Comparison of Hepatic-like Cell Production from Human Embryonic Stem Cells and Adult Liver Progenitor Cells: CAR Transduction Activates a Battery of Detoxification Genes

In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate

Altered cytokine profile under control of the serotonergic system determines the regulation of CYP2C11 and CYP3A isoforms

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The impact of serotonergic system dysfunction on the regulation of P4501A isoforms during liver insufficiency and consequences for thyroid hormone homeostasis

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Encapsulation d'hépatocytes dans un biomatériau poreux en vue d'une implantation dans un modèle animal

Pour pallier le manque de solutions thérapeutiques et le faible nombre de greffons hépatiques disponibles pour traiter certaines atteintes du foie, l ingénierie tissulaire et l implantation de tissus reconstruits représentent des solutions potentielles à explorer. Notre approche repose sur la microencapsulation de cellules hépatiques humaines dans des billes d alginate poreuses. Dans un premier temps, ce travail de thèse a permis de mettre en place de nouvelles combinaisons de matériaux a partir de l alginate et d analyser l influence de ces matériaux sur le comportement mécanique des billes et sur le comportement cellulaire de la lignée HepG2 C3A. La conclusion de cette étude montre que l ajout de collagène et/ou de coating de poly-L-lysine augmente la rigidité des billes et diminue les coefficients de transfert à l intérieur de la bille, tout en permettant une prolifération et un maintien de la fonctionnalité des cellules HepG2 C3A. Ensuite, à titre préliminaire, les billes ont été implantées chez un modèle de souris immunocompétente afin d évaluer la biocompatibilité des matériaux. Dans un deuxième temps, les hépatocytes humains primaires ont été encapsulés dans des billes d alginate mélangées ou non avec du collagène de type I et mis en culture sur des durées de 7 a 14 jours. Les résultats montrent une perte du niveau basal des expressions et des activités des cytochromes P450 3A4 et 1A1, de la fonctionnalité et de la différenciation des hépatocytes en culture statique. Dans un dernier temps, les hépatocytes humains primaires encapsules ont été mis en culture dans deux types de bioréacteurs afin d étudier l effet de la culture en condition dynamique. Les résultats montrent que la culture dynamique en lit fluidise permet d augmenter la fonctionnalité et la différenciation des hépatocytes encapsules. Cette méthode de culture pourrait donc être employée avant l implantation des hépatocytes microencapsulés, afin d assurer une viabilité optimale et un maintien des fonctions métaboliques indispensables dans un contexte de suppléance hépatique.Further to the lack of therapeutic solutions and the low number of hepatic transplants available to treat some hepatic diseases, tissue engineering and implantation of reconstructed tissue are possible alternatives to explore. Our approach is based on microencapsulation of hepatic cells in porous alginate beads. In a first time, this thesis work allowed to set up new combinations of materials based on alginate and to analyze their influence on bead mechanical behavior and on HepG2 C3A behavior. The results of this study shown that the adding of collagen and/or of poly-L-lysine coating increased the rigidity of the beads and decreased the mass transfer coefficients inside the bead while allowing a proliferation and a preservation of the functionality of HepG2 C3A. Then, the beads were implanted in small animal model of immunocompetent mice to estimate the biocompatibility of the different materials. In a second time, the primary human hepatocytes were encapsulated in beads of alginate mixed or not with type I collagen and cultivated during 7 to 14 days. The results have shown a basal loss of the level of the expressions and the activities of cytochromes P450 3A4 and 1A1, the functionality and the differentiation of encapsulated primary human hepatocytes. In a last time, encapsulated primary human hepatocytes were cultivated in two types of bioreactors in order to analyze the effect of dynamic culture conditions. The datas shown that the dynamic culture in fluidized bed bioreactor enhanced the functionality and the differentiation of encapsulated hepatocytes. This culture condition could be used before implantation of encapsulated hepatocytes in order to assure an optimal viability and a good maintenance of metabolic functions essential in a context of hepatic suppliance.COMPIEGNE-BU (601592101) / SudocSudocFranceF

Biocompatible modified water as a non-pharmaceutical approach to prevent metabolic syndrome features in obesogenic diet-fed mice

