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1
Mundiri, Nur Azmiati, Meta Maulida Damayanti, Maya Tejasari, Annisa Rahmah Furqaani, and R. A. Retno Ekowati. "Pengaruh Fraksi Air Buah Lemon terhadap Gambaran Morfologi Jaringan Hati Mencit Tua yang Diberi Pakan Tinggi Lemak." Jurnal Integrasi Kesehatan & Sains 1, no. 1 (January 31, 2019): 49–53. http://dx.doi.org/10.29313/jiks.v1i1.4321.
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Dislipidemia merupakan salah satu faktor risiko dari non alcoholic fatty liver disease (NAFLD). NAFLD mempunyai karakteristik steatosis hepatik, hepatocyte ballooning, inflamasi lobular, dan fibrosis. Kandungan flavonoid pada Citrus limon dipercaya dapat mencegah steatosis hepatik. Tujuan penelitian ini mengetahui pengaruh fraksi air buah lemon terhadap gambaran morfologi jaringan hati mencit tua yang diberi pakan tinggi lemak. Penelitian ini merupakan penelitian eksperimental dengan subjek penelitian adalah mencit (Mus musculus) jantan galur DDY tua yang dibagi menjadi empat kelompok secara acak, terdiri atas kelompok kontrol dan tiga kelompok perlakuan dengan konsentrasi fraksi air buah lemon 20,6; 41,2; 82,4 mg/20 gram BB mencit. Data jumlah hepatosit dengan droplet lemak dan hepatocyte ballooning dianalisis menggunakan uji ANOVA dan Uji Kruskal Willis. Terdapat perbedaan jumlah hepatosit dengan droplet lemak (p=0,063) dan hepatosit yang mengalami pembengkakan (p=0,109) antara kelompok kontrol dan kelompok perlakuan. Simpulan penelitian ini adalah fraksi air buah lemon dapat mencegah hepatocyte ballooning dan pembentukan droplet lemak pada hepatosit mencit tua yang diberikan pakan tinggi lemak. PROTECTIVE EFFECT OF WATER FRACTION OF LEMON ON HIGH-FAT DIET-INDUCED LIVER INJURY IN OLD MICEDyslipidemia is one of the risk factors of non alcoholic fatty liver disease (NAFLD). NAFLD is characterized by hepatic steatosis, hepatocyte ballooning, lobular inflammation, and fibrosis. Flavonoid in Citrus limon is believed to prevent hepatic steatosis. The aim of this study is to know the protective effect of lemon’s water fraction on high-fat diet-induced liver injury in old mice. This was an experimental study with old male mice (Mus musculus) DDY strain divided into four groups randomly, consisting of control group and three groups given with water fraction of lemon at concentration 20.6; 41.2; 82.4 mg/20 gram mice body weight. Total count of hepatocytes with fat droplets and hepatocytes ballooning were analyzed using ANOVA and Kruskal Willis tests. There are differences in the amount of hepatocytes with fat droplets (p=0.063) and hepatocytes ballooning (p=0.109) between the control group and the treatment group. The conclusion of this study is lemon’s water fraction can prevent the formation of hepatocyte ballooning and fat droplet in old mice’s hepatocyte fed by high-fat diet.
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Sibuea, Christine Verawaty, Enjelin Sasa Kristanti Hutabarat, and David M. T. Simangunsong. "Viabilitas Hepatosit pada Monokultur 3D Metode Hanging Drop dan Monokultur 2D." Nommensen Journal of Medicine 7, no. 2 (February 28, 2022): 36–38. http://dx.doi.org/10.36655/njm.v7i2.623.
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Background: Liver is a vital organ that has many functions including metabolism, detoxification of toxins and protein synthesis. The prevalence of liver disease is high and the treatment of liver disease continues to develop, so liver models are needed to study liver disease mechanisms and test drug toxicity. Many hepatocyte culture methods are being developed to construct an optimal liver model. The best culture method maintains high hepatocyte viability.
Objective : This study aims to determine the viability of hepatocytes in 3D hanging drop method culture and in 2D culture.
Methods: Hepatocytes were isolated from the liver of Sprague Dawley-Rats rats (n=2) and digested with Trypsin. Primary hepatocytes were cultured using the hanging drop method and conventional (2D) method. Hepatocyte viability was analyzed using the Trypan Blue Exclusion Test.
Results: Hanging drop method had higher viability (81.18%) than the 2D culture method (69.22%) with p>0,05.
Conclusion: The hanging drop method has better viability than the 2D culture method.
Keywords: Hepatocyte, viability, 3D culture, 2D culture, hanging drop
Latar belakang: Hati merupakan organ vital tubuh yang memiliki banyak fungsi diantaranya metabolisme, detoksifikasi racun dan sintesis protein. Prevalensi penyakit hati tinggi dan terapi penyakit hati terus berkembang, sehingga model hati sangat dibutuhkan untuk mempelahari mekanisme penyakit hati dan uji toksisitas obat. Banyak metode kultur hepatosit yang terus dikembangkan untuk menghasilkan suatu model hati yang optimal. Metode kultur yang terbaik adalah metode kultur dengan viabilitas hepatosit yang tinggi.
Tujuan : Penelitian ini bertujuan untuk mengetahui viabilitas hepatosit pada kultur 3D metode hanging drop dan pada kultur 2D.
Metode: Hepatosit diisolasi dari hati tikus Sprague Dawley-Rats (n=2) dan digesti dengan Tripsin. Hepatosit primer tikus dikultur dengan metode hanging drop (tetes gantung) dan metode konvensional (2D). Viabilitas hepatosit dianalisa dengan menggunakan Trypan Blue Exclusion Test.
Hasil: Viabilitas hepatosit pada metode hanging drop memiliki viabilitas lebih tinggi (81,18%) dibandingan dengan metode kultur 2D (69,22%) dengan nilai p<5.
Kesimpulan: Metode hanging drop memiliki viabilitas yang lebih baik dibandingkan metode kultur 2D.
Kata Kunci: Hepatosit, viabilitas, kultur 3D, kultur 2D, hanging drop
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Skuratov, A. G., D. R. Petrenyov, and A. N. Kondrachuk. "EXPRESSION OF MARKER GENES BY HEPATOCYTE-LIKE CELLS differentiated from mesenchymal stem cells." Health and Ecology Issues, no. 3 (September 28, 2013): 105–10. http://dx.doi.org/10.51523/2708-6011.2013-10-3-22.
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Objective: to investigate the expression of marker genes by hepatocyte-like cells differentiated from mesenchymal stem cells (MSCs). Materials and methods. Wistar white rats, bone marrow MSCs, isolated hepatocytes of the rats were obtained by enzymatic perfusion of liver; differentiation of MSCs in hepatocyte direction; light microscopy; investigation of expression of genes by polymerase chain reaction (PCR). Results. The observed changes in the gene expression profile during the stages of differentiation indicate the presence of the cells differentiated into hepatocytic direction in MSCs culture. The expression of Carbox, Krt18, Krt19 Cyt1A1 genes depends on the composition of the medium and is not permanent and inducible in nature. It is important to go on searching for the molecular markers of MSCs differentiation in the hepatocytic direction. These results demonstrate the necessity to systematize the available data on the changes in the levels of gene expression during MSCs differentiation into hepatocytes to unify the conditions of assessment of the gene expression profiling.
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Sibuea, Christine Verawaty, Jeanne Adiwinata Pawitan, and Radiana Antarianto. "Pengaruh Penggantian Medium terhadap Viabilitas Hepatosit Kultur 3D Organoid Hati." Nommensen Journal of Medicine 7, no. 2 (February 28, 2022): 39–42. http://dx.doi.org/10.36655/njm.v7i2.625.
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Background : Liver organoids can be used as materials for Bioartificial Liver, to study the mechanism of liver disease and as drug test toxicity. Reconstruction of liver organoids requires optimal culture methods, culture medium and cellular components to construct liver organoids that resemble liver microstructure in vivo with optimal function. 3D culture method using hepatocytes and stem cells with PRP supplemented William's E can reconstruct liver organoids with liver function. Medium exchange is an usual method to maintain the required nutrients and to eliminate waste products, but it requires a sufficient supply of medium and supplementation. Method and the use of effective and efficient medium with optimal hepatocyte viability are urgently needed in the reconstruction of liver organoids.
