Normal Adult Liver Research Articles

Efficient generation of biliary epithelial cells from rabbit intrahepatic bile duct by Y-27632 and Matrigel

Efficient culture of primary biliary epithelial cells (BECs) from adult liver is useful for both experimental studies and clinical applications of tissue engineering. However, an effective culture system for long-term proliferation of adult BECs is still unachieved. Laboratory rabbit has been used in a large number of studies; however, there are no reports of BECs from normal adult rabbit. As little as 5 g of normal rabbit liver tissue were minced, digested, and then clonally cultured in medium containing FBS and ITS. Cells were characterized by cell morphology, immunoassaying, and growth rate assay. Different combination of growth factors and substrates, including Y-27632 and Matrigel, were employed to assess their effect on cell proliferation. In the primary culture, the BECs cellular sheets consisting of cuboidal cells, as well as fibroblast-like cells and other hepatic cells, emerged with time of culture. The BECs cellular sheets were then manually split into cells clumps for further characterization. The subcultured cells had typical cell morphology of cholangiocytes, expressed the specific markers of BECs, including GGT, cytokeratin (CK18), and CK19, and possessed the capacity to form duct-like structure in three-dimensional Matrigel. Y-27632 and Matrigel-treated BECs had a steady growth rate as well as colony-formation capacity. The BECs were maintained in Y-27632 and Matrigel culture system for more than 3 mo. This is the first example, to our knowledge, of the successful culture of BECs from normal adult rabbit liver. Furthermore, our results indicate that treatment of BECs with Y-27632 and Matrigel is a simple method for efficient output of BECs.

Aberrant upregulation of LRRC1 contributes to human hepatocellular carcinoma

Loss of apico-basal polarity often results in a malignant phenotype in epithelial tissues. Aberrant expression of polarity mediator proteins is closely associated with this process. LRRC1/LANO, a putative cell polarity regulator, was previously screened from our gene expression profiling in which its expression was significantly upregulated in hepatocellular carcinoma (HCC). In the present study, we provide evidences that LRRC1 plays a potential oncogenic function in HCC. Consistent with the microarray data, quantitative real-time PCR results showed LRRC1 was aberrantly upregulated in 37/56 (66.1 %, more than twofolds) of HCC specimens compared with adjacent non-cancerous livers. Furthermore, the cellular expression of LRRC1 in all HCC cell lines examined exhibited much higher level than that in normal adult liver tissue. Functional analyses revealed that overexpression of LRRC1 promoted, while knockdown of LRRC1 by RNA interference inhibited the growth and colony formation of HCC cells. Importantly, enhanced expression of LRRC1 conferred NIH3T3 cells the ability of cell transformation. In a xenograft tumor model, we found LRRC1 overexpression increased the tumorigenicity of HCC cells. Thus, our collective findings suggest that LRRC1 contributes to HCC development, and may be a potential target for therapeutic intervention in this disease.

Abstract 4200: Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells.

Abstract The microRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate gene expression by degrading mRNA and by inhibiting translation of protein. Numerous studies have demonstrated the critical roles of miRNA in physiological and pathological procedures, including cell proliferation, differentiation and apoptosis, and in carcinogenesis. Recent studies have particularly reported that miR-100 is aberrantly expressed in multiple cancers, including colorectal, lung, prostate, ovarian, thyroid, pancreatic, liver, breast, cervical and nasopharyngeal cancers. It has also been reported that reduction of miRNA-100 is associated with poor prognosis. But the effects of miRNA-100 on human hepatocellular carcinoma cells and its potential mechanisms are largely unknown. In the present study, we transfected miRNA-100 analogue into human hepatocellular carcinoma cell line HepG2 cell, employed quantitative RT-PCR, immunoblotting, immunofluorescence, flow cytometry and other approaches, and found that the expression of miRNA-100 was markedly lower but polo-like kinase 1 (Plk1) was higher in the HCC cell line and fetal liver, compared to those in the normal hepatocytes and adult liver, respectively. Moreover, increasing miRNA-100 expression in HepG2 cells inhibited cell proliferation, caused cell cycle arrest and promoted apoptosis. Mechanistic and correlation studies revealed that the anti-cancer effects of miRNA-100 were associated with downregulation of Plk1 expression at mRNA and protein levels. Factually, bioinformatic analysis has predicted that Plk1 could be a target of miRNA-100. In conclusion, our results have shown that miRNA-100 has a tumor suppressor function in human hepatocellular carcinoma cells, which may be associated with downregulation of Plk1, and implicate the potential application of miR-100 in connection with Plk1 for targeted gene therapy of hepatocellular carcinoma. Citation Format: Wuling Zhu, Binglin Fan, Hongge zhang, Xiaohui Ji, Wancai Yang, Wancai Yang. Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4200. doi:10.1158/1538-7445.AM2013-4200

