Robim M. Rodrigues

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 todays 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 Whartons 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.

Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells.

When in vitro test systems are evaluated for assessment of the toxicity of chemical compounds, particular efforts are made to mimic the in vivo reality as close as possible. Cellular models with appropriate metabolic competence, i.e. with the potency to biotransform chemical compounds, are considered crucial since some metabolites have a different toxicity than their parent compounds. In this study a cell based in vitro test system is proposed to investigate the basal cytotoxicity of several reference chemicals. Both metabolic competent HepaRG cells and cells with no or low hepatic enzyme activity (undifferentiated HepaRG and proliferating HepG2) were used. The classic Neutral Red Uptake (NRU) assay proved to be robust and reliable to be applied as viability assay. The test was performed on a robotic platform, which enabled fully automated and simultaneous screening of the compounds. The outcome of these tests grouped the tested compounds in three categories following their detoxification effect (benzo(a)pyrene, valproic acid), their bio-activation effect (aflatoxin B1) and their specific effect on inhibition of cell proliferation (cycloheximide, sodium lauryl sulphate, atropine sulphate monohydrate, acetylsalicylic acid).

Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests.

Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000 mg/kg b.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration-response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained.

Metabolomics analysis of the toxicity pathways of triphenyl phosphate in HepaRG cells and comparison to oxidative stress mechanisms caused by acetaminophen.

Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50 μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72 h exposure, cells were subjected to quenching and liquid-liquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.

Human skin-derived precursor cells are poorly immunogenic and modulate the allogeneic immune response.

Human skin‐derived precursors (hSKPs) are multipotent somatic stem cells that persist within the dermis throughout adulthood and harbor potential clinical applicability. In this study, we investigated their immunogenicity and immunosuppressive features, both in vitro and in vivo. As such, this study provides a solid basis for developing their future clinical applications. We found that hSKPs express HLA‐ABC molecules, but not HLA‐DR, rendering them poorly immunogenic. Using a coculture set‐up, we could further demonstrate that hSKPs inhibit the proliferation of allogeneic activated T cells and alter their cytokine secretion profile, in a dose‐dependent manner. Cotransplantation of hSKP and human peripheral blood leukocytes (PBL) into severe combined immune‐deficient mice also showed a significant impairment of the graft‐versus‐host response 1 week post‐transplantation and a drastic increase in survival time of 60%. From a mechanistic point of view, we found that hSKPs require cell contact as well as secretion of soluble inhibitory factors in order to modulate the immune response. The expression/secretion levels of these factors further increases upon inflammation or in the presence of activated T cells. As such, we believe that these features could be beneficial in a later allogeneic clinical setting, because rejection of engrafted allogeneic hSKP might be delayed or even avoided due to their own promotion of a tolerogenic microenvironment. Stem Cells 2014;32:2215–2228

In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells

Steatosis, also known as fatty liver disease (FLD), is a disorder in which the lipid metabolism of the liver is disturbed, leading to the abnormal retention of lipids in hepatocytes. FLD can be induced by several drugs, and although it is mostly asymptomatic, it can lead to steatohepatitis, which is associated with liver inflammation and damage. Drug-induced liver injury is currently the major cause of postmarketing withdrawal of pharmaceuticals and discontinuation of the development of new chemical entities. Therefore, the potential induction of steatosis must be evaluated during preclinical drug development. However, robust human-relevant in vitro models are lacking. In the present study, we explore the applicability of hepatic cells (hSKP-HPCs) derived from postnatal skin precursors, a stem cell population residing in human dermis, to investigate the steatosis-inducing effects of sodium valproate (Na-VPA). Exposure of hSKP-HPC to sub-cytotoxic concentrations of this reference steatogenic compound showed an increased intracellular accumulation of lipid droplets, and the modulation of key factors involved in lipid metabolism. Using a toxicogenomics approach, we further compared Na-VPA-treated hSKP-HPC and Na-VPA-treated primary human hepatocytes to liver samples from patients suffering from mild and advanced steatosis. Our data show that in hSKP-HPC exposed to Na-VPA and liver samples of patients suffering from mild steatosis, but not in primary human hepatocytes, “liver steatosis” was efficiently identified as a toxicological response. These findings illustrate the potential of hSKP-HPC as a human-relevant in vitro model to identify hepatosteatotic effects of chemical compounds.

Tailored liquid chromatography–mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells

Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies.