Objective : This study was aimed to compare the viability of primary hepatocytes in culture medium exchange liver organoids and monoculture and without culture medium exchange.
Methods : Primary hepatocytes isolated from Sprague Dawley-Rats mice (250gr, n=3) were co-cultured with umbilical cord mesenchymal stem cells, cord blood CD34+ stem cells and LX2 in PRP-supplemented William's E for 14 days. The culture medium was exchanged at 48 hours, day 7 and day 14 and no culture medium exchanged in the control group. Hepatocyte viability was analyzed using the Trypan Blue Exclusion Test at 48 hours, day 7 and day 14.
Results : Hepatocyte viability in culture medium exchange liver organoids was higher than without culture medium exchange, especially in monoculture, but there was no significant difference (p value> 0.05).
Conclusion: Hepatocyte viability in culture medium exchange liver organoids was not significantly different from no culture medium exchange liver organoids. Culture medium exchange in monoculture supported hepatocyte viability up to day 14.
Keywords: hepatocytes, liver organoids, viability, culture medium
ABSTRAK
Latar belakang : Organoid hati dapat digunakan sebagai bahan Bioartificial Liver, mempelajari mekanisme penyakit hati dan uji toksisitas obat. Rekonstruksi organoid hati membutuhkan metode kultur, medium kultur dan komponen seluler yang optimal untuk menghasilkan organoid hati yang menyerupai mikrostruktur hati in vivo dengan fungsi yang optimal. Metode kultur 3D menggunakan hepatosit dan sel punca mesenkimal dengan William’s E yang disuplementasi PRP dapat merekonstruksi organoid hati dengan fungsi hati. Pergantian medium merupakan metode yang sering dilakukan untuk mempertahankan nutrisi yang dibutuhkan dan untuk membuang sisa metabolit sel, tetapi membutuhkan persediaan medium dan suplementasi yang cukup banyak. Metode dan penggunaan medium yang efektif dan efisien dengan viabilitas hepatosit yang optimal sangat dibutuhkan dalam rekonstruksi organoid hati.
Tujuan : Penelitian ini bertujuan untuk mengetahui perbandingan viabilitas hepatosit primer pada organoid hati dengan pergantian medium kultur dan tanpa pergantian medium kultur.
Metode : Hepatosit primer yang diisolasi dari tikus Sprague Dawley-Rats (250gr, n=3) diko-kultur dengan sel punca mesenkimal asal tali pusat, sel punca CD34+ asal darah tali pusat dan LX2 dalam William’s E yang disuplementasi PRP selama 14 hari. Medium kultur diganti pada 48 jam, hari ke-7 dan hari ke-14 dan tidak dilakukan pergantian medium pada kelompok kontrol. Viabilitas hepatosit dianalisa dengan menggunakan Trypan Blue Exclusion Test pada 48 jam, hari ke-7 dan hari ke-14.
Hasil : Viabilitas hepatosit pada organoid hati dengan pergantian medium kultur tampak lebih banyak dibandingkan tanpa pergantian medium kultur khususnya pada monokultur, tetapi tidak terdapat perbedaan yang signifikan (nilai p>0,05).
Kesimpulan : Viabilitas hepatosit pada organoid hati dengan pergantian medium kultur tidak berbeda secara signifikan dengan organoid hati tanpa pergantian medium kultur. Pergantian medium kultur pada monokultur mendukung viabilitas hepatosit hingga hari ke-14.
Kata Kunci : Hepatosit, organoid hati, viabilitas, medium kultur
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Gómez-Aristizábal, Alejandro, and John Edward Davies. "The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity." Biochemistry and Cell Biology 91, no. 3 (June 2013): 140–47. http://dx.doi.org/10.1139/bcb-2012-0079.
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Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC–hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC–hepatocyte interactions.
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Naruse, K., Y. Sakai, I. Nagashima, G. X. Jiang, M. Suzuki, and T. Muto. "Comparisons of Porcine Hepatocyte Spheroids and Single Hepatocytes in the Non-Woven Fabric Bioartificial Liver Module." International Journal of Artificial Organs 19, no. 10 (October 1996): 605–9. http://dx.doi.org/10.1177/039139889601901008.
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We previously developed a new bioreactor of the bioartificial liver composed of non-woven fabric. We have also experimented with hepatocyte spheroids, with the aim of improving the efficiency of this NWF bioreactor. In this study, we compared the efficiencies of NWF bioreactors employing hepatocyte spheroids versus single hepatocytes. Hepatocytes were isolated from a whole pig liver by Seglen's method. 1.0 × 1010 single hepatocytes were immobilized in the NWF bioreactor. Another 1.0 × 1010 hepatocytes were allowed to form spheroids by 24 hr suspension culture in a 4-L culture vessel, before being immobilized in the bioreactor. Hepatocyte spheroids were found to be functionally superior, on a per-cell basis, to single hepatocytes in the NWF bioreactor. However, the NWF bioreactor employing hepatocyte spheroids exhibited lower efficiency than that employing single hepatocytes, because the total number of the hepatocytes had decreased during the 24 hr suspension culture.
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Mason, William S., Chunxiao Xu, Huey Chi Low, Jeffry Saputelli, Carol E. Aldrich, Catherine Scougall, Arend Grosse, Richard Colonno, Sam Litwin, and Allison R. Jilbert. "The Amount of Hepatocyte Turnover That Occurred during Resolution of Transient Hepadnavirus Infections Was Lower When Virus Replication Was Inhibited with Entecavir." Journal of Virology 83, no. 4 (December 10, 2008): 1778–89. http://dx.doi.org/10.1128/jvi.01587-08.
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ABSTRACT Transient hepadnavirus infections can involve spread of virus to the entire hepatocyte population. In this situation hepatocytes present following recovery are derived from infected hepatocytes. During virus clearance antiviral cytokines are thought to block virus replication and formation of new covalently closed circular DNA (cccDNA), the viral transcriptional template. It remains unclear if existing cccDNA is eliminated noncytolytically or if hepatocyte death and proliferation, to compensate for killing of some of the infected hepatocytes, are needed to remove cccDNA from surviving infected hepatocytes. Interpreting the relationship between hepatocyte death and cccDNA elimination requires knowing both the amount of hepatocyte turnover and whether cccDNA synthesis is effectively blocked during the period of immune destruction of infected hepatocytes. We have addressed these questions by asking if treatment of woodchucks with the nucleoside analog inhibitor of viral DNA synthesis entecavir (ETV) reduced hepatocyte turnover during clearance of transient woodchuck hepatitis virus (WHV) infections. To estimate hepatocyte turnover, complexity analysis was carried out on virus-cell DNA junctions created by integration of WHV and present following recovery in the livers of WHV-infected control or ETV-treated woodchucks. We estimated that, on average, 2.2 to 4.8 times less hepatocyte turnover occurred during immune clearance in the ETV-treated woodchucks. Computer modeling of the complexity data suggests that mechanisms in addition to hepatocyte death were responsible for elimination of cccDNA during recovery from transient infections.
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Gupta, Sanjeev, Pankaj Rajvanshi, Emma Aragona, Chang-Don Lee, Purnachandra R. Yerneni, and Robert D. Burk. "Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion." American Journal of Physiology-Gastrointestinal and Liver Physiology 276, no. 3 (March 1, 1999): G629—G638. http://dx.doi.org/10.1152/ajpgi.1999.276.3.g629.
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To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically marked rat or mouse cells. Hosts were subjected to liver injury by carbon tetrachloride (CCl4), to liver regeneration by a two-thirds partial hepatectomy, and to hepatocellular DNA synthesis by infusion of hepatocyte growth factor for comparative analysis. Transplanted hepatocytes were documented to integrate in periportal areas of the liver. In response to CCl4 treatments after cell transplantation, the transplanted hepatocyte mass increased incrementally, with the kinetics and magnitude of DNA synthesis being similar to those of host hepatocytes. In contrast, when cells were transplanted 24 h after CCl4 administration, transplanted hepatocytes appeared to be injured and most cells were rapidly cleared. When hepatocyte growth factor was infused into the portal circulation either subsequent to or before cell transplantation and engraftment, transplanted cell mass did not increase, although DNA synthesis rates increased in cultured primary hepatocytes as well as in intact mouse and rat livers. These data suggested that procedures causing selective ablation of host hepatocytes will be most effective in inducing transplanted cell proliferation in the normal liver. The number of transplanted hepatocytes was not increased in the liver by hepatocyte growth factor administration. Repopulation of the liver with genetically marked hepatocytes can provide effective reporters for studying liver growth control in the intact animal.
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Pastor, Catherine M., and Valérie Vilgrain. "Steatosis Alters the Activity of Hepatocyte Membrane Transporters in Obese Rats." Cells 10, no. 10 (October 13, 2021): 2733. http://dx.doi.org/10.3390/cells10102733.
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Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty (fa/fa) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.
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GHIURCO, Ioan Florin, Aurel DAMIAN, Vasile Florin RUS, Cristian MARTONOS, Maria Cătălina MATEI, Victoria BUZA, Laura Cristina ȘTEFĂNUȚ, et al. "Mitochondria Load Degree in Hepatocytes of the Classical Hepatic Lobules in Chinchilla (Chinchilla lanigera)." Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Veterinary Medicine 78, no. 1 (May 4, 2021): 76. http://dx.doi.org/10.15835/buasvmcn-m:2021.0004.
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Hepatocytes represent the majority of the liver cell population and are arranged in the form of cords placed in intimate contact with the sinusoidal capillaries. The functional complexity corroborated with the intensity of the activity of hepatocytes requires large amounts of energy. The organelles involved in the production of chemical energy used in the activity of hepatocytes are the mitochondria. The purpose of this study was to verify the mitochondrial load of hepatocytes in all areas of the classical hepatic lobules, in order to indirectly assess the intensity of hepatocyte activity in each area. Materials and Methods Five fresh corpses of chinchilla (Chinchilla lanigera) from an independent breeder from Bistrița-Năsăud county were used. Liver fragments were harvested and fixated in Kolster’s solution for 24 hours, stained with Heidenhain ferric hematoxylin, and assessed using Olympus BX41 microscope. Fixation with Kolster's solution and the staining with Heidenhain's iron hematoxylin clearly shows the hepatocytic mitochondria in shades from gray to black. The liver lobules displayed an uneven distribution of mitochondria depending on the area. In zone 1 of the classical hepatic lobule, the degree of loading of hepatocytes with mitochondria is larger than in zone 2 and much larger than in zone 3. Morphological features of the hepatocytes, including the number and distribution of mitochondria in the hepatic lobules, should improve the understanding of the physiology and pathology of the liver.
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Lilja, Helene, Pierre Blanc, Achilles A. Demetriou, and Jacek Rozga. "Response of Cultured Fetal and Adult Rat Hepatocytes to Growth Factors and Cyclosporine." Cell Transplantation 7, no. 3 (May 1998): 257–66. http://dx.doi.org/10.1177/096368979800700304.
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Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about × 100 higher and after 10 days in culture ×20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was × 13.8 with EGF, ×18.5 with transforming growth factor alpha (TGF-α), and ×7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-α or HGF. EGF-, TGF-α- and HGF-dependent DNA synthesis was inhibited by transfroming growth factor beta-1 (TGF-β1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-α- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36–46%, whereas in adult hepatocytes by 19–27%. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-β1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.
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Rivas, Pedro A., Alfredo J. Fabrega, Daniel Schwartz, William Dagiantis, Michael G. Ward, Jacqueline Blanchard, and Raymond Pollak. "Transplantation of Hepatocytes: An In-Vitro and In-Vivo Study in Canines." Cell Transplantation 3, no. 2 (March 1994): 193–201. http://dx.doi.org/10.1177/096368979400300208.
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We isolated and transplanted hepatocytes in the canine, large animal model to evaluate hepatocyte yield and purity as well as the optimal site for hepatocyte engraftment (i.e., the spleen or the portal bed). We obtained viable, pure, single hepatocyte suspensions that were readily preserved at 4°C in University of Wisconsin (UW) solution for up to 3 days. Both intrasplenic and portal vein injection were well tolerated, with minimal recipient morbidity and mortality when 1-2 × 109 hepatocytes were injected into immunosuppressed allogeneic hosts. We noted the embolization of hepatocytes into the parenchyma of the native liver within 7 days of intrasplenic transplantation that produced a mild reversible derangement of liver function and histology. These results warrant consideration prior to clinical trials of hepatocyte transplantation in man.
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Garcı́a, Fabiana, Arlinet Kierbel, M. Cecilia Larocca, Sergio A. Gradilone, Patrick Splinter, Nicholas F. LaRusso, and Raúl A. Marinelli. "The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP." Journal of Biological Chemistry 276, no. 15 (January 17, 2001): 12147–52. http://dx.doi.org/10.1074/jbc.m009403200.
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We previously found that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected anN-glycosylated band of ∼34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt2cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt2cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt2cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.
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Sun, D., Y. Gong, H. Kojima, G. Wang, E. Ravinsky, M. Zhang, and G. Y. Minuk. "Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth." American Journal of Physiology-Gastrointestinal and Liver Physiology 285, no. 1 (July 2003): G12—G19. http://dx.doi.org/10.1152/ajpgi.00513.2002.
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Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized ( P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-β3 receptor mRNA expression and all cell lines were negative for GABAA-β3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-β3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-β3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-β3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.
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Aurich, Hendryk, Sarah Koenig, Christian Schneider, Jens Walldorf, Petra Krause, Wolfgang E. Fleig, and Bruno Christ. "Functional Characterization of Serum-Free Cultured Rat Hepatocytes for Downstream Transplantation Applications." Cell Transplantation 14, no. 7 (August 2005): 497–506. http://dx.doi.org/10.3727/000000005783982855.
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Although ex vivo culture of hepatocytes is known to impair functionality, it may still be considered as desirable to propagate or manipulate them in culture prior to transplantation into the host liver. The aim of this study was to clarify whether rat hepatocytes cultured over different periods of time proliferate and retain their hepatocyte-specific functions following transplantation into the recipient liver. Rat hepatocytes were cultured under serum-free conditions in the presence of hepatocyte and epidermal growth factors. Cells derived from wild-type donor livers were transplanted into the livers of CD26-deficient rats. Cell proliferation and the expression of hepatocyte-specific markers were determined before and after transplantation. Cell number increased threefold over a culture period of 10 days. The expression of connexin 32 and phosphoenolpyruvate carboxykinase declined over time, indicating the loss of hepatocyte-specific functions. Hepatocytes cultured over 4 or 7 days and then transplanted proliferated in the host parenchyma. The transplanted cells expressed connexin 32, cytokeratin 18, and phosphoenolpyruvate carboxykinase, indicating the differentiated phenotype. The loss of hepatocyte-specific functions during culture may be restored after transplantation, suggesting that the proper physiological environment is required to maintain the differentiated phenotype.
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Peng, Weng Chuan, Lianne J. Kraaier, and Thomas A. Kluiver. "Hepatocyte organoids and cell transplantation: What the future holds." Experimental & Molecular Medicine 53, no. 10 (October 2021): 1512–28. http://dx.doi.org/10.1038/s12276-021-00579-x.
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AbstractHistorically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.
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Afford, Simon C., Satinder Randhawa, Aristides G. Eliopoulos, Stefan G. Hubscher, Lawrence S. Young, and David H. Adams. "CD40 Activation Induces Apoptosis in Cultured Human Hepatocytes via Induction of Cell Surface Fas Ligand Expression and Amplifies Fas-mediated Hepatocyte Death during Allograft Rejection." Journal of Experimental Medicine 189, no. 2 (January 18, 1999): 441–46. http://dx.doi.org/10.1084/jem.189.2.441.
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We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68+ macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.
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Chang, Tian, Ji, Zhou, Hou, Zhao, Yang, Yang, and Li. "Single-Cell Transcriptomes Reveal Characteristic Features of Mouse Hepatocytes with Liver Cholestatic Injury." Cells 8, no. 9 (September 11, 2019): 1069. http://dx.doi.org/10.3390/cells8091069.