Def Functions as a Cell Autonomous Factor in Organogenesis of Digestive Organs in Zebrafish

Digestive organs originate from the endoderm. Morphogenesis of the digestive system is precisely controlled by multiple factors that dictate the cell fate and behavior so that the specific digestive organs are timely formed in the right place and develop into right size and structure. We showed previously that digestive organ expansion factor (def) is a gene whose expression is enriched in the liver, pancreas and intestine. Loss-of-function of def in the defhi429 mutant confers hypoplastic digestive organs partly due to alteration of expression of genes related to the p53 pathway. However, the molecular mechanism for the involvement of Def in the organogenesis of digestive organs is still largely unknown. For example, it is not known whether Def regulates specific pathways in a specific organ. To address this question, we generated four independent Tg(fabp10a:def) transgenic fish lines which over-expressed Def specifically in the liver. We characterized Tg-I, one of the transgenic lines, in detail with genetic, molecular and histological approaches. We found that Tg-I restored the liver but not exocrine pancreas and intestine development in the defhi429 mutant. However, Tg-I adult fish in the wild type (WT) background exhibits reduced liver-to-body ratio and all four transgenic lines conferred abnormal intrahepatic structure. Microarray data analysis showed that certain specific functional pathways were affected in the liver of Tg-I. These results demonstrate that Def functions in a cell autonomous manner during early liver development and aberrant Def protein expression might lead to disruption of the structural integrity of a normal adult liver.

315 FoxO3 REGULATES HEPATIC GLUCOSE PRODUCTION

regeneration is insufficient. We here further investigated the expression pattern of the novel oval cell marker Neighbor of Punc E 11 (Nope) in liver regeneration after chronic (Mdr2−/− mice) and/or acute liver injury (partial hepatectomy) in adult mice. Methods: For analysis of chronic liver injury, liver tissue was extracted from Mdr2−/− of different age and stage of fibrosis. Additionally, Mdr2−/− and Balb/c mice were analyzed for the impact of acute liver injury with or without pretreatment with DNAalkylating reagents to block hepatocytic proliferation. 24 hours to 6 months postoperatively, mice were sacrificed and the liver tissue was tested for expression levels of Nope via quantitative RT-PCR. Costainings were performed for Nope in combination with epithelial-specific marker E-cadherin, the biliary marker CK19 and markers of hepatic stem/progenitor cells (A6, EpCAM). Results: While normal adult liver tissue shows only negligible expression of Nope, chronic liver injury in Mdr2−/− mice leads to a considerably increased expression level of Nope. Co-stainings with CK19 demonstrated a bile-duct-specific expression of Nope in these mice. While partial hepatectomy in the normal adult liver has no effect on Nope, it leads to the development of Nope-positive hepatocytic cell clusters in the chronically injured Mdr2−/− mouse model especially if hepatocyte proliferation is blocked. These Nope-positive clusters are negative for CK19 but stain positive for E-cadherin. Oval cell markers A6 and EpCAM both show coexpression with Nope in proliferating ductular cells, but only a minor hepatocytic cell fraction within Nope-positive clusters also stains positive for A6. Conclusion: We here report the expression of the novel oval cell marker Nope in liver regeneration after chronic and/or acute liver injury. The increase of Nope in chronic liver injury is mainly limited to bile ducts, but especially after additional acute liver injury and blocked hepatocytic regeneration a small population of Nopepositive hepatocytic progenitor cells arises. We conclude that Nope detects a novel progenitor cell population within the regenerating adult liver.

