p38α deficiency and oxidative stress cause cytokinesis failure in hepatocytes.

Cytokinesis is the last step in mitosis and it implies re-organization of the actin cytoskeleton. Its failure is one of the major mechanisms of polyploidy and binucleation in mammals. Our aims were 1) to assess the role of redox-sensitive p38α MAPK in cytokinesis by studying the liver of wild type mice or liver-specific p38α knock-out mice; 2) to assess the role of oxidative stress associated with hepatocyte isolation on cytokinesis. When p38α was down-regulated in hepatocytes, MK2 phosphorylation on threonine 334 was completely abrogated. Activation of MNK-1, required for abscission of the intercellular bridge, was diminished. Key proteins of the RhoA pathway (phospho-PRK2, nuclear phosphorylated cofilin, and cytosolic p27) were assessed confirming the impairment of this pathway. The absence of p38α in aging liver also led to a decrease in HSP27 phosphorylation, which is required for actin polymerization. Indeed, a severe impairment in the F-actin filamentous structure was found in the liver of old mice upon p38α deficiency. Consequently, long-term p38α MAPK down-regulation markedly affects the RhoA pathway and actin cytoskeleton dynamics inducing actin disassembly and cytokinesis failure upon aging. On the other hand, hepatocyte isolation caused marked glutathione depletion, increased generation of reactive oxygen species, and activated cell cycle entry. Addition of N-acetyl cysteine to isolation media prevented glutathione depletion, restrained the cell cycle entry, and abrogated defective cytokinesis and the formation of binucleated hepatocytes during isolation. Our results show that hepatocytes do enter into S phase but they do not complete cell division with age upon p38α deficiency or upon oxidative stress associated with isolation leading in both cases to cytokinesis failure and binucleation.

Clinical outcome of hepatocyte transplantation in four pediatric patients with inherited metabolic diseases.

Hepatocyte transplantation (HT) has become an effective therapy for patients with metabolic inborn errors. We report the clinical outcome of four children with metabolic inborn errors that underwent HT, describing the cell infusion protocol and the metabolic outcome of transplanted patients. Cryopreserved hepatocytes were used as this allows scheduling of treatments. Functional competence (viability, cell attachment, major cytochrome P450 and UDP-glucuronosyltransferase 1A1 activities, and urea synthesis) and microbiological safety of cell batches were assessed prior to clinical use. Four pediatric patients with liver metabolic diseases [ornithine transcarbamylase (OTC) deficiency, Crigler-Najjar (CNI) syndrome, glycogen storage disease Ia (GSD-Ia), and tyrosinemia type I (TYR-I)] underwent HT. Indication for HT was based on severity of disease, deterioration of quality of life, and benefits for the patients, with the ultimate goal to improve their clinical status whenever liver transplantation (LT) was not indicated or to bridge LT. Cells were infused into the portal vein while monitoring portal flow. The protocol included antibiotic prophylaxis and immunosuppressant therapy. After HT, analytical data on the disease were obtained. The OTC-deficient patient showed a sustained decrease in plasma ammonia levels and increased urea production after HT. Further cell infusions could not be administered given a fatal nosocomial fungus sepsis 2 weeks after the last HT. The CNI and GSD-Ia patients improved their clinical status after HT. They displayed reduced serum bilirubin levels (by ca. 50%) and absence of hypoglycaemic episodes, respectively. In both cases, the HT contributed to stabilize their clinical status as LT was not indicated. In the infant with TYR-I, HT stabilized temporarily the biochemical parameters, resulting in the amelioration of his clinical status while diagnosis of the disease was unequivocally confirmed by full gene sequencing. In this patient, HT served as a bridge therapy to LT.

Steatotic liver: a suitable source for the isolation of hepatic progenitor cells.

BACKGROUND: Alternative and/or complementary sources of cells such as hepatic progenitor cells (HPC) are under investigation for hepatic cell therapy purposes. Steatotic livers are those most commonly rejected for clinical transplantation and are also unsuitable for good quality hepatocyte isolation. AIM: Taken together these two facts, our aim was to investigate whether they could represent a suitable source for the isolation of progenitor cells. METHODS: Rats fed for 7 weeks with methionine-choline deficient diets showing proved steatotic signs (i.e. increase in hepatic lipids; macrovesicular steatosis) and steatotic and normal human liver samples were used to study the expression of HPC markers and to isolate these cells. RESULTS: In the liver of the steatotic rats there was a significant increase in HPC (known as oval cells in rodents) markers such as Thy-1, epithelial cell adhesion molecule (EpCAM) and OV-6 (2-, 3- and 5-fold increase respectively). Additionally, there was an increase in the yield of isolated oval cells compared to control rats. Similarly, studies using human livers clearly confirmed an increase in the expression of HPC markers in the steatotic tissue and a significant rise in the number of isolated progenitor cells (EpCAM+, Thy-1+, OV-6+) (10, 12 and 11.6 × 10(4)  cells/g of tissue respectively). CONCLUSIONS: These data suggest that steatotic livers, discarded for orthotopic liver transplantation and hepatocyte isolation, could be a suitable source for large scale isolation of HPC which might be potential candidates in liver cell therapy.