Human stem cell-derived hepatocytes: breakthrough of an expedient tool for preclinical assessment of drug-induced liver injury?

human hepatocytes are also prohibitively expensive for high-throughput screening purposes. Problems are also encountered when human hepatic cell lines are used, as these cells are mostly derived from liver cancer patients and do not adequately represent the normal population diversity (Guguen-Guillouzo and Guillouzo 2010). Consequently, innovative approaches to predict adverse liver responses in humans are urgently needed. New developments coming from the rapidly advancing human stem cell research field are in the pipeline (McGivern and Ebert 2013). In fact, due to the biological flexibility of stem cells, biologists and toxicologists strongly believe that once the in vivo mechanisms driving cell differentiation and dedifferentiation are fully understood, stem cells can be modulated a la carte. As a consequence, these cells will obtain the appropriate functionalities that are required for evaluating a particular toxicological mode of action (MoA). As such, stem cells could represent a valuable “fit for purpose” tool in the unraveling of the MoA of a chemical substance at the molecular level. Integration of stem cells in an in vitro setting to screen for toxicity could significantly increase the predictive capacity of the testing platform. Indeed, starting from embryonic and postnatal stem cells, functional hepatocyte-like cells can be generated that possess the required machinery to predict hepatotoxicity and provide valuable human-based toxicological information (Baxter et al. 2010; Snykers et al. 2009; Sullivan et al. 2010). As an immediate consequence of the enormous advancement of “omics” technology during the last decade, an overwhelming amount of biologically relevant information can be generated and thanks to the availability of appropriate computational tools, the obtained data can be relatively easy and quickly interpreted. The first reports on accurate prediction of hepatotoxicity in response to specific Detection of drug toxicity, often occurring late during the drug development process, is of major concern for the pharmaceutical industry and jeopardizes the potential marketing of new chemical entities (Pammolli et al. 2011; Goldkind and Laine 2006). In addition, one of the leading causes of post-marketing withdrawal of pharmaceuticals is drug-induced liver injury (DILI) (Russmann et al. 2009), suggesting that the currently used in vitro and in vivo preclinical models for assessing liver toxicity are not effective enough (Pammolli et al. 2011; Godoy et al. 2013; Olson et al. 2000). In line with recent regulatory developments in Europe for chemicals (EU 2006) and cosmetics (EU 2009), global consensus is growing to introduce, whenever possible, physiologically relevant alternative methods to animal testing, also in the pharmaceutical industry (Bouvier d’Yvoire et al. 2012). Current in vitro approaches for hepatotoxicity screening are mainly based on primary cells of different species, including man, or human hepatomaderived cell lines. Primary human hepatocyte cultures represent the gold standard, but their use is hampered by their low availability and their inability to proliferate in culture (Fraczek et al. 2013). Due to intensive transplantation programs, hepatocytes can be seldom isolated from healthy human livers. Instead, they are obtained from patients suffering from severe liver injuries or coping with a multidrug regimen and therefore often display poor cell quality (Guguen-Guillouzo and Guillouzo 2010). Due to their scarcity, primary

Metabolomics profiling of steatosis progression in HepaRG® cells using sodium valproate

Non-alcoholic Fatty Liver Disease (NAFLD) is a frequently encountered Drug-Induced Liver Injury (DILI). Although this stage of the disease is reversible, it can lead to irreversible damage provoked by non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Therefore, the assessment of NAFLD is a paramount objective in toxicological screenings of new drug candidates. In this study, a metabolomic fingerprint of NAFLD induced in HepaRG® cells at four dosing schemes by a reference toxicant, sodium valproate (NaVPA), was obtained using liquid-liquid extraction followed by liquid chromatography and accurate mass-mass spectrometry (LC-AM/MS). The combination of a strict design of experiment with a robust detection method, applied on sodium valproate, validated the possibilities of untargeted metabolomics in hepatic toxicological research. Distinctive patterns between exposed and control cells were consistently observed, multivariate analyses selected up to 200 features of interest, revealing hallmark NAFLD-biomarkers, such as diacylglycerol and triglyceride accumulation and carnitine deficiency. Initial toxic responses show increased levels of S-adenosylmethionine and mono-acetylspermidine in combination with only a moderate increase in triglycerides. New specific markers of toxicity have been observed, such as spermidines, creatine, and acetylcholine. The described design of experiment provides a valuable metabolomics platform for mechanistic research of toxicological hazards and identified new markers for steatotic progression.

Optimisation of in vitro sample preparation for LC-MS metabolomics applications on HepaRG cell cultures

Untargeted metabolomics studies are designed to observe as many metabolites as possible. A reliable protocol should be fast, efficient, unspecific and it should introduce as little variance as possible. Liquid–liquid extraction (LLE) is often applied during sample preparation, since it leads to a polar (aqueous) and a non-polar (solvent) fraction. In the present study, we have applied for the first time pH buffers and other additives during the LLE to prevent degradation of the metabolites and to improve the precision of the method. The use of chamber slides in comparison to well plates improved the washing and quenching step and reduced the average quench time. Extraction efficiency and usability improved as well. The stabilisation of the pH of the LLE-solvent and the addition of anti-oxidants and chelators improved the precision of the method. Optimal results were observed at buffer strength of 10 mM at pH 4.4. Ascorbic acid (0.5 mM) and butyl-hydroxytoluene (1 mM) in combination with EDTA (1 mM) prevented oxidation and degradation.