Preexisting epithelial diversity in normal human livers: A tissue-tethered cytometric analysis in portal/periportal epithelial cells

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here.

Betaine homocysteine methyltransferase (BHMT)-dependent remethylation pathway in human healthy and tumoral liver

Carcinogenesis is a multi-step and multifactorial process. It includes genetic, epigenetic, nutritional and environmental factors, which are closely interconnected. Human hepatocellular carcinoma (HCC) is among the most frequent and lethal cancers. Imbalance in the S-adenosylmethionine (SAM) concentration, the main methyl group donor, strongly influences the development of HCC. Key enzymes of carbon metabolism are greatly reduced in patients with cirrhosis and HCC. These alterations play a role in genetic instability and epigenetic modifications (DNA methylation, and histone modifications), however, the molecular underlying mechanisms are still poorly understood. We aimed to investigate betaine homocysteine methyltransferase (BHMT) expression in HepG2 cells and human hepatocarcinoma tissues. Tumor and surrounding healthy tissue were compared. HepG2 cells and tumor samples showed a strong decrease in BHMT transcripts resulting from the transcription of a splicing variant that contained a frameshift mutation generating a premature termination codon and gene loss of function. This splicing variant, not detected in normal adult and fetal liver, cannot be explained by any mechanism involving the known splicing consensus sequences. BHMT activity was abolished in HepG2 cells and protein expression was detected neither in HepG2 cells nor in five of the six tumor samples investigated. Further investigation is needed to elucidate whether this abnormal BHMT transcription is part of cause or consequence of liver carcinogenesis.

Participation of peribiliary glands in biliary tract pathophysiologies

To investigate the roles of peribiliary glands around the bile ducts in the pathophysiology of the biliary tract. The expression of fetal pancreatic markers, pancreatic duodenal homeobox factor 1 (PDX1) and hairy and enhancer of split 1 (HES1) and endodermal stem/progenitor (S/P) cell markers [CD44s, chemokine receptor type 4 (CXCR4), SOX9 and epithelial cell adhesion molecule (EpCAM)] were examined immunohistochemically in 32 normal adult livers (autopsy livers) and 22 hepatolithiatic livers (surgically resected livers). The latter was characterized by the proliferation of the peribiliary glands. Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections after deparaffinization. Although PDX1 and HES1 were expressed in both the nucleus and cytoplasm of epithelial cells, only nuclear staining was evaluated. SOX9 was expressed in the nucleus, while CD44s, CXCR4 and EpCAM were expressed in the cell membranes. The frequency and extent of the expression of these molecules in the lining epithelia and peribiliary glands were evaluated semi-quantitatively based on the percentage of positive cells: 0, 1+ (focal), 2+ (moderate) and 3+ (extensive). In normal livers, PDX1 was infrequently expressed in the lining epithelia, but was frequently expressed in the peribiliary glands. In contrast, HES1 was frequently expressed in the lining epithelia, but its expression in the peribiliary glands was focal, suggesting that the peribiliary glands retain the potential of differentiation toward the pancreas and the lining epithelia exhibit properties to inhibit such differentiation. This unique combination was also seen in hepatolithiatic livers. The expression of endodermal S/P cell markers varied in the peribiliary glands in normal livers: SOX9 and EpCAM were frequently expressed, CD44s infrequently, and CXCR4 almost not at all. The expression of these markers, particularly CD44s and CXCR4, increased in the peribiliary glands and lining epithelia in hepatolithiatic livers. This increased expression of endodermal S/P cell markers may be related to the increased production of intestinal and gastric mucin and also to the biliary neoplasia associated with the gastric and intestinal phenotypes reported in hepatolithiasis. The unique expression pattern of PDX1 and HES1 and increased expression of endodermal S/P cell markers in the peribiliary glands may be involved in biliary pathophysiologies.