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Hepatocytes are the main parenchymal cells of the liver and play important roles in liver homeostasis and disease process. The heterogeneity of normal hepatocytes has been reported, but there is little knowledge about hepatocyte subtype and distinctive functions during liver cholestatic injury. Bile duct ligation (BDL)-induced mouse liver injury model was employed, and single-cell RNA sequencing was performed. Western blot and qPCR were used to study gene expression. Immunofluoresence was employed to detect the expressions of marker genes in hepatocytes. We detected a specific hepatocyte cluster (BDL-6) expressing extracellular matrix genes, indicating these hepatocytes might undergo epithelia-mesenchymal transition. Hepatocytes of BDL-6 also performed tissue repair functions (such as angiogenesis) during cholestatic injury. We also found that four clusters of cholestatic hepatocytes (BDL-2, BDL-3, BDL-4, and BDL-5) were involved in inflammatory process in different ways. To be specific, BDL-2/3/5 were inflammation-regulated hepatocytes, while BDL-4 played a role in cell chemotaxis. Among these four clusters, BDL-5 was special. because the hepatocytes of BDL-5 were proliferating hepatocytes. Our analysis provided more knowledge of hepatocyte distinctive functions in injured liver and gave rise to future treatment aiming at hepatocytes.
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Kobayashi, Naoya, and Noriaki Tanaka. "Engineering of Human Hepatocyte Lines for Cell Therapies in Humans: Prospects and Remaining Hurdles." Cell Transplantation 11, no. 5 (July 2002): 417–20. http://dx.doi.org/10.3727/000000002783985693.
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Hepatocyte-based biological therapies are increasingly envisioned for temporary support in acute liver failure and provision of specific-liver functions in liver-based metabolic deficiency. One of the hurdles to develop such therapies is severe shortage of human livers for hepatocyte isolation. To address the issue, we have focused on reversible immortalization of human hepatocytes. Such technology can allow rapid preparation of functional and uniform human hepatocytes. Here we present our strategy to construct transplantable human hepatocyte cell lines.
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Lu, Na, Yun Liu, An Tang, Lulu Chen, Dengshun Miao, and Xiaoqin Yuan. "Hepatocyte-Specific Ablation of PP2A Catalytic SubunitαAttenuates Liver Fibrosis Progression via TGF-β1/Smad Signaling." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/794862.
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Protein phosphatase 2A (PP2A), a family of the major serine/threonine phosphatases in cells, regulates many aspects of physiological processes. However, isoform-specific substrates and the biological role of each specific member of the PP2A family remain largely unknown. In this study, we investigated whether PP2A catalytic subunit Cα(PP2Acα) is involved in chronic hepatic injury and fibrosis. A hepatocyte-specific PP2Acαablation mice model was established to examine the effect of PP2Acαon carbon tetrachloride- (CCl4-) induced chronic hepatic injury and fibrosis. Our results showed that PP2Acαknockout mice were less susceptible to chronic CCl4-induced liver injury as evidenced by lower levels of serum alanine aminotransferase and aspartate aminotransferase, decreased hepatocyte proliferation, and increased rate of apoptotic removal of the injured hepatocytes. PP2Acαknockout mice also displayed a lesser extent of liver fibrosis as a significant decrease in the proportion ofα-smooth muscle actin-expressing cells and collagen deposition was observed in their liver tissues. Furthermore, the levels of serum TGF-β1 and hepatocytic Smad phosphorylation were reduced in the PP2Acαknockout mice. These data suggest that hepatocyte-specific ablation of PP2Acαprotects against CCl4-induced chronic hepatic injury and fibrogenesis and the protective effect is mediated at least partially through the impaired TGF-β1/Smad signaling.
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Aoki, Takeshi, Tomotake Koizumi, Yasuna Kobayashi, Daisuke Yasuda, Yoshihiko Izumida, Zhenghao Jin, Nobukazu Nishino, et al. "A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation." Cell Transplantation 14, no. 9 (October 2005): 609–20. http://dx.doi.org/10.3727/000000005783982710.
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Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37°C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.
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Mason, William S., Allison R. Jilbert, and Samuel Litwin. "Hepatitis B Virus DNA Integration and Clonal Expansion of Hepatocytes in the Chronically Infected Liver." Viruses 13, no. 2 (January 30, 2021): 210. http://dx.doi.org/10.3390/v13020210.
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Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.
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Kang, Sun Woo Sophie, Victoria C. Cogger, David G. Le Couteur, and Dong Fu. "Multiple cellular pathways regulate lipid droplet homeostasis for the establishment of polarity in collagen sandwich-cultured hepatocytes." American Journal of Physiology-Cell Physiology 317, no. 5 (November 1, 2019): C942—C952. http://dx.doi.org/10.1152/ajpcell.00051.2019.
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Hepatocyte polarization is energy dependent. The establishment of polarization in collagen sandwich culture of hepatocytes requires utilization of lipid droplets and mitochondrial β-oxidation to supply ATP. Multiple cellular pathways are involved in lipid droplet homeostasis; however, mechanistic insights of how hepatocytes utilize lipid droplets during polarization remain elusive. The current study investigated the effects of various pathways involved in lipid droplet homeostasis on bioenergetics during hepatocyte polarization. The results showed that hepatocytes were dependent on lipolysis of lipid droplets to release fatty acids for β-oxidation. Inhibition of lipolysis significantly decreased cellular fatty acid and ATP levels and inhibited hepatocyte polarization, revealing that lipolysis was an important mechanism for providing energy for hepatocyte polarization. The results also demonstrated that autophagic degradation of lipid droplets (lipophagy) was not essential for breaking down lipid droplets. Conversely, autophagy contributed to lipid droplet formation and played a key role in sustaining lipid droplet stores for energy production. In addition, cholesterol biosynthesis/cholesterol esterification and de novo fatty acid synthesis also contributed to maintaining lipid droplet stores for bioenergetics during hepatocyte polarization. In summary, multiple cellular pathways are coordinated to maintain lipid droplet homeostasis and sustain fatty acid β-oxidation during hepatocyte polarization.
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Tolosa, Laia, Eugenia Pareja-Ibars, M. Teresa Donato, Miriam Cortés, Silvia López, Nuria Jiménez, José Mir, José V. Castell, and M. José Gómez-Lechón. "Neonatal Livers: A Source for the Isolation of Good-Performing Hepatocytes for Cell Transplantation." Cell Transplantation 23, no. 10 (October 2014): 1229–42. http://dx.doi.org/10.3727/096368913x669743.
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Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (β1-integrin, β-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug-metabolizing capability (cytochrome P450 and UDP-glucuronosyltransferase enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM+ staining), which could also participate in regeneration of liver parenchyma after transplantation. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human hepatocyte transplantation applicability.
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Ju, Wenjun, Atsushi Ogawa, Joerg Heyer, Dirk Nierhof, Liping Yu, Raju Kucherlapati, David A. Shafritz, and Erwin P. Böttinger. "Deletion of Smad2 in Mouse Liver Reveals Novel Functions in Hepatocyte Growth and Differentiation." Molecular and Cellular Biology 26, no. 2 (January 15, 2006): 654–67. http://dx.doi.org/10.1128/mcb.26.2.654-667.2006.
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ABSTRACT Smad family proteins Smad2 and Smad3 are activated by transforming growth factor β (TGF-β)/activin/nodal receptors and mediate transcriptional regulation. Although differential functional roles of Smad2 and Smad3 are apparent in mammalian development, the relative functional roles of Smad2 and Smad3 in postnatal systems remain unclear. We used Cre/loxP-mediated gene targeting for hepatocyte-specific deletion of Smad2 (S2HeKO) in adult mice and generated hepatocyte-selective Smad2/Smad3 double knockouts by intercrossing AlbCre/Smad2f/f (S2HeKO) and Smad3-deficient Smad3ex8/ex8 (S3KO) mice. All strains were viable and had normal adult liver. However, necrogenic CCL4-induced hepatocyte proliferation was significantly increased in S2HeKO compared to Ctrl and S3KO livers, and transplanted S2HeKO hepatocytes repopulated recipient liver at dramatically increased rates compared to Ctrl hepatocytes in vivo. Using primary hepatocytes, we found that TGF-β-induced G1 arrest, apoptosis, and epithelial-to-mesenchymal transition in Ctrl and S2HeKO but not in S3KO hepatocytes. Interestingly, S2HeKO cells spontaneously acquired mesenchymal features characteristic of epithelial-to-mesenchymal transition (EMT). Collectively, these results demonstrate that Smad2 suppresses hepatocyte growth and dedifferentiation independent of TGF-β signaling. Smad2 is not required for TGF-β-stimulated apoptosis, EMT, and growth inhibition in hepatocytes.
