Genetic and Epigenetic Modification of Rat Liver Progenitor Cells via HNF4α Transduction and 5' Azacytidine Treatment: An Integrated miRNA and mRNA Expression Profile Analysis.

Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors, including the hepatocyte nuclear factors (HNF) 3ß and HNF6, but not the hepatic master regulator HNF4α. In this study, we first investigated the impact of the ectopic expression of HNF4α in rLEC on both mRNA and microRNA (miR) level by means of microarray technology. We found that HNF4α transduction did not induce major changes to the rLEC phenotype. However, we next investigated the influence of DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated naïve rLEC by exposure to 5' azacytidine (AZA), which was found to have a significant impact on rLEC gene expression. The transduction of HNF4α or AZA treatment resulted both in significantly downregulated C/EBPα expression levels, while the exposure of the cells to AZA had a significant effect on the expression of HNF3ß. Computationally, dysregulated miRNAs were linked to target mRNAs using the microRNA Target Filter function of Ingenuity Pathway Analysis. We found that differentially regulated miRNA-mRNA target associations predict ectopic HNF4α expression in naïve rLEC to interfere with cell viability and cellular maturation (miR-19b-3p/NR4A2, miR30C-5p/P4HA2, miR328-3p/CD44) while it predicts AZA exposure to modulate epithelial/hepatic cell proliferation, apoptosis, cell cycle progression and the differentiation of stem cells (miR-18a-5p/ESR1, miR-503-5p/CCND1). Finally, our computational analysis predicts that the combination of HNF4α transduction with subsequent AZA treatment might cause changes in hepatic cell proliferation and maturation (miR-18a-5p/ESR1, miR-503-5p/CCND1, miR-328-3p/CD44) as well as the apoptosis (miR-16-5p/BCL2, miR-17-5p/BCL2, miR-34a-5p/BCL2 and miR-494-3p/HMOX1) of naïve rLEC.

Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.

Measurement of Albumin Secretion as Functionality Test in Primary Hepatocyte Cultures.

One of the most important functions of hepatocytes is the synthesis of serum proteins, more specifically of serum albumin. Albumin secretion in serum is essential, since it maintains the oncotic pressure in the body. Measurement of albumin secretion is used as a liver function test to indicate potential liver injury and liver pathology. In this chapter, a protocol for the measurement of albumin secretion in the supernatant of cultured rat hepatocytes is described. The procedure relies on an enzyme-linked immunosorbent assay allowing rat albumin to be quantitatively measured.

Functionality Testing of Primary Hepatocytes in Culture by Measuring Urea Synthesis.

One of the mechanisms of healthy hepatocytes to detoxify ammonia in the liver consists of converting it into urea. When liver function is impaired, this detoxification capacity decreases and may cause severe pathologies, such as hepatic encephalopathy. Consequently, urea synthesis is a parameter that can be used to monitor liver functionality. In this chapter, a protocol for the measurement of urea synthesis in the culture medium of cultured rat hepatocytes is described. The procedure relies on a chromogenic reagent that specifically forms a colored complex with urea. The latter can be measured colorimetrically and is directly proportional to the urea concentration in the sample.

MicroRNAs as key regulators of xenobiotic biotransformation and drug response.

In the last decade, microRNAs have emerged as key factors that negatively regulate mRNA expression. It has been estimated that more than 50% of protein-coding genes are under microRNA control and each microRNA is predicted to repress several mRNA targets. In this respect, it is recognized that microRNAs play a vital role in various cellular and molecular processes and that, depending on the biological pathways in which they intervene, distorted expression of microRNAs can have serious consequences. It has recently been shown that specific microRNA species are also correlated with toxic responses induced by xenobiotics. Since the latter are primarily linked to the extent of detoxification in the liver by phase I and phase II biotransformation enzymes and influx and efflux drug transporters, the regulation of the mRNA levels of this particular set of genes through microRNAs is of great importance for the overall toxicological outcome. Consequently, in this paper, an overview of the current knowledge with respect to the complex interplay between microRNAs and the expression of biotransformation enzymes and drug transporters in the liver is provided. Nuclear receptors and transcription factors, known to be involved in the transcriptional regulation of these genes, are also discussed.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential.

Human adult stem cells (hASCs) have become an attractive source for autologous cell transplantation, tissue engineering, developmental biology, and the generation of human-based alternative in vitro models. Among the 3 germ cell layers, the mesoderm is the origin of today's most widely used and characterized hASC populations. A variety of isolated nonhematopoietic mesoderm-derived stem cell populations exist, and all of them show important differences in terms of function, efficacy, and differentiation potential both in vivo and in vitro. To better understand whether the intrinsic properties of these cells contribute to the overall differentiation potential of hASCs, we compared the global gene expression profiles of 4 mesoderm-derived stem cell populations: human adipose tissue-derived stromal cells, human bone marrow-derived stromal cells (hBMSCs), human (fore)skin-derived precursor cells (hSKPs), and human Wharton's jelly-derived mesenchymal stem cells (hWJs). Significant differences in gene expression profiles were detected between distinct stem cell types. hSKPs predominantly expressed genes involved in neurogenesis, skin, and bone development, whereas hWJs and, to some extent, hBMSCs showed an increased expression of genes involved in cardiovascular and liver development. Interestingly, the observed differential gene expression of distinct hASCs could be linked to existing differentiation data in which hASCs were differentiated toward specific cell types. As such, our data suggest that the intrinsic gene expression of the undifferentiated stem cells has an important impact on their overall differentiation potential as well as their application in stem cell-based research. Yet, the factors that define these intrinsic properties remain to be determined.

Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes.

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.