Influence of platelet lysate on the recovery and metabolic performance of cryopreserved human hepatocytes upon thawing.

BACKGROUND: Storage of human hepatocytes is essential for their use in research and liver cell transplantation. However, cryopreservation and thawing (C/T) procedures have detrimental effects on the viability and functionality compared with fresh cells. The aim of this study was to upgrade the standard C/T methodology to obtain better quality hepatocytes for cell transplantation to improve the overall clinical outcome. METHODS: Human hepatocytes isolated from donor livers were cryopreserved in University of Wisconsin solution with 10% dimethyl sulfoxide (standard medium), which was supplemented with 10% or 20% of platelet lysate. Thawing media supplemented with up to 30 mM glucose was also investigated. The effects on cell viability, adhesion proteins (e-cadherin, ß-catenin, and ß1-integrin) expression, attachment efficiency, apoptotic indicators, Akt signaling, ATP levels, and cytochrome P450 activities have been evaluated. RESULTS: The results indicate that the hepatocytes cryopreserved in a medium supplemented with platelet lysate show better recovery than those preserved in the standard medium: higher expression of adhesion molecules, higher attachment efficiency and cell survival; decreased number of apoptotic nuclei and caspase-3 activation; maintenance of ATP levels; and drug biotransformation capability close to those in fresh hepatocytes. Supplementation of thawing media with glucose led to a significant decrease in caspase-3 activation and to increased adhesion molecules preservation and Akt signal transduction after C/T. Minor nonsignificant changes in cell viability and attachment efficiency were observed. CONCLUSIONS: These promising results could lead to a new cryopreservation procedure to improve human hepatocyte cryopreservation outcome.

[Adipose tissue-derived stem cells: hepatic plasticity]. / Células madre del tejido adiposo: plasticidad hepática.

Currently, the only effective treatment for end-stage liver disease is liver transplantation. The number of patients on the waiting list increases considerably each year, giving rise to a wide imbalance between supply and demand for healthy livers. Knowledge of stem cells and their possible use have awakened great interest in the field of hepatology, these cells being one of the most promising short-term alternatives. Hepatic stem cell therapy consists of the implantation of healthy cells capable of performing the functions that damaged cells are unable to carry out. Recent observations indicate that several stem cells can differentiate into distinct cell lineages. Hepatic differentiation of adult stem cells from several origins has yielded highly promising results. Adipose tissue in adults contains a reservoir of stem cells that can be induced and differentiated into different types of cells, showing a high degree of plasticity. Because of its abundance and easy access, adipose tissue is a promising source of adult stem cells for hepatic stem cell therapy. The present article reviews the progress made in the differentiation of adult stem cells from adipose tissue into cells with hepatic phenotype. We also discuss the potential application of this technique as a therapy for temporary metabolic support in patients with end-stage liver failure awaiting whole organ transplantation, as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and as a treatment in patients with genetic metabolic defects in vital liver functions.

Células madre del tejido adiposo: plasticidad hepática / Adipose tissue-derived stem cells: Hepatic plasticity

Actualmente el único tratamiento efectivo para las enfermedadeshepáticas en estadio terminal es el trasplante de hígado.El número de pacientes en lista de espera aumentaconsiderablemente cada año, dando lugar a una mayor desproporciónentre la oferta y la demanda de un hígado sano.El conocimiento y el posible uso de las células madre ha despertadoun gran interés en el campo de la hepatología, haciendode ellas una de las alternativas más prometedoras acorto plazo. La terapia celular hepática permitiría suplir alhígado de células sanas capaces de llevar a cabo las funcionesque las células dañadas no son capaces de desarrollar.Observaciones recientes han puesto de manifiesto la capacidadde las células madre de diferenciarse hacia diferenteslinajes celulares. La diferenciación hepática de células madreadultas de diversos orígenes ha dado resultados muyprometedores. El tejido adiposo contiene en el individuoadulto un reservorio de células madre capaces de ser inducidasy diferenciadas hacia diferentes estirpes celulares, presentandoun elevado grado de plasticidad celular. La abundanciade este tejido y su fácil accesibilidad hacen de él unaprometedora fuente de células madre adultas para su uso enterapia celular hepática. Se presenta una revisión de losavances obtenidos en la diferenciación de células madre procedentesdel tejido adiposo hacia células de fenotipo hepáticoy sus posible aplicaciones como un método terapéuticocon la finalidad de mantener la función hepática del pacientedurante el período de espera hasta recibir el trasplante, opara facilitar la regeneración hepática en casos de fallo hepáticofulminante, y para el tratamiento de pacientes conmetabolopatías congénitas (AU)