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Bluhme, Emil, Ewa Henckel, Roberto Gramignoli, Therese Kjellin, Christina Hammarstedt, Greg Nowak, Ahmad Karadagi, et al. "Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death." Cell Transplantation 31 (January 2022): 096368972110699. http://dx.doi.org/10.1177/09636897211069900.
Full textAbstract:
Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015–2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80–210] minutes. Median viability was 86% (IQR: 71%–91%). Median yield was 6.9 (IQR: 3.4–12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.
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Ambrosino, Giovanni, Stefano M. M. Basso, Sergio Varotto, Enrico Zardi, Antonio Picardi, and Davide F. D'amico. "Isolated Hepatocytes versus Hepatocyte Spheroids: In Vitro Culture of Rat Hepatocytes." Cell Transplantation 14, no. 6 (July 2005): 397–401. http://dx.doi.org/10.3727/000000005783982954.
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The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 ± 4.4%), while in G2 it was 72.1 ± 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.
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Wilson, D., and J. D. Yarbrough. "Autoradiographic analysis of hepatocytes in mirex-induced adaptive liver growth." American Journal of Physiology-Gastrointestinal and Liver Physiology 255, no. 1 (July 1, 1988): G132—G139. http://dx.doi.org/10.1152/ajpgi.1988.255.1.g132.
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The relationships between [3H]thymidine incorporation into hepatocyte nuclei, cell enlargement, and mitotic index were studied in intact (INT) and adrenalectomized (ADX) mirex-dosed rats. In INT mirex-dosed rats the sequence of events included the following: a biphasic response in nuclear labeling of mononuclear hepatocytes with peaks at 48 and 66 h postmirex dose, a peak in mitotic activity 66 h postmirex dose, and a significant increase in binuclear hepatocyte size 48 h postmirex dose. In ADX mirex-dosed rats the sequence of events included the following: a biphasic response in nuclear labeling of mononuclear hepatocytes with peaks at 24 and 48 h postmirex dose, a peak in mitotic activity 60 h postmirex dose, and a marginal increase in binuclear hepatocyte size 48 h postmirex dose. Corticosterone supplements to ADX mirex-dosed rats significantly reduced nuclear labeling of the mononuclear hepatocytes and increased the size of binuclear hepatocytes to that observed in INT mirex-dosed rats. This study demonstrates that adaptive liver growth consists of a hyperplastic response that involves mononuclear hepatocytes and a hypertrophic response that involves binuclear hepatocytes. Both responses appear to be modulated by corticosterone.
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Catapano, G., L. DE Bartolo, C. P. Lombardi, and E. Drioli. "The Effect of Catabolite Concentration on the Viability and Functions of Isolated Rat Hepatocytes." International Journal of Artificial Organs 19, no. 4 (April 1996): 245–50. http://dx.doi.org/10.1177/039139889601900407.
Full textAbstract:
The treatment of patients with hepatic failure by means of hybrid liver support devices using primary xenogeneic hepatocytes is currently hindered by the rapid loss of cell metabolic functions. Similarly to what happens with other mammalian cells, accumulation of catabolites in the neighborhood of cultured hepatocytes might significantly affect their viability and functions. In this paper, we investigated the effects of high concentrations of catabolites, such as ammonia and lactic acid, on the viability and functions of rat hepatocytes cultured on collagen coated Petri dishes. The effects on hepatocyte functions were established with respect to their ability to synthesize urea and to eliminate ammonia. Indeed, high catabolite concentrations effected both hepatocyte viability and functions. The number of viable hepatocytes decreased with increasing ammonia concentrations in the culture medium. High ammonia concentrations had also both an inhibitory and a toxic effect on hepatocyte functions. In fact, the hepatocytes synthesized urea and eliminated ammonia at rates that decreased with increasing ammonia concentrations. Similarly, high lactic acid concentrations were toxic to the cells and also inhibited their synthetic functions.
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Cui, Changhao, Shin Enosawa, Hitomi Matsunari, Hiroshi Nagashima, and Akihiro Umezawa. "Natural Flavonol, Myricetin, Enhances the Function and Survival of Cryopreserved Hepatocytes In Vitro and In Vivo." International Journal of Molecular Sciences 20, no. 24 (December 4, 2019): 6123. http://dx.doi.org/10.3390/ijms20246123.
Full textAbstract:
To improve the therapeutic potential of hepatocyte transplantation, the effects of the mitogen-activated protein kinase kinase 4 (MKK4) inhibitor, myricetin (3,3′,4′,5,5′,7-hexahydroxylflavone) were examined using porcine and human hepatocytes in vitro and in vivo. Hepatocytes were cultured, showing the typical morphology of hepatic parenchymal cell under 1–10 µmol/L of myricetin, keeping hepatocyte specific gene expression, and ammonia removal activity. After injecting the hepatocytes into neonatal Severe combined immunodeficiency (SCID) mouse livers, cell colony formation was found at 10–15 weeks after transplantation. The human albumin levels in the sera of engrafted mice were significantly higher in the recipients of myricetin-treated cells than non-treated cells, corresponding to the size of the colonies. In terms of therapeutic efficacy, the injection of myricetin-treated hepatocytes significantly prolonged the survival of ornithine transcarbamylase-deficient SCID mice from 32 days (non-transplant control) to 54 days. Biochemically, the phosphorylation of MKK4 was inhibited in the myricetin-treated hepatocytes. These findings suggest that myricetin has a potentially therapeutic benefit that regulates hepatocyte function and survival, thereby treating liver failure.
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Kaushansky, Alexis, Laura S. Austin, Sebastian A. Mikolajczak, Fang Y. Lo, Jessica L. Miller, Alyse N. Douglass, Nadia Arang, Ashley M. Vaughan, Malcolm J. Gardner, and Stefan H. I. Kappe. "Susceptibility to Plasmodium yoelii Preerythrocytic Infection in BALB/c Substrains Is Determined at the Point of Hepatocyte Invasion." Infection and Immunity 83, no. 1 (October 13, 2014): 39–47. http://dx.doi.org/10.1128/iai.02230-14.
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After transmission byAnophelesmosquitoes,Plasmodiumsporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible toPlasmodium yoeliipreerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitatePlasmodiumliver infection.
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Staricoff, M. A., R. D. Cohen, and J. P. Monson. "Carrier-mediated lactate entry into isolated hepatocytes from fed and starved rats: Zonal distribution and temperature dependence." Bioscience Reports 15, no. 2 (April 1, 1995): 99–109. http://dx.doi.org/10.1007/bf01200144.
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We examined the possibility of quantitative differences in lactate entry into periportal and perivenous hepatocytes under different nutritional states. The rate of14C-L(+)-lactate uptake was determined after 15-second incubations with freshly isolated zonally separated hepatocytes using a centrifuge stop technique at 37 °C and 4 °C, in the presence or absence of either differing amounts of unlabelled lactate or of a hepatocyte lactate transport inhibitor,α-cyano-3-hydroxycinnamate. Total entry as well as carrier mediated entry of14C-L(+)-lactate into the isolated cell populations was found to be similar in periportal and perivenous hepatocytes, irrespective of the nutritional state of the animal. Periportal and perivenous hepatocytes showed a greater tendency to transport lactate when isolated from starved animals, in agreement with previously reported data from non-zonally separated isolated hepatocytes. The activity of the hepatocyte plasma-membrane lactate transporter was diminished between fourfold and eightfold in transport studies conducted at 4 °C; similar results were obtained in unseparated and zonally separated suspensions. Temperature dependence of the hepatocyte transporter is markedly less than that reported for the erythrocyte transporter.
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Nussler, A. K., Z. Z. Liu, M. Di Silvio, M. A. Sweetland, D. A. Geller, J. R. Lancaster, T. R. Billiar, P. D. Freeswick, C. L. Lowenstein, and R. L. Simmons. "Hepatocyte inducible nitric oxide synthesis is influenced in vitro by cell density." American Journal of Physiology-Cell Physiology 267, no. 2 (August 1, 1994): C394—C401. http://dx.doi.org/10.1152/ajpcell.1994.267.2.c394.