Preparation and Characterization of Monoclonal Antibody Against Glypican-3

Glypican-3 (GPC3) has been reported as a novel serum and histochemical marker for hepatocellular carcinoma (HCC) by several groups. As an oncofetal protein, it is expressed abundantly in the fetal liver, inactive in the normal adult liver, and frequently reactivated in HCC. Immunology reagents are urgently needed to proceed with mechanism-related research, clinical validation, and application. In this report, monoclonal antibodies (MAbs) against GPC3 were made from hyperimmune BALB/c mice by injecting 100 μg of purified antigen intraperitoneally. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Finally 13 mouse hybridomas producing MAbs to GPC3 were established. The MAbs obtained were fully characterized using Western blot analysis, immunofluorescence, and immunohistochemistry. The results showed that these antibodies could be used for preliminary application of the next step mechanism-related research and GPC3 expression level analysis.

CITED1 Expression in Liver Development and Hepatoblastoma

Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF) and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1), a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT). In this current study, CITED1 expression is detected in mouse embryonic liver initially on post-coitum day 10.5 (e10.5), begins to taper by e14.5, and is undetectable in e18.5 and adult livers. CITED1 expression is detected in regenerating murine hepatocytes following liver injury by partial hepatectomy and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Importantly, while CITED1 is undetectable in normal human adult livers, 36 of 41 (87.8%) hepatoblastoma specimens express CITED1, where it is enriched in EMEF specimens compared to specimens of pure fetal histology. CITED1 overexpression in Hep293TT human hepatoblastoma cells induces cellular proliferation and upregulates the Wnt inhibitors Kringle containing transmembrane protein 1 (KREMEN1) and CXXC finger protein 4 (CXXC4). CITED1 mRNA expression correlates with expression of CXXC4 and KREMEN1 in clinical hepatoblastoma specimens. These data show that CITED1 is expressed during a defined time course of liver development and is no longer expressed in the adult liver but is upregulated in regenerating hepatocytes following liver injury. Moreover, as in WT, this embryonic marker is reexpressed in hepatoblastoma and correlates with embryonal histology. These findings identify CITED1 as a novel marker of hepatic progenitor cells that is re-expressed following liver injury and in embryonic liver tumors.

Ectopic Overexpression of COTE1 Promotes Cellular Invasion of Hepatocellular Carcinoma

Family with sequence similarity 189, member B (FAM189B), alias COTE1, a putative oncogene selected by microarray, for the first time was here found to be significantly up-regulated in hepatocellular carcinoma (HCC) specimens and HCC cell lines. mRNA expression of COTE1 in HCC samples and cell lines was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR, while protein expression of COTE1 in HCC tissues was assessed by immunohistochemistry. In addition, invasion of HCC cells was observed after overexpressing or silencing COTE1. In the total of 48 paired HCC specimens, compared with the adjacent non-cancer tissues, the expression of COTE1 was up-regulated in 31 (p<0.01). In HCC cell lines, COTE1 expression was significantly higher than in normal human adult liver (p<0.01). Overexpression of COTE1 enhanced HCC-derived LM6 and MHCC-L cellular invasion in vitro. In contrast, COTE1 knockdown via RNAi markedly suppressed these phenotypes, as documented in LM3 and MHCC-H HCC cells. Mechanistic analyses indicated that COTE1 could physically associate with WW domain oxidoreductase (WWOX), a tumor suppressor. COTE1 may be closely correlated with invasion of hepatocellular carcinoma (HCC) cells and thus may serve as an effective target for gene therapy.

