplantation of Cytotoxic T-Lymphocyte Associated Protein 4-Fas Ligand--Modified Hepatic Oval Cells for Acute Liver Injury in Rats

BackgroundIntrasplenic transplantation of xenogeneic hepatic oval cells (HOCs) may provide metabolic support for acute liver injury. However, xenoreactive lymphocyte-mediated immune response hinders HOCs' survival in the xeno-spleen parenchyma. Cytotoxic T-lymphocyte associated protein 4-Fas ligand (CTLA4.FasL), a fusion product integrating 2 inhibitory elements against lymphocytes into 1 molecule, effectively inhibited the proliferation of allogeneic and autoimmune lymphocytes. The purpose of this study was to explore the effect of CTLA4.FasL on the proliferation of xenoreactive lymphocytes and evaluate the therapeutic efficacy of CTLA4.FasL-modified HOC transplantation on acute liver injury in rats. MethodsThe effect of CTLA4.FasL-modified mouse liver epithelial progenitor cells (CTLA4.FasL-LEPCs) on the proliferation of rat lymphocytes in xeno-mixed lymphocyte reaction was investigated. Furthermore, CTLA4.FasL-LEPCs were intrasplenically transplanted in carbon tetrachloride- and partial hepatectomy-treated rats, and the therapeutic effect was evaluated using hematoxylin and eosin staining and alanine aminotransferase and aspartate aminotransferase assays. The hepatocytic differentiation of CTLA4.FasL-LEPCs in xenogeneic spleen was monitored by immunohistochemical staining for albumin. ResultsIn xeno-mixed lymphocyte reaction, CTLA4.FasL-LEPCs substantially inhibited the rat lymphocytes proliferation. CTLA4.FasL-LEPC transplantation significantly ameliorated liver injury compared with mCherry-modified LEPC and LEPC transplantation, as assessed by hematoxylin and eosin staining, alanine aminotransferase, and aspartate aminotransferase assays. Albumin positive cells appeared only in CTLA4.FasL-LEPCs group, but not in the mCherry-modified LEPCs group and LEPCs group. ConclusionsOur results indicate CTLA4.FasL-LEPCs substantially improved liver function and structure in carbon tetrachloride- and partial hepatectomy-induced acute liver injury rats through long-term hepatocytic differentiation.

HISTOLOGICAL AND IMMUNO-HISTOCHEMICAL STUDY ON THE POSSIBLE EFFECT OF VITAMIN K2 ON THE HEPATIC OVAL CELLS IN DIABETIC AND NON-DIABETIC ALBINO RATS

Background: Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from hypoinsulinemia, defective insulin action, or both. s-cells regeneration from stem cells or pancreatic progenitor cells is an attractive method for diabetic treatment. Hepatic oval cells (HOCs), intra-hepatic cells of bi-potent progenitor capabilities, can proliferate and differentiate into both hepatocytes and bile duct epithelial cells. They had the potentiality to transdifferentiate into pancreatic endocrine cells, as both liver and pancreas have the same origin (upper primitive foregut endoderm). Vitamin K2 can promote recovery in patients with liver cirrhosis and prevent insulin resistance reducing the risk of type 2 diabetes.Aim of the work: Evaluating the histological and immunohistochemical changes in hepatocytes and oval cells in diabetic and non-diabetic rats’ liver under treatment with vitamin K2.Materials and methods: 88 adult male albino rats were divided into 3 groups: Group I (control group), Group II (diabetic group); where diabetes was induced using single intraperitoneal (IP) dose of streptozotocin (STZ) following 2 weeks of high fat diet and Group III (treatment group) where diabetic rats were given daily oral vitamin K2 for 20 days (5 days on and 5 days off). Random blood sugar was measured for each group at day 0, 16 and then every 5 days till the end of the experiment (36 days). All groups’ sections were subjected to H&E and immunohistochemical stains for CD34, CK19 and insulin. Optical density for CD34 positive immunoreaction and mean area percent for CK19 and insulin immunoreaction were measured and data were statistically analyzed. Results and Conclusion: HOCs gave a positive reaction of changing into insulin producing cells in diabetic rats thus they may be modified to change into extra pancreatic source of insulin. Additionally, administration of vitamin K2 helped increase the proliferative ability of the HOCs in case of diabetes.

