Desarrollo, análisis y optimización de modelos celulares hepáticos para estudios de fármaco-toxicología y terapia celular

Given the importance of the liver in the metabolism and maintenance of the homeostasis of the organism, many studies have been conducted in the area of toxicology and, more recently, in hepatic cellular therapy. However, the main drawback is the limited availability of viable and functional hepatocytes due to the scarcity of liver tissue. The purpose of this work was based on the development and characterization of hepatic cellular models to become an alternative to hepatocytes in toxicology studies and cellular therapy. To this end, three main objectives have been investigated: 1) to adopt a procedure of hepatocyte isolation from discarded organs for transplantation which determines the optimal conditions for the isolation and culture of hepatocytes, 2) to characterize the cells from the hepatoblastome HepG2, and 3) to develop a hepatogenic differentiation protocol to induce the hepatic differentiation in adipose-derived stem cells (ADSC). In particular, the hepatogenic differentiation of stem cells opens a wide range of possibilities to facilitate the establishment of an adult differentiated cellular model useful for pharmaco-toxicological studies and for hepatic cellular therapy. The use of adult stem cells may allow the establishment of an adult cellular model with properties that others cellular models, like HepG2, do not show. However, it is necessary to optimise the isolation and cryopreservation procedures, as well as the differentiation protocols from adult stem cells and try to acquire a wide knowledge of the cellular and molecular mechanisms that control the transdifferentiation to hepatocytes.Key Words: cellular transplant, esteatosis, human hepatocytes, hepatoblastomes, stem cells.El hígado juega un papel fundamental en el metabolismo de medicamentos y en el mantenimiento de la homeostasis del organismo y, por tanto los modelos celulares hepáticos desempeñan un papel clave para estudios fármaco-toxicológicos y más recientemente en el campo de la terapia celular. Sin embargo, la limitada disponibilidad de hepatocitos viables y funcionales debido a la falta de tejido hepático es la principal limitación para utilizar estos recursos celulares. El objetivo del presente trabajo se ha basado en el desarrollo y caracterización de modelos celulares hepáticos que puedan constituir una alternativa a los hepatocitos para este tipo de aplicaciones. Para ello se han abordado tres estrategias diferentes: 1) optimización del proceso de obtención de hepatocitos a partir de hígados enteros descartados para transplante, determinando las condiciones adecuadas para el aislamiento y cultivo de hepatocitos; 2) caracterización funcional de las células del hepatoblastoma HepG2 y 3) desarrollo de un protocolo para inducir la diferenciación hepatogénica de células madre mesenquimales adultas derivadas de tejido adiposo (ADSC). Para conseguir un buen aprovechamiento de hígados descartados para transplante resulta necesario optimizar los protocolos de aislamiento y criopreservación de hepatocitos. El estudio con células madre adultas se presenta como una alternativa muy válida para la obtención de hepatocitos-like viables y funcionalmente activos útiles a corto plazo en estudios de fármaco-toxicología y en un futuro para terapia celular hepática. El uso de células madre abre un gran abanico de posibilidades facilitando el establecimiento de un modelo celular diferenciado adulto con características que otros modelos celulares, como son el hepatoma humano HepG2, no presentan. No obstante, es necesario adquirir un mayor conocimiento de los mecanismos celulares y moleculares que controlan la transdiferenciación a hepatocitos.Palabras clave: trasplante celular, esteatosis, hepatocitos humanos, hepatoblastomas, células madre

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development

Comparison of basal cytotoxicity data between mammalian and fish cell lines A literature survey

During the last 20 years, in vitro assays with fish cells have been used in ecotoxicology for testing chemicals, effluents and in toxicity identification evaluations (TIE) studies. Due to the good correlation found between the in vitro data and the in vivo fish data in ranking chemical toxicity, they have been proposed as an alternative to acute fish bioassays for toxicity screening of chemicals. Nevertheless, they are recognized to be less sensitive when compared with in vivo fish bioassays. Fish cells have slower cell cycles than mammalian cells, so it was suggested that mammalian cells could be more sensitive than fish cells for testing chemicals. However, as fish cells offer some advantages over mammalian cells, mainly related to their ease of handling, some authors found them preferable to use for routine testing. The present study was undertaken to analyze whether mammalian cell lines are more, less, or equally sensitive, compared to fish cells lines. To this end, basal cytotoxicity data from a literature survey of mammalian and fish cell lines have been compared. There was a good linear correlation of IC50 values (r = 0.915) between fish and mammalian cells for a set of 51 chemicals after 24-h of treatment. The correlation coefficient of IC50 values (r = 0.888) decreased when 24-h treated mammalian cells were correlated against 48-h treated fish cell lines. For the dataset selected for this study, fish and mammalian cells showed a similar sensitivity for most of the chemicals after being treated for 24 h. We conclude that fish and mammalian cells can equally well predict the basal toxicity of the set of chemicals used in this study, and that increased exposure periods did not increase the correlation between midpoint toxicities nor sensitivities for any of the cell lines used. The more practical handling of fish cells favors their recommendation as a better alternative for replacing fish bioassay than mammalian cells. © 2005 Elsevier Ltd. All rights reserved

Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies.

A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes to monitor the efficiency of the RT step. The degree of amplification is estimated using appropriate cDNA standards and quantitation of the amplified products by fluorescent measurement. This assay can be used to quantify the most relevant CYPs in human liver and cultured human hepatocytes. CYPs 3A4 and 2E1 were the most abundant mRNAs in human liver (2.5 and 1.7 x 10(8) molecules/microgram of total RNA respectively), whereas 1A1 and 2D6 were the least abundant isoforms (1.2 and 2.1 x 10(6) molecules/microgram of total RNA). A similar pattern was also found in short-term cultured human hepatocytes. This technique is also suitable for assessing CYP mRNA induction by xenobiotics. Cells exposed to 3-methylcholanthrene showed a characteristic increased expression of CYP1A2 and 1A1 mRNAs. Upon incubation with phenobarbital and rifampin (rifampicin), human hepatocytes increased CYP 2B6, 3A4, and 3A5 among others.Part of this research was conducted with the support of EUProjects BMH4-CT96-0254 and BIO4-CT96-0052. We acknowledge the support of Fondo de Investigaciones Sanitarias of the Spanish Ministry of Health (97/1061). C. Rodrı´guez-Antona was a recipient of a fellowship of Generalitat Valenciana.N

Cytochrome P-450 mRNA expression in human liver and its relationship with enzyme activity.

CYP activity and protein contents have been measured in human liver using different techniques. In contrast, CYP mRNA data are scarce and the relationships between CYP mRNA contents and activities have not been established. These studies deserve further attention because mRNA determinations by RT-PCR require a very small amount of material (e.g., liver needle biopsy) and could provide important data regarding CYP expression regulation. In this study we measured in 12 human liver samples the mRNA contents of 10 CYPs by quantitative RT-PCR and the metabolic activities using specific substrates. mRNA contents and activities showed high correlation coefficients for CYP1A1, CYP1A2, CYP3A4, CYP2D6, and CYP2B6 (0.96, 0.94, 0.69, 0.61, and 0.52, respectively), but no significant correlations were found for CYP2C9, CYP2A6, and CYP2E1. The results suggest that the regulation of CYP1A1, CYP1A2, CYP3A4, CYP2D6, and CYP2B6 expression is essentially pretranslational and that their mRNA levels could allow a good estimate of their activity.N

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells.

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP-beta: LAP and LIP, HNF-3alpha, HNF-4alpha, RXR-alpha) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.N