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Hepatocyte plating density is known to affect cell function. Human and rat hepatocytes have been shown to express the inducible nitric oxide synthase (INOS) in response to cytokines plus lipopolysaccharide (LPS). The following studies were performed to determine the effects of hepatocyte plating density on the regulation of INOS. Rat hepatocytes were plated at densities from 10(4) to 20 x 10(4) hepatocytes/cm2 and stimulated with a combination of LPS, interferon-gamma, interleukin-1, and tumor necrosis factor. We found that NO2- plus NO3- released from stimulated hepatocytes declines with increasing hepatocyte density. Similar effects were seen for 3',5'-cyclic monophosphate release into supernatants and in the amount of nonheme iron-nitrosyl signals measured by electron paramagnetic resonance spectroscopy. Limitations of substrate (L-arginine) and 5,6,7,8-tetrahydrobiopterin were excluded as cause of the reduced nitric oxide generation at higher densities. Although mRNA levels for INOS were not influenced when measured at 24 h, there was a marked reduction in INOS enzyme activity and INOS protein detectable by Western blotting at higher cell density. Total protein synthesis decreased as hepatocyte density increased in both nonstimulated and stimulated hepatocytes at higher cell densities. These data suggest that reduced INOS translation may account for the density-dependent reduction in INOS activity in cultured hepatocytes. The importance of this phenomenon remains to be determined in vivo but has important implications for the in vitro study of INOS expression.
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Minnis-Lyons, Sarah E., Sofía Ferreira-González, Niya Aleksieva, Tak Yung Man, Victoria L. Gadd, Michael J. Williams, Rachel V. Guest, et al. "Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation." Science Signaling 14, no. 688 (June 22, 2021): eaay9185. http://dx.doi.org/10.1126/scisignal.aay9185.
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In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver. Here, we used a temporally restricted model of hepatic repair in which large-scale hepatocyte injury and regeneration are initiated through the acute loss of Mdm2 in hepatocytes, resulting in the rapid, coordinated proliferation of BECs. We found that transient, early activation of Notch1- and Notch3-mediated signaling and entrance into the cell cycle preceded the phenotypic expansion of BECs into hepatocytes. Notch inhibition reduced BEC proliferation, which resulted in failure of BECs to differentiate into hepatocytes, indicating that Notch-dependent expansion of BECs is essential for hepatocyte regeneration. Notch signaling increased the abundance of the insulin-like growth factor 1 receptor (IGF1R) in BECs, and activating IGFR signaling increased BEC numbers but suppressed BEC differentiation into hepatocytes. These results suggest that different signaling mechanisms control BEC expansion and hepatocyte differentiation.
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Kato, Kazuya, W. John B. Hodgson, Nader G. Abraham, Kazhuiko Onodera, Masato Imai, Shinichi Kasai, and Michio Mito. "Expression and Inducibility of Cytochrome P450 Iiia Family within Intrasplenically Transplanted Fetal Hepatocytes." Cell Transplantation 5, no. 1 (January 1996): 117–22. http://dx.doi.org/10.1177/096368979600500116.
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With the development of transplantation of hepatocytes into the spleen, interest has focused on the metabolic changes associated with hepatocyte proliferation. As these changes are important for drug metabolism in hepatocytes, we examined the expression and inducibility of the cytochrome P450 IIIA family within transplanted hepatocytes. Fetal hepatocytes were harvested at 20 days of gestation from spontaneously hypertensive rats (SHRs) and transplanted into recipient adult SHR spleens. Microscopic examination of the recipient spleens 4 and 10 wk after transplantation revealed masses of hepatocytes with cordlike structures in the red pulp. Proliferating hepatocytes were detected with a bromodeoxyuridine (BrdU) immunohistochemical stain. Immunochemical studies detected cytochromes (cytos) P450 p and P450 HLp in fetal hepatocytes before transplantation without prior induction. And although these cytos were not detected by 10 wk after transplantation, they were induced with dexamethasone. These results demonstrated that fetal hepatocytes can be transplanted successfully into recipient spleens and suggested that fetal hepatocytes grow in the spleen, similar to the adult hepatocyte response.
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Sheng, Liang, Bijie Jiang, and Liangyou Rui. "Intracellular lipid content is a key intrinsic determinant for hepatocyte viability and metabolic and inflammatory states in mice." American Journal of Physiology-Endocrinology and Metabolism 305, no. 9 (November 1, 2013): E1115—E1123. http://dx.doi.org/10.1152/ajpendo.00401.2013.
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The liver is an essential metabolic organ. In addition to metabolizing glucose and lipids, hepatocytes also secrete various cytokines that modulate both hepatocyte metabolism and liver inflammation. Hepatocyte injury and death and liver inflammation are the major contributors to liver diseases, including nonalcoholic steatohepatitis (NASH). Anatomic locations have a profound effect on hepatocyte metabolism, and liver zonation describes the metabolic heterogeneity of hepatocytes along the portovenous axis. However, it is unclear whether hepatocyte heterogeneity is affected by intrinsic factors and whether dietary fat, a risk factor for NASH, has distinct detrimental effects on different hepatocyte subpopulations. Here, we showed that mouse livers contained both high-lipid and low-lipid subpopulations of hepatocytes. The high-lipid subpopulation was more susceptible to injury and apoptosis and produced more proinflamatrory cytokines after treatment with endotoxin and saturated fatty acids. Dietary fat consumption further increased fatty acid uptake, intracellular lipid levels, hepatocyte injury and death, and the expression of proinflammatory cytokines in the high-lipid subpopulation. In contrast, dietary fat slightly increased lipid levels, cell death, and expression of proinflammatory cytokines in the low-lipid subpopulation. The low-lipid subpopulation produced more glucose. Fat consumption further activated the gluconeogenic program in the low-lipid, but not the high-lipid, subpopulations. These data suggest that intracellular lipid content is a key intrinsic determinant for hepatocyte heterogeneity of metabolic, inflammatory, and survival states.
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Johnson, Lynt B., John Aiken, David Mooney, Betsy L. Schloo, Linda Griffith-Cima, Robert Langer, and Joseph P. Vacanti. "The Mesentery as a Laminated Vascular Bed for Hepatocyte Transplantation." Cell Transplantation 3, no. 4 (July 1994): 273–81. http://dx.doi.org/10.1177/096368979400300403.
Full textAbstract:
The small bowel mesentery provides a unique structure of a large vascularized surface area to support hepatocyte transplantation. Cell-seeded polymeric matrices can be juxtaposed in a relatively atraumatic manner between leaves of mesentery such that adequate exchange of nutrients and diffusion of gases can proceed in the interim while neovascularization occurs. Hepatocytes obtained from (RHA) Wistar rats by collagenase perfusion were seeded onto non-woven filamentous sheets of polyglycolic acid 1 × 3 cm in size and 2 mm thickness to a density of 500,000 cells/cm2. Twenty-six recipient Gunn rats (UDP-glucuronyl transferase deficient) underwent laparotomy. Hepatocyte-ladened polymer sheets were placed between leaves of mesentery. Eight sheets were placed per animal and the leaves were approximated, creating a functional implant 1 × 3 × 2 cm. Biopsies between 5-99 days after implantation revealed neovascularization, moderate inflammatory reaction and the presence of viable hepatocytes in 96% (25/26). Immunoperoxidase studies using anti-albumin antibody substantiated hepatocyte specific function in implants. HPLC profiles of bile from Gunn rats transplanted with hepatocytes from congeneic (RHA) rats demonstrated the presence of bilirubin conjugates. There were no conjugation fractions seen in control gunn rats without hepatocyte transplantation. Although total serum bilirubin did not significantly decrease, conjugated bilirubin was identified in 46% (12/26) animals after transplantation with congeneic hepatocytes. We conclude that the mesentery of the small bowel provides a large vascularized surface for cell transplantation. Large numbers of metabolically active hepatocytes can engraft, vascularize, and show function. The mesentery may be a potential bed for clinical hepatocyte transplantation.
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Borkham-Kamphorst, Erawan, Ute Haas, Eddy Van de Leur, Anothai Trevanich, and Ralf Weiskirchen. "Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response." International Journal of Molecular Sciences 21, no. 15 (July 23, 2020): 5230. http://dx.doi.org/10.3390/ijms21155230.