A histopathological study on adrenal cysts in ferrets.

Adrenal disorders are common in ferrets, but there are few studies on cystic lesions of the adrenal gland. The present study describes pathological and immunohistochemical features of adrenal cysts in eleven ferrets and discusses their histogenesis. In nine of eleven cases examined, which included seven, one, and one right, left, and bilateral cases, respectively, cysts were in the adrenal cortex and lined with epithelial cells. These epithelial cells contained an Alcian blue-negative/PAS-positive material and were positive for cytokeratin (CK) 7. The staining pattern was similar to that of biliary epithelial cells in the ferret. In five of the cases, there were small ducts adjacent to the cysts that were positive for CK7 and CK20 and negative for CK19. Based on the anatomical proximity between the right adrenal and liver, the immunohistochemical features of the small duct cells were comparable to those of hepatic oval cells. These results indicate the possibility that these adrenocortical cysts in the ferret originated from the biliary system. In the other two cases, the cysts lacked an epithelial cell lining, and there were dilated lymphoid vessels around the cysts. These cysts were assumed to have developed in the adrenal medulla, because the cyst wall was positive for glial fibrillary acidic protein and there were adrenal medullary cells positive for synaptophysin in the cyst wall. Therefore, the medullary cysts may have been associated with dilated vasculatures.

Pharmacological Mobilization of Endogenous Bone Marrow Stem Cells Promotes Liver Regeneration after Extensive Liver Resection in Rats

Rapid regeneration of the remnant liver is critical for preventing liver failure and promoting recovery after extensive liver resection. Numerous studies have demonstrated the involvement of bone marrow-derived stem cells in liver regeneration and the potential benefits of bone marrow stem cell therapy. To avoid the preparation of stem cells, we proposed in this study to mobilize endogenous bone marrow stem cells pharmacologically with a combination of AMD3100 (A), an antagonist of CXCR4 and low-dose FK506 (F). Here we show that AF combination therapy significantly increased lineage negative (Lin-) CD34+ and Lin-CD133+ stem cells in peripheral blood and enhanced recruitment of CD133+ cells into the remnant liver in a rat model of 85% partial hepatectomy. Recruiting CD133+ stem cells in the remnant liver was associated with increased proliferation of hepatic oval cells and paralleled the increased SDF-1, CXCR4 and HGF expression. Importantly, AF combination therapy increased the number of Ki67 positive hepatocytes and BrdU incorporation in the remnant liver and improved serum levels of albumin. Our results demonstrate that pharmacological mobilization of endogenous bone marrow stem cells with AF combination therapy can enhance endogenous stem cell mobilization to promote liver regeneration and improve liver function after extensive hepatectomy.

Embedded Disposable Functionalized Electrochemical Biosensor with a 3D-Printed Flow Cell for Detection of Hepatic Oval Cells (HOCs).

Hepatic oval cells (HOCs) are considered the progeny of the intrahepatic stem cells that are found in a small population in the liver after hepatocyte proliferation is inhibited. Due to their small number, isolation and capture of these cells constitute a challenging task for immunosensor technology. This work describes the development of a 3D-printed continuous flow system and exploits disposable screen-printed electrodes for the rapid detection of HOCs that over-express the OV6 marker on their membrane. Multiwall carbon nanotube (MWCNT) electrodes have a chitosan film that serves as a scaffold for the immobilization of oval cell marker antibodies (anti-OV6-Ab), which enhance the sensitivity of the biomarker and makes the designed sensor specific for oval cells. The developed sensor can be easily embedded into the 3D-printed flow cell to allow cells to be exposed continuously to the functionalized surface. The continuous flow is intended to increase capture of most of the target cells in the specimen. Contact angle measurements were performed to characterize the nature and quality of the modified sensor surface, and electrochemical measurements (cyclic voltammetry (CV) and square wave voltammetry (SWV)) were performed to confirm the efficiency and selectivity of the fabricated sensor to detect HOCs. The proposed method is valuable for capturing rare cells and could provide an effective tool for cancer diagnosis and detection.