Differentiation of intrahepatic peribiliary glands and pancreatic acinar cells from the remodeling ductal plate in human fetuses

In a recent article published in Gastroenterology, Carpentier et al.1 suggested that embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells. Herein, the authors demonstrated that the ductal plate also gives rise to pancreatic acinar cells and intrahepatic peribiliary glands.2 The authors previously reported that clusters of pancreatic acinar cells are present in normal adult livers.3 The ductal plate is a double-layered cylindrical structure located in the periportal regions of the fetal liver (Fig. 1A).4-8 The ductal plate undergoes remodeling (Fig. 1B,C), leading to the normal cholangiocytes and intrahepatic peribiliary glands.4-8 The remodeling involves apoptosis and cell proliferation of the ductal plate. Several molecules, such as glycoconjugates, Levis y, Bcl-2, C-myc, tenascin, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, trypsin, pancreatic digestive enzymes, E-cadherin, and catenin, are involved in the process of ductal plate remodeling.2-10 Pancreatic acinar cell clusters develop from the remodeling ductal plate.4, 8 The authors again reviewed 42 fetal livers of various gestational ages and 32 postnatal livers, and observed that intrahepatic peribiliary glands developed from the remodeling ductal plate at 35 to 40 gestational weeks for fetal livers as well as in the infant livers. The authors also found that pancreatic acinar cells developed from remodeling and remodeled ductal plate at 38 to 40 gestational weeks for fetal livers (Fig. 1D) as well as infant livers of 1 to 3 months (Fig. 1E). Immunohistochemically, the pancreatic acinar cells contained pancreatic amylase, trypsinogen, and lipase. The development of intrahepatic bile ducts. (A) The ductal plate at 12 gestational weeks. Cytokeratin immunostaining, ×100. (B) Remodeling or remodeled ductal plate at 18 gestational weeks. Cytokeratin immunostaining, ×100. (C) The remodeling ductal plate (arrow) at 17 gestational weeks. The cells of the ductal plate invade into the portal mesenchyma (arrow). Hematoxylin and eosin, ×200. (D) The development of pancreatic acinar cells (arrow) at 39 gestational weeks. Hematoxylin and eosin, ×50. (E) Pancreatic acinar cells (arrow) develop from the remodeled ductal plate in a 2-month-old infant liver. Hematoxylin and eosin, ×200. Tadashi Terada M.D., Ph.D.*, * Department of Pathology, Shizuoka City Shimizu Hospital, Shizuoka, Japan.

Hepatic Extramedullary Hematopoiesis and Macrophages in the Adult Mouse: Histometrical and Immunohistochemical Studies

Fetal liver hematopoiesis in mice disappears approximately 2 weeks after birth; however, under experimental acute anemia extramedullary hematopoiesis occurs in the livers of adult mice. The hematopoietic foci in the liver during extramedullary hematopoiesis contain erythroblasts and macrophages. In this study, the extent of involvement of macrophages in the development and involutional process of the hematopoietic foci in adult mice livers was clarified by experimentally inducing extramedullary hematopoiesis. Hematopoietic cells appeared in the livers 2 days after phenylhydrazine (PHZ) injections. The number and area of the foci increased rapidly, reaching peak values on the sixth day. F4/80-positive macrophages were observed in the sinusoids as well as the hematopoietic foci, and were tightly surrounded by erythroblasts. Sinusoidal macrophages in normal adult livers were positive for F4/80 but negative for ER-HR3. However, in extramedullary hematopoiesis-induced livers, sinusoidal macrophages became positive for ER-HR3 antibodies. The number of ER-HR3-positive macrophages was 9.2 ± 2.9/mm² on the second day after PHZ was administered, and increased to 200.3 ± 4.2/mm² on the sixth day. On the seventh day after the PHZ injections, the number decreased and they were no longer detected at 30 days after PHZ was injected. The present study revealed that erythroblasts accumulate around sinusoidal macrophages to form an erythroblastic island with a central macrophage similar to erythropoiesis in the fetal liver. Furthermore, in line with development and regression of extramedullary hematopoiesis, macrophages express ER-HR3, a hematopoiesis related antigen.