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The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2−/− mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2−/− mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2−/− mice. Lcn2−/− mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2−/−, and Chop null (Chop−/−) primary hepatocytes and found Chop−/− hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop−/− hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop−/− mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.
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Rogler, Charles E., Remon Bebawee, Joe Matarlo, Joseph Locker, Nicole Pattamanuch, Sanjeev Gupta, and Leslie E. Rogler. "Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver." Journal of Histochemistry & Cytochemistry 65, no. 1 (November 24, 2016): 33–46. http://dx.doi.org/10.1369/0022155416675153.
Full textAbstract:
Recent investigations have reported many markers associated with human liver stem/progenitor cells, “oval cells,” and identified “niches” in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as “micro-niches.” Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.
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Weerasinghe, Sujith V. W., You-Jin Jang, Robert J. Fontana, and M. Bishr Omary. "Carbamoyl phosphate synthetase-1 is a rapid turnover biomarker in mouse and human acute liver injury." American Journal of Physiology-Gastrointestinal and Liver Physiology 307, no. 3 (August 1, 2014): G355—G364. http://dx.doi.org/10.1152/ajpgi.00303.2013.
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Several serum markers are used to assess hepatocyte damage, but they have limitations related to etiology specificity and prognostication. Identification of novel hepatocyte-specific biomarkers could provide important prognostic information and better pathogenesis classification. We tested the hypothesis that hepatocyte-selective biomarkers are released after subjecting isolated mouse hepatocytes to Fas-ligand-mediated apoptosis. Proteomic analysis of hepatocyte culture medium identified the mitochondrial matrix protein carbamoyl phosphate synthetase-1 (CPS1) among the most readily detected proteins that are released by apoptotic hepatocytes. CPS1 was also detected in mouse serum upon acute challenge with Fas-ligand or acetaminophen and in hepatocytes upon hypoosmotic stress, independent of hepatocyte caspase activation. Furthermore, CPS1 was observed in sera of mice chronically fed the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mouse CPS1 detectability was similar in serum and plasma, and its half-life was 126 ± 9 min. Immune staining showed that CPS1 localized to mouse hepatocytes but not ductal cells. Analysis of a few serum samples from patients with acute liver failure (ALF) due to acetaminophen, Wilson disease, or ischemia showed readily detectable CPS1 that was not observed in several patients with chronic viral hepatitis or in control donors. Notably, CPS1 rapidly decreased to undetectable levels in sera of patients with acetaminophen-related ALF who ultimately recovered, while alanine aminotransferase levels remained elevated. Therefore, CPS1 becomes readily detectable upon hepatocyte apoptotic and necrotic death in culture or in vivo. Its abundance and short serum half-life, compared with alanine aminotransferase, suggest that it may be a useful prognostic biomarker in human and mouse liver injury.
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Jones, B. A., Y. P. Rao, R. T. Stravitz, and G. J. Gores. "Bile salt-induced apoptosis of hepatocytes involves activation of protein kinase C." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 5 (May 1, 1997): G1109—G1115. http://dx.doi.org/10.1152/ajpgi.1997.272.5.g1109.
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Toxic bile salts induce hepatocyte apoptosis, a model relevant to liver injury during cholestasis. However, the signaling mechanisms culminating in bile salt-induced apoptosis remain unclear. Because protein kinase C (PKC) is activated by bile salts in hepatocytes and causes apoptosis in other cells, we tested the hypothesis that bile salt-induced hepatocyte apoptosis is mediated by PKC. The PKC inhibitors chelerythrine and Go-6976 reduced, whereas a PKC agonist, phorbol 12-myristate 13-acetate (PMA), increased glycochenodeoxycholate (GCDC)-induced hepatocyte apoptosis. Membrane-associated total PKC activity was increased in GCDC-treated hepatocytes. Quantitative immunoblot analysis demonstrated membrane translocation of PKC-alpha, PKC-delta, and PKC-epsilon to hepatocyte membranes after administration of GCDC. Direct activation of PKC-alpha and PKC-delta by GCDC was also demonstrated using recombinant, baculovirus-expressed PKC isoforms in a medium of defined lipid composition. Chelerythrine and Go-6976 reduced, whereas PMA enhanced, cathepsin B activity during treatment of hepatocytes with GCDC, demonstrating coupling of PKC activity to the protease effector mechanisms of apoptosis. In conclusion, our data suggest for the first time that PKC-dependent signaling pathways play a critical role in bile salt-induced hepatocyte apoptosis.
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Anderson, K., R. Andrews, L. Yin, R. McLeod, C. MacDonald, J. D. Hayes, and M. H. Grant. "Cytotoxicity of xenobiotics and expression of glutathione-S-transferases in immortalised rat hepatocyte cell lines." Human & Experimental Toxicology 17, no. 3 (March 1998): 131–37. http://dx.doi.org/10.1177/096032719801700301.
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1 Immortalised rat hepatocyte cell lines are more sensitive to the cytotoxicity of 1-chloro-2,4-dinitroben-zene and ethacrynic acid than primary cultures of hepatocytes. 2 Class alpha glutathione S-transferases are not expressed in immortalised hepatocyte cell lines. Class pi glutathione S-transferase expression is elevated in the immortalised cell lines compared with freshly isolated hepatocytes, but it is not as high as in the HTC rat hepatoma cell line. 3 Immortalised hepatocyte cell lines may provide a sensitive model system for detecting cytotoxicity associated with xenobiotics which are detoxified by glutathione S-transferases.
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Chouhan, Manil, Juliana Puppi, Estela Solanas, Ragai R. Mitry, Anil Dhawan, and Robin D. Hughes. "Hepatocyte Labeling with 99mTc-GSA: A Potential Non-Invasive Technique for Tracking Cell Transplantation." International Journal of Artificial Organs 35, no. 6 (May 4, 2012): 450–57. http://dx.doi.org/10.5301/ijao.5000096.
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Background: Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation, however, the fate of transplanted hepatocytes is not well defined. 99mTc-galactosyl-serum albumin (99mTc-GSA) is a clinical scintigraphic agent which is specifically taken up by the hepatocyte asialoglycoprotein receptor (ASGPR). Aims: To investigate labeling of fresh and cryopreserved human hepatocytes and fresh rat hepatocytes in vitro using 99mTc-GSA Methods: Human and rat hepatocytes were isolated from liver tissue by collagenase perfusion. The ASGPR were characterized using immunohistochemistry and RT-PCR. Hepatocytes were incubated with 99mTc-GSA in suspension at 4°C and 37°C. Cell viability and function was determined using cell mitochondrial dehydrogenase (MTS) and sulphorhodamine B (SRB) assays. Results: Fresh and cryopreserved human hepatocytes expressed the ASGPR. Incubation of hepatocytes in suspension with 99mTc-GSA reduced the viability of hepatocytes, but this was similar to unlabeled control cells. Greater loss of viability was seen on incubation at 37°C compared to 4°C, but there was a significantly greater uptake of 99mTc-GSA at the physiological temperature (6.6 ± SE 0.6-fold increase, p<0.05) consistent with ASGPR-mediated endocytosis. MTS and SRB assays were not significantly affected by labeling with 99mTc-GSA in all three cell types. A mean of 18.5% of the radioactivity was released over 120 min when 99mTc-GSA - labeled hepatocytes were shaken in vitro at 37°C. Conclusions: Human and rat hepatocytes can be labeled with 99mTc-GSA, which may have potential application for in vivo imaging after hepatocyte transplantation.
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Voieikova, D., L. Stepanova, O. Savchuk, L. Ostapchenko, and M. Kondro. "Protein analysis of rat hepatocytes under conditions glutamate-induced obesity and its correction." Bulletin of Taras Shevchenko National University of Kyiv. Series: Biology 70, no. 2 (2015): 81–84. http://dx.doi.org/10.17721/1728_2748.2015.70.81-84.
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We had characterized low-, medium- and high-molecular protein fractions of hepatocytes under development of glutamate-induced obesity and correction of nanocrystalline cerium dioxide and pioglitazone. Protein fractions were separated by electrophoresis using a 10 % Laemmli SDS-PAGE sodium dodecyl sulfate. Protein hepatocytes change under glutamate-induced obesity: high-protein reduced, and low-protein increased. Changes in hepatocyte proteins are consistent with previously established changes in protein content of hepatocytes under the influence HCD rich in fats and carbohydrates. We had noticed similar changes in protein of hepatocytes under correction cerium dioxide, but compared with glutamate-induced obesity, low proteins were lower. Pioglitazone didn't show a positive effect on hepatocyte proteins that may be associated with short-term administration.