Currently, the only effective treatment for end-stage liverdisease is liver transplantation. The number of patients onthe waiting list increases considerably each year, giving riseto a wide imbalance between supply and demand for healthylivers. Knowledge of stem cells and their possible use haveawakened great interest in the field of hepatology, these cellsbeing one of the most promising short-term alternatives. Hepaticstem cell therapy consists of the implantation of healthycells capable of performing the functions that damagedcells are unable to carry out. Recent observationsindicate that several stem cells can differentiate into distinctcell lineages. Hepatic differentiation of adult stem cells fromseveral origins has yielded highly promising results. Adiposetissue in adults contains a reservoir of stem cells that can beinduced and differentiated into different types of cells, showinga high degree of plasticity. Because of its abundanceand easy access, adipose tissue is a promising source of adultstem cells for hepatic stem cell therapy. The present articlereviews the progress made in the differentiation of adultstem cells from adipose tissue into cells with hepatic phenotype.We also discuss the potential application of thistechnique as a therapy for temporary metabolic support inpatients with end-stage liver failure awaiting whole organtransplantation, as a method to support liver function andfacilitate regeneration of the native liver in cases of fulminanthepatic failure, and as a treatment in patients with geneticmetabolic defects in vital liver functions (AU)

[Alternative sources of hepatocytes for cell therapy]. / Fuentes alternativas de hepatocitos para la terapia celular.

There is an urgent need to search for alternatives to whole organ transplantation. Several methods have been proposed. Among these strategies, cell transplantation is currently one of the most promising. To achieve this aim, in addition to highly differentiated adult hepatocytes, the use of stem cells is considered a highly attractive therapeutic method for the treatment of liver disease and for temporary support of hepatic function until a liver becomes available for organ transplantation. This strategy is based on the ability of stem cells to differentiate into different cellular types according to their environment. Therefore, stem cells could be an unlimited source of hepatic cells for transplantation and gene therapy. Bone marrow is considered the most promising source of adult stem cells, partly due to the versatility of the cells obtained in repairing damaged tissues of several lineages. Several different types of stem cells have been described in bone marrow: hematopoietic, mesenchymal, side population and multipotent adult stem cells. Bone marrow cells have been hypothesized as a third recruitment source in liver regeneration in addition to hepatocytes and endogenous liver stem cells. Consequently, attempts have been made to differentiate them into hepatic lineage for their subsequent use in hepatic cell therapy. The present article reviews the progress made in this field or research.

Fuentes alternativas de hepatocitos para la terapia celular / Alternative sources of hepatocytes for cell therapy

Existe una necesidad urgente de buscar alternativas al trasplante de órgano entero. Diversos métodos han sido propuestos como alternativas al trasplante hepático. Entre ellos, el trasplante celular es actualmente uno de los más prometedores. Para ello, alternativamente al uso de hepatocitos adultos plenamente diferenciados, se considera el uso de «células madre» como método terapéutico muy atractivo para las enfermedades hepáticas y para el mantenimiento de la función hepática hasta la obtención de un injerto adecuado para trasplante. Esta estrategia está basada en la capacidad de las células madre de diferenciarse en varios tipos celulares en función del entorno en que se encuentren. Así, las células madre constituirían un recurso inagotable de células hepáticas para trasplante y terapia génica. La médula ósea se considera el tejido fuente de células troncales adultas más prometedor debido, en parte, a la versatilidad de las células obtenidas para reparar tejidos dañados de muy diversas estirpes. Se han descrito diferentes tipos de células madre en la médula ósea: hematopoyéticas, mesenquimales, la población lateral y las células progenitoras adultas multipotenciales. Se cree que las células de la médula ósea son la tercera fuente de reclutamiento en la regeneración hepática después de los hepatocitos y las células madre exógenas del hígado. Por este motivo se ha intentado diferenciarlas a linaje hepático para su posterior uso en la terapia celular hepática. En este trabajo se hace una revisión de los avances alcanzados en este sentido

There is an urgent need to search for alternatives to whole organ transplantation. Several methods have been proposed. Among these strategies, cell transplantation is currently one of the most promising. To achieve this aim, in addition to highly differentiated adult hepatocytes, the use of stem cells is considered a highly attractive therapeutic method for the treatment of liver disease and for temporary support of hepatic function until a liver becomes available for organ transplantation. This strategy is based on the ability of stem cells to differentiate into different cellular types according to their environment. Therefore, stem cells could be an unlimited source of hepatic cells for transplantation and gene therapy. Bone marrow is considered the most promising source of adult stem cells, partly due to the versatility of the cells obtained in repairing damaged tissues of several lineages. Several different types of stem cells have been described in bone marrow: hematopoietic, mesenchymal, side population and multipotent adult stem cells. Bone marrow cells have been hypothesized as a third recruitment source in liver regeneration in addition to hepatocytes and endogenous liver stem cells. Consequently, attempts have been made to differentiate them into hepatic lineage for their subsequent use in hepatic cell therapy. The present article reviews the progress made in this field or research