Neighbor of Punc E 11: Expression Pattern of the New Hepatic Stem/Progenitor Cell Marker During Murine Liver Development

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

The Phenotypic Characteristic of Liver-Derived Stem Cells From Adult Human Deceased Donor Liver

Liver transplantation is the only effective treatment for end-stage liver disease. Because of the limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult liver contains hepatic stem cells is highly controversial. Several studies have suggested the presence of stem cells in the adult normal human liver. However, a population with stem cell properties has not yet been isolated. The purpose of this study was to identify and characterize progenitor cells in normal adult human liver. We isolated and expanded human liver stem cells (HLSCs) from a donated liver not suitable for liver transplantation or characterizing them by fluorescence-activated cell sorter, polymerase chain reaction, and immunofluorescence assay. HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, CD90, CD105, and CD166 but not the hematopoietic stem cell markers CD34, CD45, and CD117. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed CD26, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. We concluded that HLSCs expressed several mesenchymal but not hematopoietic stem cell markers as well as CD26 and CK18, indicating a partial commitment to hepatic cells.

Histone H4 histidine phosphorylation: kinases, phosphatases, liver regeneration and cancer

Phosphorylation of histone H4 on one or both of its two histidine residues has been known to occur in liver cells for nearly 40years and has been associated with proliferation of hepatocytes during regeneration of the liver following mechanical damage. More recently, large increases in histone H4 histidine kinase activity have been found to occur associated with proliferation and differentiation of liver progenitor cells following chemical damage that prevents hepatocyte proliferation. In addition, it has been shown this histone H4 histidine kinase activity is elevated nearly 100-fold in human foetal liver and several hundredfold in hepatocellular carcinoma tissue compared with normal adult liver. In the present paper, we review what is currently known about histone H4 histidine phosphorylation, the kinase(s) responsible and the phosphatases capable of catalysing its dephosphorylation, and briefly summarize the techniques used to detect and measure the histidine phosphorylation of histone H4 and the corresponding kinase activity.

A Novel Technique for Hepatic Progenitor Cell Isolation From Normal Adult Rat Livers

The transplantation of hepatic progenitor cells (HPCs) is a promising alternate approach to liver transplantation for patients with end-stage liver disease. Here, we report a novel technique for HPCs isolation from normal adult rat livers (no preexposure to chemicals and no injury). HPCs were isolated from normal adult rat livers using a novel four-step collagenase perfusion method followed by density gradient centrifugation. The phenotypic properties of HPCs were characterized by morphological observation, reverse-transcription polymerase chain reaction (RT-PCR), and immunocytochemistry. The results showed that HPCs formed loose colonies and possessed a round or oval shape at culture day 3. These cells proliferated slowly and exhibited progenitor-like characteristics during the 30-day culture period. RT-PCR analysis indicated that the cultured cells were positive for several HPC-specific genes, such as albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), cytokeratin 19 (CK19), CD45, hepatocyte nuclear factor 1-alpha (HNF-1α), hepatocyte nuclear factor 4-alpha (HNF-4α), and Thy-1. Immunocytochemical staining showed that these cells were consistently positive for ALB, AFP, CK18, CK19, Thy-1, and OV-6. HPCs can be isolated from normal adult rat livers using a simple and effective technique involving four-step collagenase perfusion, further confirming their potential as a strong candidate for hepatocyte therapy.

