Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition.

According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2(+) neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations.

Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy.

BACKGROUND: mRNA electroporation of dendritic cells (DCs) facilitates processing and presentation of multiple peptides derived from whole antigen, tailored to different HLA molecules. Clinical responses to electroporated moDC vaccines, however, have been suboptimal. Human Langerhans-type DCs (LCs) are the most potent conventional DC subtype for inducing CD8+ cytotoxic T lymphocytes (CTLs) in vitro. We recently demonstrated that Wilms' tumor 1 (WT1) mRNA-electroporated LCs are superior to moDCs as stimulators of tumor antigen-specific CD8+ CTLs, even though they are comparable stimulators of allogeneic T cell proliferative responses. A detailed comparative evaluation of the effects of mRNA electroporation on LCs versus moDCs, however, is needed. METHODS: Immature and partially-matured human moDCs and LCs electroporated with mRNA were compared for transfection efficiency, phenotypic changes, viability, retention of transgene expression after cryopreservation, and immunogenicity. Student t test was used for each pairwise comparison. One-way analysis of variance was used for multiple group comparisons. RESULTS: Transfection efficiency after electroporation with enhanced green fluorescent protein (eGFP) mRNA was higher for immature than for partially-matured moDCs. In contrast, transfection efficiency was higher for partially-matured than for immature LCs, with the additional benefit that electroporation itself increased maturation and activation of CD83+HLA-DRbright LCs but not moDCs. Electroporation did not impair final maturation and activation of either DC subtype, after which both mRNA-electroporated LCs and moDCs were functionally similar in stimulating allogeneic T cell proliferation, a standard assay of DC immunogenicity. CONCLUSIONS: These findings support mRNA electroporation of DCs, and in particular LCs, as an effective non-viral method to stimulate specific, potent CD8+ CTL responses. The differences between LCs and moDCs regarding this form of antigen-loading have important implications for DC-based immunotherapies.

Adverse outcome pathway-based screening strategies for an animal-free safety assessment of chemicals.

Currently, the assessment of risk to human health from exposure to manufactured chemicals is mainly based on experiments performed on living animals (in vivo). Substantial efforts are being undertaken to develop alternative solutions to in vivo toxicity testing. This new paradigm, based on the Mode-of-Action (MoA) framework, postulates that any adverse human health effect caused by exposure to an exogenous substance can be described by a series of causally-linked biochemical or biological key events with measurable parameters. The elaboration of mechanistic knowledge through literature research is necessary for a MoA-driven design of integrated testing strategies using in vitro methods for in vivo predictions. The objective of our ongoing research is to demonstrate the feasibility of an integrated approach to predict human toxicity following the Adverse Outcome Pathway (AOP) framework. In our previous work on MoA with the HepaRG cell model, we developed a strategy to identify chemicals that were hepatotoxic. This pioneered an innovative way of using data from in vitro experiments to group chemicals based on their MoA, which is likely to be an important step in a toxicity testing strategy.

Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Sex differences in liver toxicity-do female and male human primary hepatocytes react differently to toxicants in vitro?

There is increasing amount of evidence for sex variation in drug efficiency and toxicity profiles. Women are more susceptible than men to acute liver injury from xenobiotics. In general, this is attributed to sex differences at a physiological level as well as differences in pharmacokinetics and pharmacodynamics, but neither of these can give a sufficient explanation for the diverse responses to xenobiotics. Existing data are mainly based on animal models and limited data exist on in vitro sex differences relevant to humans. To date, male and female human hepatocytes have not yet been compared in terms of their responses to hepatotoxic drugs. We investigated whether sex-specific differences in acute hepatotoxicity can be observed in vitro by comparing hepatotoxic drug effects in male and female primary human hepatocytes. Significant sex-related differences were found for certain parameters and individual drugs, showing an overall higher sensitivity of female primary hepatocytes to hepatotoxicants. Moreover, our work demonstrated that high content screening is feasible with pooled primary human hepatocytes in suspension.

Iron oxide nanoparticle toxicity testing using high-throughput analysis and high-content imaging.

Applying validated in vitro assays to the study of nanoparticle toxicity is a growing trend in nanomaterial risk assessment. Precise characterisation of reference nanomaterials and a well-regulated in vitro testing system are required to determine the physicochemical descriptors which dictate the toxic potential of nanoparticles. The use of automated, high-throughput technologies to facilitate the identification and prioritisation of nanomaterials which could pose a risk is desirable and developments are underway. In this study, two mammalian fibroblast lines (Balb/c 3T3 and COS-1 cells) were treated with a range of concentrations of iron oxide nanomaterials manufactured for use in medical diagnostics, using an automated platform and high-content-imaging endpoints for cell viability, oxidative stress and DNA damage (double-strand breaks). At the same time, the high-throughput comet assay was employed to measure DNA strand breaks and oxidised bases. Our results show that these methods provide a fast way to determine the toxicity of coated and uncoated iron oxide nanoparticles and, furthermore, to predict the mechanism of toxicity in vitro.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: effects of methylmercury, lead and aluminum evaluated by gene expression studies.

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

Three-dimensional HepaRG model as an attractive tool for toxicity testing.

The culture of HepaRG cells as three dimensional (3D) structures in the spinner-bioreactor may represent added value as a hepatic system for toxicological purposes. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine use in the drug testing industry. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG system, the induction of CYP450 enzymes (i.e., CYP1A2, 2B6, 2C9, and 3A4) and the activity of the phase II enzyme, uridine diphosphate glucuronoltransferase (UGT), were tested. The long-term functionality of the system was demonstrated by 7-week stable profiles of albumin secretion, CYP3A4 induction, and UGT activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters. The use of in silico models to analyze the in vitro data related to hepatotoxic activity of acetaminophen (APAP) demonstrated the advantage of the integration of kinetic and dynamic aspects for a better understanding of the in vitro cell behavior. The bioactivation of APAP and its related cytotoxicity was assessed in a system compatible to high-throughput screening. The approach also proved to be a good strategy to reduce the time necessary to obtain fully differentiated cell cultures. In conclusion, HepaRG cells cultured in 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.

Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase.

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.