International audienceThe prevalence of metabolic syndrome (MetS), elevating cardiovascular risks, is increasing worldwide, with no available global therapeutic options. The intake of plain, mineral or biocompatible modified waters was shown to prevent some MetS features. This study was designed to analyze, in mice fed a high fat and sucrose diet (HFSD), the effects on MetS features of the daily intake of a reverse osmosed, weakly remineralized, water (OW) and of an OW dynamized by a physical processing (ODW), compared to tap water (TW). The HFSD was effective at inducing major features of MetS such as obesity, hepatic steatosis and inflammation, blood dyslipidemia, systemic glucose intolerance and muscle insulin resistance. Compared to TW, OW intake decreased hepatic fibrosis and inflammation, and mitigated hepatic steatosis and dyslipidemia. ODW intake further improved skeletal muscle insulin sensitivity and systemic glucose tolerance. This study highlights the deleterious metabolic impacts of the daily intake of TW, in combination with a high energy diet, and its possible involvement in MetS prevalence increase. In addition, it demonstrates that biocompatible modified water may be promising non-pharmaceutical, cost-effective tools for nutritional approaches in the treatment of MetS

Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes.

Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway

AhR is required for skatole-mediated CYP1A1 and CYP1A2 up-regulation.

<p>(A) RT-qPCR analysis of <i>AhR</i>, <i>CYP1A1</i> and <i>CYP1A2</i> mRNA expression in PHHs (Donor #391) after siRNA-mediated <i>AhR</i> down-regulation. (B) RT-qPCR analysis of <i>CYP1A1</i> (B) and <i>CYP1A2</i>(C) mRNA expression in PHHs after siRNA-mediated <i>AhR</i> down-regulation and incubation with 50 or 100 μM skatole, or 10 nM TCDD. * significantly different from cells transfected with non-target siRNA.</p

Improving Prediction of Metabolic Clearance Using Quantitative Extrapolation of Results Obtained From Human Hepatic Micropatterned Cocultures Model and by Considering the Impact of Albumin Binding

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Adult-derived human liver stem/progenitor cells infused 3 days post-surgery improve liver regeneration in a mouse model of extended hepatectomy.

There is growing evidence that cell therapy constitutes a promising strategy for liver regenerative medicine. In the setting of hepatic cancer treatments, cell therapy could prove a useful therapeutic approach for managing the acute liver failure that occurs following extended hepatectomy. In this study, we examined the influence of delivering adult human liver mesenchymal stem/progenitor cells (ADHLSCs) at two different early time points in an immunodeficient mouse model (Rag(2-/-)IL2Rγ(-/-) ) that had undergone a 70%-hepatectomy procedure. The hepato-mesenchymal cells were intrasplenically infused either immediately after surgery (n= 26) or following a critical 3-day period (n=26). We evaluated the cells' capacity to engraft at Day 1 and Day 7 following transplantation by means of human Alu qPCR quantification, along with histological assessment of human albumin and alpha-smooth muscle actin. In addition, cell proliferation (anti-mouse and human Ki67 staining) and murine liver weight were measured in order to evaluate liver regeneration. At Day 1 post-transplantation, the ratio of human to mouse cells was similar in both groups, whereas 1 week post-transplantation, this ratio was significantly improved (p <0.016) in mice receiving ADHLSC injection at Day 3 post-hepatectomy (1.7%), compared to those injected at the time of surgery (1%). Based on liver weight, mouse liver regeneration was more extensive 1 week post-transplantation in mice transplanted with ADHLSCs (+65.3%) compared to that of mice from the sham vehicle group (+42.7%). In conclusion, infusing ADHLSCs 3 days after extensive hepatectomy improves the cell engraftment and murine hepatic tissue regeneration, thereby confirming that ADHLSCs could be a promising cell source for liver cell therapy and hepatic tissue repair

Evidence for an important role of host microRNAs in regulating hepatic fibrosis in humans infected with &ITSchistosoma japonicum&IT

International audienceMicroRNAs (miRNAs) are short, non-coding RNAs that repress the translation of target gene transcripts. They have been implicated in various activities such as cell proliferation, survival, differentiation, migration and metabolism. We report here the first known miRNome and transcriptome analysis of human livers displaying advanced fibrosis due to Schistosoma japonicum infection. We present evidence that hsa-miR-150-5p, hsa-miR-10a-5p, hsa-miR-199a-3p, hsa-miR-4521, hsa-miR-222/221, hsa-miR-663b and hsa-miR-143-3p (associated without correction) play an important role in hepatic fibrosis by acting on metabolism, organization of the extracellular matrix proteins, lipid mobilization and limitation of oxidative damage stress. (C) 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved