Journal articles on the topic 'Hepatocyte'
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1
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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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.
3
Fan, J. Y., G. Dama, Y. L. Liu, W. Y. Guo, and J. T. Lin. "Combinational Overexpression of <i>Foxa3</i> and <i>Hnf4a</i> Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes." Молекулярная биология 57, no. 4 (July 1, 2023): 668–70. http://dx.doi.org/10.31857/s0026898423040031.
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In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4a, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.
4
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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Flendrig, L. M., D. Sommeijer, N. C. J. J. Ladiges, A. A. Te Velde, M. A. W. Maas, G. G. A. Jörning, J. Daalhuisen, and R. A. F. M. Chamuleau. "Commercially Available Media for Flushing Extracorporeal Bioartificial Liver Systems Prior to Connection to the Patient's Circulation: An in vitro Comparative Study in Two and Three Dimensional Porcine Hepatocyte Cultures." International Journal of Artificial Organs 21, no. 8 (August 1998): 467–72. http://dx.doi.org/10.1177/039139889802100803.
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Extracorporeal bioartificial liver (BAL) systems based on hepatocytes need to be flushed before clinical application, as hepatocyte culture media are not approved for medical use. Commercially available 0.9% NaCl solution and hemofiltration solution (both supplemented with 10% human albumin) were investigated in vitro to test their potential to wash BAL systems with minimal stress for the cultured hepatocytes. After a 2 hour incubation, the lidocaine metabolising capacity and release of liver enzymes were assessed. As hepatocytes have been cultured in bioreactors in either two or three dimensional cell configurations, we tested the media in respectively hepatocyte monolayers cultures and in our newly developed bioreactor in which hepatocytes reorganise as small hepatocyte aggregates. The three dimensional hepatocyte cultures tolerated both media well, and no significant differences were seen compared with hepatocytes cultured in Williams’ E (reference hepatocyte culture medium). The two dimensional hepatocyte cultures tolerated the supplemented hemofiltration solution and the reference medium equally well, but the condition of the porcine hepatocytes monolayer cultures was significantly impaired when incubated with the supplemented physiological saline solution. In conclusion, as a supplemented physiological saline solution may have detrimental effects on the condition of the hepatocytes, the more complex hemofiltration solution (bicarbonate buffered, glucose, essential minerals) was considered the better alternative for flushing bioartificial liver systems.
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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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Zhu, Y. X., L. Zhu, Y. F. Chen, J. M. Xu, Z. L. Shen, R. J. Liu, J. Zou, Mingqing Yuan, Fan Ye, and Qingqi Zeng. "Luteoloside Ameliorates Palmitic Acid-Induced in Vitro Model of Non-alcoholic Fatty Liver Disease via Activating STAT3-Triggered Hepatocyte Regeneration." Folia Biologica 67, no. 3 (2021): 126–33. http://dx.doi.org/10.14712/fb2021067030126.
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Luteoloside (Lute), a bioactive natural ingredient, widely exists in nature and possesses hepatoprotective and hepatocyte proliferation-promoting properties. This study aimed to investigate whether Lute could counteract non-alcoholic fatty liver disease (NAFLD)-caused hepatocyte damage via its stimulation of hepatocyte regeneration efficacy and to explore the involved mechanism. LO2 cells and primary hepatocytes were used to examine the hepatocyte proliferation effects of Lute under physiological conditions and in the palmitic acid (PA)- induced in vitro model of NAFLD. STAT3 and cell cycle-related proteins (cyclin D1, c-myc and p21) were evaluated by Western blot. Under physiological conditions, LO2 cells and primary hepatocytes treated with various concentration of Lute for 12 and 24 h showed increased hepatocyte proliferation, especially with 20 μM treatment for 24 h. More notably, under the model conditions, co-incubation with 20 μM of Lute also markedly reversed PA-induced inhibition of cell proliferation and viability in primary hepatocytes. Mechanistically, Lute could activate STAT3 and subsequently increase cyclin D1 and cmyc expression, which positively regulates cell cycle progression, and decrease expression of p21, an inhibitor of cell cycle progression. Furthermore, Luteinduced hepatocyte proliferation-promoting efficacy was abolished by STAT3 inhibitor stattic. Collectively, Lute can alleviate PA-induced hepatocyte damage via activating STAT3-mediated hepatocyte regeneration.
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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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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.