Granulin-Epithelin Precursor Is an Oncofetal Protein Defining Hepatic Cancer Stem Cells

Background and AimsIncreasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers.MethodsProtein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity.ResultsWe demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEPhigh cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEPlow counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice.ConclusionsOur findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co-expression of GEP and ABCB5 further enriches a subpopulation with enhanced CSC properties. The current data provide new insight into the therapeutic strategy.

Ultrasound measurement and clinical analysis on the caudate lobe of adult liver

Objective To measure the imaging data of the caudate lobe of liver with the ultrasound measurement for the normal adults and patients with liver disease to provide morphological evidence for clinical ultrasound adult caudate lobe lesions.Methods Though the method of forward-looking ultrasound measurements,we analyzed the differences between gender,age,height,weight,caudate lobe of patients with liver disease form 134 cases of normal adult liver caudate lobe and 34 cases of patients ? liver caudate lobe.Results The front and back diameter:man (2.174 ± 0.416) cm,woman (2.010 ± 0.350) cm and the left and right diameter:man(2.899 ± 0.322)cm,woman (2.683 ± 0.306)cm,the men liver caudate lobe were longer than those of the women.The up and down diameter and the left and right diameter:(5.115 ± 0.583) cm,(2.212 ± 0.367)cm of the young-age group liver caudate lobe were longer than those of the mid-age group:(4,814 ± 0.570)cm,(2.038 ± 0.384)cm and the old-age group:(4.781 ± 0.914)cm,(1.955 ± 0.460)cm.The differences were statistically significant.Liver caudate lobe diameters of too light,moderate,overweight,obese had no significant difference (P＞ 0.05).The up and down diameter,the front and back diameter and the left and right diameter of the normal adult liver caudate lobe were longer than those of the patients,with statistical significant differences (P＜ 0.01).Conclusions The front and back diameter and the left and right diameter of the men liver caudate lobe are longer than those of the women.The up and down diameter and the left and right diameter of the young people liver caudate lobe are longer than those of the mid-age group and the old-age group.The every diameter of adult liver caudate has no connects with their weights and heights.The patient liver caudate lobe diameters are longer than those of normal adults.We should care the differences of the liver caudate lobe in physiology and disease characteristics ways when we use ultrasound and its diagnosis to avoid missed diagnosis or misdiagnosis. Key words: Adult; Caudate lobe of liver; Ultrasound measurement; Clinic

A splicing variant leads to complete loss of function of betaine–homocysteine methyltransferase (BHMT) gene in hepatocellular carcinoma

The remethylation of homocyteine into methionine is catalyzed either by methionine synthase (MTR) or by betaine–homocysteine methyltransferase (BHMT), in the liver. Choline/betaine deficiency and impaired BHMT pathway have been associated with hepatocellular carcinogenesis, in animal models. The molecular mechanisms that impair the BHMT pathway are unknown. We aimed to investigate BHMT, BHMT2, and MTR expression in HepG2 cells and human hepatocarcinoma tissues. Transcripts were quantified by RT-qPCR and splicing was assessed by analysis of exon junctions and sequencing of variants. Protein expression was studied by Western Blot, immunohistochemistry and enzyme activity. Tumor tissue was compared with surrounding healthy tissue. RT-qPCR of HepG2 cells and of tumor samples showed a strong decrease of transcripts of BHMT and BHMT2, compared to normal. MTR transcript levels were not different. The decreased BHMT expression resulted from the transcription of a splicing variant that produced a frameshift in exon 4, with a premature termination codon in exon 5 and a loss of function of the gene. This splicing variant did not fit with any mechanism resulting from known splicing consensus sequences and was not detected in normal adult and fetal liver. Consistently, BHMT activity was abolished in HepG2 and protein expression was not detectable in HepG2 and in 5 of the 6 tumor samples, compared to normal tissues. In conclusion, a transcription variant of exon 4 produces a loss of function of BHMT in human hepatocarcinoma. Whether this abnormal transcription of BHMT is part or consequence of liver carcinogenesis should deserve further investigations.