Oxidative stress triggers cytokinesis failure in hepatocytes upon isolation.

Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX(®) Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation.

Human hepatocyte transplantation in patients with hepatic failure awaiting a graft.

BACKGROUND: Hepatocyte transplantation (HT) has the potential to become a promising treatment to temporarily support liver function in patients with liver failure. METHODS: Two patients, who had already received a liver transplant (LT) in the past, with an end-stage liver disease due to recurrent hepatitis C virus cirrhosis, suffering acute-on-chronic liver failure while on the waiting list for an LT, received HT as a bridge to whole-organ retransplantation. After HT and during intensive care unit admission, blood tests and ammonia levels were determined every 12 and 24 h, respectively, before and after each hepatocyte infusion. RESULTS: The present study describes monitoring of analytical and clinical parameters and improvement of liver function following HT. In both patients, we managed to lower the blood ammonia levels and clinically improve the degree of hepatic encephalopathy, thus serving as a bridge to liver retransplantation in 1 patient. CONCLUSIONS: We believe that this therapy may be an alternative treatment in patients with chronic liver disease who suffer episodes of acute decompensation as a bridge to conventional LT.

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Sequential hepatogenic transdifferentiation of adipose tissue-derived stem cells: relevance of different extracellular signaling molecules, transcription factors involved, and expression of new key marker genes.

Adipose tissue contains a mesenchymal stem cell (MSC) population known as adipose-derived stem cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding protein alpha and beta (CEBPalpha and beta), peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1alpha), and hepatocyte nuclear factor 4 alpha (HNF4alpha)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the step-by-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), alpha-2-macroglobuline (alpha2M), complement protein C3 (C3), and selenoprotein P1 (SEPP1); late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stem cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4alpha and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.

Desarrollo, análisis y optimización de modelos celulares hepáticos para estudios de fármacotoxicología y terapia celular / Development and optimization of hepatic cell models for pharmaco-toxicology and cell therapy research

El hígado juega un papel fundamental en el metabolismo de medicamentos y enel mantenimiento de la homeostasis del organismo y, por tanto los modelos celulareshepáticos desempeñan un papel clave para estudios fármaco-toxicológicos ymás recientemente en el campo de la terapia celular. Sin embargo, la limitada disponibilidadde hepatocitos viables y funcionales, debido a la falta de tejido hepático,es la principal limitación para utilizar estos recursos celulares. El objetivo delpresente trabajo se ha basado en el desarrollo y caracterización de modelos celulareshepáticos que puedan constituir una alternativa a los hepatocitos para estetipo de aplicaciones. Para ello se han abordado tres estrategias diferentes: 1) optimizacióndel proceso de obtención de hepatocitos a partir de hígados enterosdescartados para transplante, determinando las condiciones adecuadas para elaislamiento y cultivo de hepatocitos; 2) caracterización funcional de las células delhepatoblastoma HepG2, y 3) desarrollo de un protocolo para inducir la diferenciaciónhepatogénica de células madre mesenquimales adultas derivadas de tejidoadiposo (ADSC). Para conseguir un buen aprovechamiento de hígados descartadospara transplante resulta necesario optimizar los protocolos de aislamiento y criopreservaciónde hepatocitos. El estudio con células madre adultas se presenta comouna alternativa muy válida para la obtención de hepatocitos-like viables y funcionalmenteactivos, útiles a corto plazo en estudios de fármaco-toxicología y en unfuturo para terapia celular hepática. El uso de células madre abre un gran abanicode posibilidades, facilitando el establecimiento de un modelo celular diferenciadoadulto con características que otros modelos celulares, como son el hepatomahumano HepG2, no presentan. No obstante, es necesario adquirir un mayor conocimientode los mecanismos celulares y moleculares que controlan la transdiferenciacióna hepatocitos

Given the importance of the liver in the metabolism and maintenance of thehomeostasis of the organism, many studies have been conducted in the area oftoxicology and, more recently, in hepatic cellular therapy. However, the maindrawback is the limited availability of viable and functional hepatocytes due tothe scarcity of liver tissue. The purpose of this work was based on the developmentand characterization of hepatic cellular models to become an alternative tohepatocytes in toxicology studies and cellular therapy. To this end, three mainobjectives have been investigated: 1) to adopt a procedure of hepatocyte isolationfrom discarded organs for transplantation which determines the optimal conditions for the isolation and culture of hepatocytes, 2) to characterize the cells from thehepatoblastome HepG2, and 3) to develop a hepatogenic differentiation protocolto induce the hepatic differentiation in adipose-derived stem cells (ADSC). Inparticular, the hepatogenic differentiation of stem cells opens a wide range ofpossibilities to facilitate the establishment of an adult differentiated cellular modeluseful for pharmaco-toxicological studies and for hepatic cellular therapy. The useof adult stem cells may allow the establishment of an adult cellular model withproperties that others cellular models, like HepG2, do not show. However, it isnecessary to optimise the isolation and cryopreservation procedures, as well as thedifferentiation protocols from adult stem cells and try to acquire a wide knowledgeof the cellular and molecular mechanisms that control the transdifferentiation tohepatocytes

Terapia celular hepática: tipos celulares potencialmente útiles para el trasplante / No disponible

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