Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells.

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity.

Differences in the Early Development of Human and Mouse Embryonic Stem Cells.

We performed a systematic analysis of gene expression features in early (10-21 days) development of human vs mouse embryonic cells (hESCs vs mESCs). Many development features were found to be conserved, and a majority of differentially regulated genes have similar expression change in both organisms. The similarity is especially evident, when gene expression profiles are clustered together and properties of clustered groups of genes are compared. First 10 days of mESC development match the features of hESC development within 21 days, in accordance with the differences in population doubling time in human and mouse ESCs. At the same time, several important differences are seen. There is a clear difference in initial expression change of transcription factors and stimulus responsive genes, which may be caused by the difference in experimental procedures. However, we also found that some biological processes develop differently; this can clearly be shown, for example, for neuron and sensory organ development. Some groups of genes show peaks of the expression levels during the development and these peaks cannot be claimed to happen at the same time points in the two organisms, as well as for the same groups of (orthologous) genes. We also detected a larger number of upregulated genes during development of mESCs as compared to hESCs. The differences were quantified by comparing promoters of related genes. Most of gene groups behave similarly and have similar transcription factor (TF) binding sites on their promoters. A few groups of genes have similar promoters, but are expressed differently in two species. Interestingly, there are groups of genes expressed similarly, although they have different promoters, which can be shown by comparing their TF binding sites. Namely, a large group of similarly expressed cell cycle-related genes is found to have discrepant TF binding properties in mouse vs human.

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.

Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons.

Fam40b is required for lineage commitment of murine embryonic stem cells.

FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-ß (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.

Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+).

Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP(+)) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP(+) (5 µM). At 18-24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP(+). Thus, combined 'Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery.

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-ß and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay.

A tanscriptomics study to elucidate the toxicological mechanism of methylmercury chloride in a human stem cell based in vitro test.

Traditional approaches in evaluating the hazard of drug candidates on the developing offspring are often time-consuming and cost-intensive. Moreover, variations in the toxicological response of different animal species to the tested substance cause severe problems when extrapolating safety dosages for humans. Therefore, more predictive and relevant toxicological systems based on human cell models are required. In the presented study the environmental toxicant methylmercury chloride (MeHgCl), known to cause structural developmental abnormalities in the brain, was used as reference compound to develop a concept contributing to a mechanistic understanding of the toxicity of an investigated substance. Despite the fact, that there are significant data available from animal studies and from poisonings in Japan and Iraq, uncertainties on the mechanism of MeHgCl during human development are still remaining and qualify the substance for further analysis. Transcriptomics analysis in combination with a human cell based in vitro model has been used in order to elucidate the toxicity of MeHgCl at molecular level. Differentiating neural precursor cells that have been exposed continuously to non- and low-cytotoxic concentrations of MeHgCl were investigated. Quantitative change in the mRNA expression profiles of selected genes demonstrated the sensitivity of the cell model and its qualification for a transcriptomics study screening changes in the expression profile of the complete human genome of MeHgCl-treated human neural cells. Potential biomarkers were identified and these candidate marker genes as well as their involvement in a possible toxic mechanism of MeHgCl during the human neurulation process are hereby introduced. The study confirmed the hypothesis that a cellular model based on a human stem cell line can be applied for elucidating unknown mode of actions of developmental toxicants.

Characterisation of a neural teratogenicity assay based on human ESCs differentiation following exposure to valproic acid.

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity.

A liver-derived rat epithelial cell line from biliary origin acquires hepatic functions upon sequential exposure to hepatogenic growth factors and cytokines.