10
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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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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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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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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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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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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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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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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Hou, Yung-Te, Chia-Chun Wu, Wen-Ting Wang, Wen-Tse Yang, Ying-Hsiu Liao, and Chien-Yu Chen. "Monitoring Cultured Rat Hepatocytes Using RNA-Seq In Vitro." International Journal of Molecular Sciences 24, no. 8 (April 19, 2023): 7534. http://dx.doi.org/10.3390/ijms24087534.
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Compared to other techniques, RNA sequencing (RNA-Seq) has the advantage of having details of the expression abundance of all transcripts in a single run. In this study, we used RNA-Seq to monitor the maturity and dynamic characteristics of in vitro hepatocyte cultures. Hepatocytes, including mature hepatocytes and small hepatocytes, were analyzed in vitro using RNA-Seq and quantitative polymerase chain reaction (qPCR). The results demonstrated that the gene expression profiles measured by RNA-Seq showed a similar trend to the expression profiles measured by qPCR, and can be used to infer the success of in vitro hepatocyte cultures. The results of the differential analysis, which compared mature hepatocytes against small hepatocytes, revealed 836 downregulated and 137 upregulated genes. In addition, the success of the hepatocyte cultures could be explained by the gene list screened from the adopted gene enrichment test. In summary, we demonstrated that RNA-Seq could become an effective method for monitoring the whole transcriptome of hepatocyte cultures and provide a more comprehensive list of factors related to the differentiation of small hepatocytes into mature hepatocytes. This monitoring system not only shows high potential in medical applications but may also be a novel method for the clinical diagnosis of liver-related diseases.
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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.
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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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Lee, Ji-Hyun, Hey-Jung Park, Young-A. Kim, Doo-Hoon Lee, Jeong-Kwon Noh, Jong-Gab Jung, Hee-Hoon Yoon, Suk-Koo Lee, and Sanghoon Lee. "Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an “Off-the-Shelf” Bioartificial Liver System." Bioengineering 9, no. 12 (November 29, 2022): 738. http://dx.doi.org/10.3390/bioengineering9120738.
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To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.
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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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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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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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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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Wei, Miaomiao, Xinlong Yan, Xin Xin, Haiqiang Chen, Lingling Hou, and Jinhua Zhang. "Hepatocyte-Specific Smad4 Deficiency Alleviates Liver Fibrosis via the p38/p65 Pathway." International Journal of Molecular Sciences 23, no. 19 (October 2, 2022): 11696. http://dx.doi.org/10.3390/ijms231911696.
Full textAbstract:
Liver fibrosis is a wound-healing response caused by the abnormal accumulation of extracellular matrix, which is produced by activated hepatic stellate cells (HSCs). Most studies have focused on the activated HSCs themselves in liver fibrosis, and whether hepatocytes can modulate the process of fibrosis is still unclear. Sma mothers against decapentaplegic homologue 4 (Smad4) is a key intracellular transcription mediator of transforming growth factor-β (TGF-β) during the development and progression of liver fibrosis. However, the role of hepatocyte Smad4 in the development of fibrosis is poorly elucidated. Here, to explore the functional role of hepatocyte Smad4 and the molecular mechanism in liver fibrosis, a CCl4-induced liver fibrosis model was established in mice with hepatocyte-specific Smad4 deletion (Smad4Δhep). We found that hepatocyte-specific Smad4 deficiency reduced liver inflammation and fibrosis, alleviated epithelial-mesenchymal transition, and inhibited hepatocyte proliferation and migration. Molecularly, Smad4 deletion in hepatocytes suppressed the expression of inhibitor of differentiation 1 (ID1) and the secretion of connective tissue growth factor (CTGF) of hepatocytes, which subsequently activated the p38 and p65 signaling pathways of HSCs in an epidermal growth factor receptor-dependent manner. Taken together, our results clearly demonstrate that the Smad4 expression in hepatocytes plays an important role in promoting liver fibrosis and could therefore be a promising target for future anti-fibrotic therapy.
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Jóźawiak, A., W. Karlik, M. Wiechetek, and A. Weryńaski. "Attachment and Metabolic Activity of Hepatocytes Cultivated on Selected Polymeric Membranes." International Journal of Artificial Organs 21, no. 8 (August 1998): 460–66. http://dx.doi.org/10.1177/039139889802100807.
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Plasma or hormones added to hepatocyte incubation media mask the function of membranes as substrata per se for hepatocyte adhesion. This hypothesis was verified with hepatocyte cultures on various membranes in serum and hormone free medium. Freshly isolated rat hepatocytes were seeded on flat sheet membranes made of Cellulose Acetate (CA), aminated Cellulose Acetate (aCA), polysulfone (PSf) and sulfonated polysulfone (sPSf) and incubated in Hank's Balanced Salts Solutions (HBSS) as well as in William's E medium supplemented with newborn calf serum. It was found that PSf promoted hepatocyte adhesion most effectively. Good properties of PSf as a biomaterial for hepatocyte culture were confirmed in both media cultures. Urea synthesis and ammonia utilization measured in hepatocytes cultured on PSf were higher compared with other membrane cultures. PSf secured longer viability for a higher number of cells seeded on membrane compared with other investigated membranes, which is the reason for higher metabolic activity in PSf culture.
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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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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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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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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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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.
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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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Won, Jong-Ho, Dong-Ho Choi, Jung-Hoon Kim, Sook-Ja Kim, Hee-Jeong Cheung, Nam-Su Lee, and Hee-Sook Park. "Human Mesenchymal Stem Cells Injected Via Portal Vein Are Differentiated into Human Hepatocytes in Regenerating Rat Liver." Blood 106, no. 11 (November 16, 2005): 1694. http://dx.doi.org/10.1182/blood.v106.11.1694.1694.
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Abstract Objectives: Human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Evidence has been accumulated to indicate that certain compartments of bone marrow cells are capable to differentiating into hepatocytes in vitro. In this study we attempted to examine the differentiation ability of human MSCs into hepatocytes in vitro and in vivo by injected them into rat portal vein in partially resected rat liver model. Materials and Methods: MSCs were isolated from human bone marrow and induced differentiation with our protocol containing hepatocyte growth factor in vitro. Four - to - 5 week-old female Sprague Dawley rats were used for xenotransplantation model. Culture expanded MSCs (5 X 106 cells/rat) were injected into the portal vein and 70% hepatectomy was performed on the subsequent day. All rats were immunosuppressed with a daily intraperitoneal injection of cyclosporine A. Results: The morphology of the MSCs was changed into hepatocyte-like cells after in vitro culture for 28days and expression of hepatocyte specific genes also confirmed with RT-PCR and immunohistochemical stain. Transplanted MSCs differentiated into hepatocytes and they surprisingly composed hepatic cords with expression of the human albumin and human hepatocyte specific genes at 21 days after infusion. Conclusion: We have demonstrated that human MSCs can differentiate into functional hepatocyte-like cells in vitro and in vivo. Therefore, human MSCs may become an alternative source to hepatocyte regeneration or liver cell transplantation.
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Hooser, S. B., V. R. Beasley, E. J. Basgall, W. W. Carmichael, and W. M. Haschek. "Microcystin-LR-induced Ultrastructural Changes in Rats." Veterinary Pathology 27, no. 1 (January 1990): 9–15. http://dx.doi.org/10.1177/030098589002700102.
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The ultrastructure of hepatic, pulmonary, and renal lesions was evaluated in rats injected intraperitoneally with a lethal dose of microcystin-LR (MCLR, 160 μg/kg), a cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Hepatic lesions were first Seen at 10 minutes post-dosing and consisted of mild widening of hepatocyte intercellular spaces centrilobularly. At 20 minutes post-dosing, hepatocyte plasma membrane alterations were more pronounced, consisting of plasma membrane invagination with formation of variably sized and shaped intracytoplasmic vacuoles, loss of microvilli along the sinusoidal face, and widespread, pronounced hepatocyte separation. By 30 minutes, the space of Disse was markedly widened. At 60 minutes post-dosing, centrilobular areas contained necrotic cells and apparently intact, isolated, organelles intermingled with erythrocytes and platelets. In less severely affected regions there was prominent hepatocyte rounding, and erythrocytes and platelets were present in the widened space of Disse. Large amounts of hepatocellular debris and intact hepatocytes were present in the pulmonary vasculature, while smaller amounts of debris were also seen in the glomerular and peritubular capillaries of the renal cortex. This study shows that initial lesions are confined to Shape changes in the plasma membrane of hepatocytes. These changes are consistent with the hypothesis that microcystin-LR induces alterations in the hepatocyte cytoskeleton. Later changes consist of hepatocyte disassociation and necrosis, as well as endothelial damage, which allow release of hepatocytes and debris into the circulation with microembolism in lungs and kidneys.
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Damo, Martina, D. Scott Wilson, and Jeffrey A. Hubbell. "Hepatocyte-dependent cross-presentation of soluble antigens induces antigen-specific CD8+ T cell tolerance." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 197.19. http://dx.doi.org/10.4049/jimmunol.196.supp.197.19.
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Abstract The ability of hepatocytes to cross-present soluble extracellular antigens and the related immunological outcomes remain poorly investigated. Our work shows that murine primary hepatocytes actively uptake and process extracellular antigens in EEA1+ and TAP1+ phagosomes, which are functionally related to cross-presentation. In fact EEA1+TAP1+ organelles, which are one of the signatures of professional cross-presenting cells, are also found in the cytoplasm of hepatocytes. Moreover, in vitro hepatocyte-dependent cross-presentation of OVA to OT-I cells is sensitive to specific drug inhibitors of endosomal and proteasomal activity, providing direct evidence that endocytosed antigens enter the cross-presentation pathway. In vivo, cross-presenting hepatocytes induce tolerance of adoptively transferred OT-I cells by clonal deletion, as pro-apoptotic markers are upregulated on hepatocyte-educated OT-I cells. Non-deleted OT-I cells show instead significantly reduced response to vaccination, indicative of anergy. Active engulfment or emperipolesis of antigen cross-presenting hepatocytes by OT-I cells is also detected in vitro, as an additional means of T cell deletion. PD-1/PD-L1 interactions participate in the induction of hepatocyte-dependent cross-tolerance, as specific blockage of PD-L1 on hepatocytes significantly reduces the development of hepatocyte-dependent cross-tolerance. Due to their physiologic metabolic functions and their capacity to cross-present extracellular antigens here described, we propose hepatocytes as one of the major players in the establishment of antigen-specific CD8+ T cell peripheral tolerance.
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Donato, María Teresa, Agustín Lahoz, Sandra Montero, Ana Bonora, Eugenia Pareja, José Mir, José V. Castell, and María José Gómez-Lechón. "Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation." Cell Transplantation 17, no. 10-11 (October 2008): 1211–19. http://dx.doi.org/10.3727/096368908787236620.
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Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM ClNH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.
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Tackett, Bryan C., Hongdan Sun, Yu Mei, Janielle P. Maynard, Sayuri Cheruvu, Arunmani Mani, Andres Hernandez-Garcia, Nadarajah Vigneswaran, Saul J. Karpen, and Sundararajah Thevananther. "P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy." American Journal of Physiology-Gastrointestinal and Liver Physiology 307, no. 11 (December 1, 2014): G1073—G1087. http://dx.doi.org/10.1152/ajpgi.00092.2014.
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Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2−/−) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24–72 h) in response to 70% PH were impaired in P2Y2−/− mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2−/− remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2−/− mice were treated with ATP or ATPγS for 5–120 min and 12–24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.
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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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Shibuya, Kazuaki, Masaaki Watanabe, Ryoichi Goto, Masaaki Zaitsu, Yoshikazu Ganchiku, and Akinobu Taketomi. "The Efficacy of the Hepatocyte Spheroids for Hepatocyte Transplantation." Cell Transplantation 30 (January 1, 2021): 096368972110000. http://dx.doi.org/10.1177/09636897211000014.
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The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes ( p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 106 cells) or isolated hepatocytes (fSH, 3.6 × 106 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.
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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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Jitraruch, Suttiruk, Anil Dhawan, Robin D. Hughes, Celine Filippi, Sharon C. Lehec, Leanne Glover, and Ragai R. Mitry. "Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation." Cell Transplantation 26, no. 8 (August 2017): 1341–54. http://dx.doi.org/10.1177/0963689717720050.
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Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.
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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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Malhi, Harmeet, Maria Eugenia Guicciardi, and Gregory J. Gores. "Hepatocyte Death: A Clear and Present Danger." Physiological Reviews 90, no. 3 (July 2010): 1165–94. http://dx.doi.org/10.1152/physrev.00061.2009.
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The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including drugs and alcohol, participation in lipid and fatty acid metabolism, its unique role in the enterohepatic circulation of bile acids, the widespread prevalence of hepatotropic viruses, and its existence within a milieu of innate immune responding cells. Apoptosis and necrosis are the most widely recognized forms of hepatocyte cell death. The hepatocyte displays many unique features regarding cell death by apoptosis. It is quite susceptible to death receptor-mediated injury, and its death receptor signaling pathways involve the mitochondrial pathway for efficient cell killing. Also, death receptors can trigger lysosomal disruption in hepatocytes which further promote cell and tissue injury. Interestingly, hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-xL and Mcl-1, which have nonredundant functions. Endoplasmic reticulum stress or the unfolded protein response contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury. All of these processes contributing to hepatocyte injury are discussed in the context of potential therapeutic strategies.