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Ernst, Linda M., Nancy B. Spinner, David A. Piccoli, Joanne Mauger, and Pierre Russo. "Interlobular Bile Duct Loss in Pediatric Cholestatic Disease is Associated with Aberrant Cytokeratin 7 Expression by Hepatocytes." Pediatric and Developmental Pathology 10, no. 5 (September 2007): 383–90. http://dx.doi.org/10.2350/06-09-0171.1.
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The objective of this study was to determine whether aberrant hepatic expression of cytokeratin 7 (CK7) and/or other putative stem cell markers is seen in pediatric cholestatic diseases. Eighteen liver biopsies and 14 liver explants from pediatric patients with extrahepatic biliary atresia (EHBA), Alagille syndrome (AGS), primary sclerosing cholangitis (PSC), inborn errors of bile acid synthesis, and progressive familial intrahepatic cholestasis (PFIC) were examined along with 5 histologically normal control liver biopsies. Immumohistochemical stains (CK7, CD56, and OV6) were performed on paraffin-embedded tissue. Staining of interlobular bile ducts (ILBD), proliferating bile ductules, and hepatocytes was scored using a semiquantitative scale. There were significant differences in CK7 staining of hepatocytes among the cholestatic diseases ( P < 0.006). All cases with AGS showed CK7 hepatocyte staining, while EHBA and PSC had variable hepatocyte staining. Patients with PFIC had prominent CK7 hepatocyte staining, while those with inborn errors of bile acid synthesis had little. Control biopsies showed rare hepatocyte staining. Analysis based on the presence or absence of ILBD revealed significantly more CK7 hepatocyte staining in cases with loss of ILBD ( P < 0.001). CD56 staining of hepatocytes was also present more frequently in cases with absent or reduced ILBD. Regardless of underlying disease, loss of ILBD is a major determinant of aberrant expression of CK7 by hepatocytes. Aberrant CK7 expression may reflect a metaplastic change to a “stem cell” phenotype induced by loss of contact with the more distal biliary tree.
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Okumura, Nobuaki, Tomohiko Koh, Yuichi Hasebe, Taiichiro Seki, and Toyohiko Ariga. "A Novel Function of Thrombin-activatable Fibrinolysis Inhibitor during Rat Liver Regeneration and in Growth-promoted Hepatocytes in Primary Culture." Journal of Biological Chemistry 284, no. 24 (April 22, 2009): 16553–61. http://dx.doi.org/10.1074/jbc.m109.011452.
Full textAbstract:
Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl4)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.
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Hisaka, Toru, Bernard Lardeux, Thierry Lamireau, Torsten Wüestefeld, Patricia Lalor, Véronique Neaud, Patrick Maurel, et al. "Expression of tissue factor pathway inhibitor-2 in murine and human liver regulation during inflammation." Thrombosis and Haemostasis 91, no. 03 (2004): 569–75. http://dx.doi.org/10.1160/th03-06-0358.
Full textAbstract:
SummaryTissue factor pathway inhibitor-2 (TFPI-2) is a recently described serine proteinase inhibitor. Human and murine TFPI-2 share about 50% homology. The aim of this study was to investigate the cellular localization of human and murine TFPI-2 in the liver and the regulation of their expression during acute inflammation. Northern blot, in situ hybridization and studies on isolated hepatocytes demonstrated a high-level expression of TFPI-2 in murine hepatocytes. On the other hand, very little TFPI-2 mRNA expression could be detected in human liver. Studies with isolated human liver cells suggested that TFPI-2 expression in human liver was mainly observed in liver sinusoidal endothelial cells rather than hepatocytes. Liver murine TFPI-2 expression was greatly increased after lipopolysaccharide administration with a delayed kinetics as compared to α1-acid glycoprotein, a classical acute-phase reactant. Accordingly, studies with isolated cells showed that the increase in TFPI-2 transcripts occurred in non-hepatocytic cells. Moreover, the LPS response was abolished in mice with a hepatocyte-specific KO for the gp130 receptor, thus indicating that a mediator from hepatocytes is involved in the up-regulation of TFPI-2 in non-parenchymal cells. In conclusion, murine TFPI-2 is highly expressed in hepatocytes in the normal murine liver and is upregulated in non-parenchymal cells in the context of inflammation. The large difference in the level of liver expression of human and murine TFPI-2 suggests that despite significant sequence similarities, these proteins presumably have different functions in the two species.
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Grant, M. H., S. J. Smith, and M. D. Burke. "Strain differences in the maintenance of cytochrome P-450 and mixed-function-oxidase activities in cultured rat hepatocytes Effect of prostaglandins." Biochemical Journal 239, no. 3 (November 1, 1986): 785–88. http://dx.doi.org/10.1042/bj2390785.
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The mixed-function-oxidase (MFO) activities, ethoxyresorufin and pentoxyphenoxazone O-dealkylase, of cultured Hooded-Lister(HL)-rat hepatocytes declined rapidly during 72 h of culture, whereas in Sprague-Dawley(SD)-rat hepatocytes the MFO activities increased between 24 and 72 h in culture. Cytochrome P-450 content declined at the same rate in both HL- and SD-rat hepatocyte cultures. NADPH:cytochrome c reductase and NADH:cytochrome b5 reductase were more stable in SD- than in HL-rat hepatocyte cultures. 16,16-Dimethylprostaglandins E2 and F2 alpha improved the maintenance of cytochrome P-450 content, MFO activity and NADPH:cytochrome c reductase in the HL-rat hepatocyte cultures. In SD-rat hepatocytes, the prostaglandins had no effect on cytochrome P-450 content or NADPH:cytochrome c reductase activity, whereas they prevented the increase observed in MFO activities between 24 and 72 h after culture.
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Wu, Zhi-Tao, Dan Yao, Shu-Yi Ji, Xuan Ni, Yi-Meng Gao, Li-Jian Hui, and Guo-Yu Pan. "Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology." Cellular Physiology and Biochemistry 38, no. 5 (2016): 1815–30. http://dx.doi.org/10.1159/000443120.
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Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.
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Billiar, T. R., R. D. Curran, B. G. Harbrecht, J. Stadler, D. L. Williams, J. B. Ochoa, M. Di Silvio, R. L. Simmons, and S. A. Murray. "Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes." American Journal of Physiology-Cell Physiology 262, no. 4 (April 1, 1992): C1077—C1082. http://dx.doi.org/10.1152/ajpcell.1992.262.4.c1077.
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Hepatocytes are known to synthesize nitric oxide (NO) from L-arginine via an inducible NO synthase. Studies were performed to determine the relationship between hepatocyte NO production and the stimulation of hepatocyte soluble guanylate cyclase. A combination of lipopolysaccharide (LPS), interferon-gamma, tumor necrosis factor, and interleukin-1 stimulates the biosynthesis of large quantities of nitrite and nitrate (NO2- + NO3-). Hepatocyte NO2- + NO3- production was associated with only small increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels but much greater increases in extracellular cGMP release over an 18-h time period. This cGMP synthesis was dependent on the L-arginine concentration and was inhibited in a reversible manner by NG-monomethyl-L-arginine. The cytokines or LPS added alone induced small increases in nitrogen oxide production and concomitant minor elevations in cGMP release. Atrial natriuretic peptide also stimulated the release of cGMP by hepatocytes which appeared to be independent of the cytokine+LPS-induced cGMP release. The addition of probenecid reduced the cGMP release by 66%, while cell damage was excluded as a cause for the extracellular release. Addition of 3-isobutyl-1-methylxanthine, but not M&B 22948, increased hepatocyte intra- and extracellular cGMP levels after cytokine+LPS stimulation. Induction of nitrogen oxide synthesis by hepatocytes in vivo by injecting rats with killed Corynebacterium parvum resulted in increased cGMP levels in freshly isolated hepatocytes and increased cGMP release by the hepatocytes when placed in culture.(ABSTRACT TRUNCATED AT 250 WORDS)