Histology, immunohistochemistry, and in situ hybridization reveal overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model

Survivors of Ebola virus infection may become subclinically infected, but whether animal models recapitulate this complication is unclear. Using histology in combination with immunohistochemistry and in situ hybridization in a retrospective review of a guinea pig confirmation-of-virulence study, we demonstrate for the first time Ebola virus infection in hepatic oval cells, the endocardium and stroma of the atrioventricular valves and chordae tendinae, satellite cells of peripheral ganglia, neurofibroblasts and Schwann cells of peripheral nerves and ganglia, smooth muscle cells of the uterine myometrium and vaginal wall, acini of the parotid salivary glands, thyroid follicular cells, adrenal medullary cells, pancreatic islet cells, endometrial glandular and surface epithelium, and the epithelium of the vagina, penis and, prepuce. These findings indicate that standard animal models for Ebola virus disease are not as well-described as previously thought and may serve as a stepping stone for future identification of potential sites of virus persistence.

Autophagy regulates proliferation and biliary differentiation of hepatic oval cells via the MAPK/ERK signaling pathway.

Hepatic oval cells (HOCs) are thought to possess self‑renewal ability and a bipotential capacity for differentiation, which allows them to differentiate into hepatocytes and cholangiocytes. Autophagy serves an important role in self‑renewal and differentiation of stem cells; however, how autophagy contributes to proliferation and differentiation of hepatic progenitor cells has yet to be elucidated. In the present study, autophagy was regulated by rapamycin (Rapa) and chloroquine (Chlo) administration. The results demonstrated that Chlo‑treated HOCs exhibited decreased autophagic activity alongside a decreased tendency to proliferate, as determined by Cell Counting Kit‑8. In addition, activation of autophagy by Rapa enhanced the biliary differentiation of HOCs. Furthermore, increased phosphorylated (p)‑extracellular signal‑regulated kinase (ERK)/p‑p38 expression was observed following the induction of autophagy, thus indicating that the mitogen‑activated protein kinase (MAPK)/ERK signaling pathway was activated by autophagy to exert effects on the stimulation of HOC proliferation and differentiation. In conclusion, the present study demonstrated that autophagy regulates proliferation and biliary differentiation of HOCs via the MAPK/ERK signaling pathway. These results suggest a role for autophagy in stimulating the proliferation and differentiation of HOCs.

Inhibitory effects of HNF4\u03b1 on migration/maltransformation of hepatic progenitors: HNF4\u03b1-overexpressing hepatic progenitors for liver repopulation

BackgroundAlthough they are expandable in vitro, hepatic progenitors are immature cells and share many immunomarkers with hepatocellular carcinoma, raising potential concerns regarding maltransformation after transplantation. This study investigated the effects of hepatic nuclear factor (HNF) 4α on the proliferation, migration, and maltransformation of hepatic progenitors and determined the feasibility of using these manipulated cells for transplantation.MethodsThe effects of HNF4α on rat hepatic progenitors (i.e. hepatic oval cells) were analyzed by HNF4α overexpression and HNF4α shRNA. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice injured by carbon chloride (CCl4) were then transplanted with control, HNF4α-overexpressing or HNF4α-suppressing hepatic oval cells. Finally, the engraftment of these cells in the recipient liver was analyzed.ResultsRat hepatic progenitors (i.e. hepatic oval cells) expressed HNF4α, although less than that in hepatocytes. When HNF4α was overexpressed in these cells, the proliferation and migration of hepatic oval cells were reduced; but when HNF4α was suppressed by shRNA, the proliferation and migration, and even anchorage-independent growth, of these cells were accelerated. RNA microarray and gene functional analysis revealed that suppressing HNF4α not only impaired many biosynthesis and metabolism pathways of hepatocytes but also increased pathways for cancer. When transplanted into CCl4-injured NOD/SCID mice, few HNF4α-suppressing hepatic oval cells localized into the liver, while control cells and HNF4α-overexpressing cells engrafted into the liver and differentiated into albumin-positive hepatocytes. Interestingly, the hepatocytes derived from HNF4α-overexpressing cells were less migrative and expressed less c-Myc than the cells derived from control cells.ConclusionHNF4α constrains proliferation, migration, and maltransformation of hepatic progenitors, and HNF4α-overexpressing hepatic progenitors serve as an optimal candidate for cell transplantation.