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Bronnikova, G. Z., and E. N. Skovorodin. "ULTRASTRUCTURE AND HEPATOCYTE KARYOCYTOMETRY OF QUAILS." VESTNIK OF THE BASHKIR STATE AGRARIAN UNIVERSITY 51, no. 3 (September 20, 2019): 36–41. http://dx.doi.org/10.31563/1684-7628-2019-51-3-36-41.
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The paper studies the functional morphology of quail hepatocytes. To conduct the research there was an electron-microscopic examination of quail liver. The structure of the nucleus and nucleolus were quantitatively assessed using objective methods of karyocytometry. Quail hepatocytes are found to have high synthetic activity. Hepatocyte karyocytometry enables to reveal hidden morphological characteristics of the cell ultrastructure being hardly detected under normal qualitative description of electron-diffraction photos. Quail hepatocyte karyocytometry indicates a pronounced structural and functional heterogeneity of hepatocytes. Therefore, electron-microscopic examination using karyocytometric methods is a highly informative method for assessing the morphological and functional state of the body and it should be used in assessing the impact of pharmacological agents on the body.
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Russell, Jacquelyn O., Sungjin Ko, Satdarshan P. Monga, and Donghun Shin. "Notch Inhibition Promotes Differentiation of Liver Progenitor Cells into Hepatocytes via sox9b Repression in Zebrafish." Stem Cells International 2019 (March 12, 2019): 1–11. http://dx.doi.org/10.1155/2019/8451282.
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Liver regeneration after most forms of injury is mediated through the proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) arising from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC-to-hepatocyte differentiation in patients with chronic liver disease could serve as a potentially new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development and has also been implicated in regulating LPC differentiation during regeneration. SRY-related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown in a zebrafish model of LPC-driven liver regeneration that inhibition of Hdac1 activity through MS-275 treatment enhances sox9b expression in LPCs and impairs LPC-to-hepatocyte differentiation. Therefore, we hypothesized that inhibition of Notch signaling would promote LPC-to-hepatocyte differentiation by repressing sox9b expression in zebrafish. We ablated the hepatocytes of Tg(fabp10a:CFP-NTR) larvae and blocked Notch activation during liver regeneration through treatment with γ-secretase inhibitor LY411575 and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC-specific Sox9b overexpression impaired LPC-to-hepatocyte differentiation. Our results establish the Notch-Sox9b signaling axis as inhibitory to LPC-to-hepatocyte differentiation in a well-established in vivo LPC-driven liver regeneration model.
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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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Yin, Sha, Qianwen Shi, Wenwei Shao, Chi Zhang, Yixiao Zhang, Xiaoyan Qiu, and Jing Huang. "Hepatocyte-Derived Igκ Exerts a Protective Effect against ConA-Induced Acute Liver Injury." International Journal of Molecular Sciences 21, no. 24 (December 9, 2020): 9379. http://dx.doi.org/10.3390/ijms21249379.
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Immunoglobulin (Igκ) has been reported to be expressed in sorted liver epithelial cells of μMT mice, and the sequence characteristics of hepatocyte-derived Igκ were different from those of classical B-cell-derived Igκ. However, the physiological function of hepatocyte-derived Igκ is still unclear. The expression of Igκ was firstly identified in primary hepatocytes and normal liver cell line (NCTC1469), and hepatocyte-derived Igκ expression was elevated and displayed unique localization in hepatocytes of concanavalin A (ConA)-induced hepatitis model. Moreover, Igκ knockout mice were more sensitive to ConA-induced hepatitis and had higher serum aspartate aminotransferase (AST) levels, more severe histological injury and a greater number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells as compared with littermate controls. Furthermore, knockdown of Igκ in primary hepatocytes and NCTC1469 cells led to accelerated activation of the mitochondrial death pathway and caspase-3 cleavage in vitro, which might be related to inhibition of NF-κB signaling pathway and activation of JNK via the cytoskeleton dynamics. Taken together, these results indicate that hepatocyte-derived Igκ mediates cellular resistance to ConA-induced liver injury by inhibiting activation of caspase-3 and the mitochondrial death pathway, suggesting that Igκ plays an important role in hepatocyte survival and exerts a protective effect against ConA-induced liver injury in mice.
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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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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.
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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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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.