Exosomes from mesenchymal stem cells induce the conversion of hepatocytes into progenitor oval cells

BackgroundWe previously reported that mesenchymal stem cells (MSCs) possess therapeutic effects in a murine model of carbon tetrachloride-induced acute liver failure. In the study, we observed that the majority of repopulated hepatocytes were of recipient origin and were adjacent to transplanted MSCs; only a low percentage of repopulated hepatocytes were from transplanted MSCs. The findings indicate that MSCs guided the formation of new hepatocytes. Exosomes are important messengers for paracrine signaling delivery. The aim of this study is to investigate the paracrine effects, in particular, the effects of exosomes from MSCs, on hepatocytes.MethodsMature hepatocytes were isolated from murine liver by a two-step perfusion method with collagenase digestion. MSCs were obtained from murine bone marrow, and conditioned medium (CM) from MSC culture was then collected. Time-lapse imaging was used for observation of cell morphological change induced by CM on hepatocytes. In addition, expression of markers for hepatic progenitors including oval cells, intrahepatic stem cells, and hepatoblasts were analyzed.ResultsTreatment with the CM promoted the formation of small oval cells from hepatocytes; time-lapse imaging demonstrated the change from epithelial to oval cell morphology at the single hepatocyte level. Additionally, expression of EpCAM and OC2, markers of hepatic oval cells, was upregulated. Also, the number of EpCAMhigh cells was increased after CM treatment. The EpCAMhigh small oval cells possessed colony-formation ability; they also expressed cytokeratin 18 and were able to store glycogen upon induction of hepatic differentiation. Furthermore, exosomes from MSC-CM could induce the conversion of mature hepatocytes to EpCAMhigh small oval cells.ConclusionsIn summary, paracrine signaling through exosomes from MSCs induce the conversion of hepatocytes into hepatic oval cells, a mechanism of action which has not been reported regarding the therapeutic potentials of MSCs in liver regeneration. Exosomes from MSCs may therefore be used to treat liver diseases. Further studies are required for proof of concept of this approach.

Tg737 regulates epithelial–mesenchymal transition and cancer stem cell properties via a negative feedback circuit between Snail and HNF4α during liver stem cell malignant transformation

Determining the origin of liver cancer stem cells is important for treating hepatocellular carcinoma. Tg737 deficiency plays an important role in the malignant transformation of liver stem cells, but the underlying mechanism remains unclear. Here we established a chemical-induced mouse hepatoma model and found that Tg737 and hepatocyte nuclear factor 4-alpha (HNF4α) expression decreased and epithelial–mesenchymal transition (EMT)-related marker expression increased during liver cancer development. To investigate the underlying mechanism, we knocked down Tg737 in WB-F344 (WB) rat hepatic oval cells. Loss of Tg737 resulted in nuclear β-catenin accumulation and activation of the Wnt/β-catenin pathway, which further promoted EMT and the malignant phenotype. XAV939, a β-catenin inhibitor, attenuated WB cell malignant transformation due to Tg737 knockdown. To clarify the relationships of Tg737, the β-catenin pathway, and HNF4α, we inhibited Snail and overexpressed HNF4α after Tg737 knockdown in WB cells and found that Snail and HNF4α comprise a negative feedback circuit. Taken together, the results showed that Tg737 regulates a Wnt/β-catenin/Snail-HNF4α negative feedback circuit, thereby blocking EMT and the malignant transformation of liver stem cells to liver cancer stem cells.