Withdrawal of promising drug candidates is often due to the detection of liver toxicity. In particular the parenchymal liver cells or hepatocytes are targeted since they are the major sites of drug transport and of metabolite formation and thus also the place where not only detoxification, but also activation of new chemical (NCE) and biological (NBE) entities may occur. Therefore, primary hepatocyte- based cultures are currently the preferred in vitro model to screen for liver toxicity. However, within a few days, they undergo dedifferentiation with loss of liver-specific functionality, including xenobiotic biotransformation capacity, making them only suitable for short-term applications. A plausible alternative to primary hepatocyte cultures that can be maintained for longer periods of time could be the use of liver-derived epithelial cell lines and their optimized derivatives. Therefore, in the present study, we evaluated the stability and the hepatic differentiation potential of a neonatal liver-derived rat epithelial cell line from biliary origin (rLEC). Undifferentiated rLEC stably express the hepatic progenitor markers CEBPA, FOXA2, GJA1, ONECUT1, KRT18 and KRT19 for at least 15 consecutive passages after cryopreservation. Upon sequential exposure to hepatogenic growth factors and cytokines, rLEC generate functional hepatic progeny, expressing mature hepatic markers including Alb, Ahr, Car, C/ebpα, Cx32, Foxa2, Hnf1α, Hnf1ß and Onecut1. Furthermore, an active polarization is observed for the hepatic drug transporters Oatp4 and Ntcp. rLEC-derived hepatic cells also acquire the ability to store glycogen, express genes encoding for key hepatic enzymes as shown by Affymetrix microarray data, and display stable CYP1A1/2- and CYP2B1/2-dependent activities for several weeks at levels comparable to those observed in cultured primary rat hepatocytes. The acquisition of such a stable and active biotransformation capacity is key for the applicability of liver-based in vitro models for long-term toxicity testing.

Derivation of es Cells From Early Stage Preimplantation Embryos and Characterisation of their Cardiac Differentiation Potential in Mice.

Most murine embryonic stem cell lines have been derived from the inner cell mass of blastocysts and extensively studied in different aspects including generation of organ specific cells. However, no detailed studies have been made on cardiac specific gene expression, immunocytochemical and electrophysiological characterisation of cardiomyocytes generated from early stage (preimplantation) embryo derived embryonic stem cells in mice. In the present study, new embryonic stem cell lines were derived from early stage preimplanatation embryos in mice. In vitro differentiation of such cell lines readily generated cardiomyocytes, which expressed different cardiac specific genes in a temporally regulated manner as well as cardiac cells specific proteins. This is probably the first report, which showed the temporal pattern of cardiac specific genes as well as protein expression in cardiac cells generated from in vitro differentiation of preimplantation embryo derived ES cells.

Effective surface-based cryopreservation of human embryonic stem cells by vitrification.

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing. Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments.

Induced pluripotent stem cells as a model for accelerated patient- and disease-specific drug discovery.

Human induced pluripotent stem (iPS) cells hold great promise for therapy of a number of degenerative diseases such as ischemic heart failure, Parkinson's disease, Alzheimer's disease, diabetes mellitus, sickle cell anemia and Huntington disease. They also have the potential to accelerate drug discovery in 3 ways. The first involves the delineation of chemical components for efficient reprogramming of patient's blood cells or cells from biopsies, obviating the need for cellular delivery of reprogramming exogenous transgenes, thereby converting hope into reality for patients suffering from degenerative diseases. Patients worldwide stand to benefit from the clinical applicability of iPS cell-based cell replacement therapy for a number of degenerative diseases. The second is the potential for discovering novel drugs in a high throughput manner using patient-specific iPS cell-derived somatic cells possessing the etiology of the specific disease. The third is their suitability for toxicological testing of drugs and environmental factors. This review focuses on these potential applications of iPS cells with special emphasis on recent updates of iPS cell research contributing to the accelerated drug discovery.

The potential of embryonic stem cells combined with -omics technologies as model systems for toxicology.

The derivation of pluripotent embryonic stem (ES) cell lines has opened up new areas of research in basic and applied science, most significantly in developmental biology and regenerative medicine. While application-oriented research has for the most part focussed on obtaining differentiated, organotypic cells from ES cells for future cell grafting therapies, ES cells have more immediate potential for use in toxicological in vitro assays used during drug development. ES cells are derived from blastocyst-stage embryos and offer an in vitro model for early development, thus enabling tests for teratogenicity testing in a human cell culture system and avoiding the pitfalls of inter-species differences. Differentiated, organotypic cells obtained from ES cells can potentially replace the primary cells and cell lines currently used for in vitro toxicology by offering a more consistent and potentially limitless source of differentiated cells. This can facilitate the establishment of comprehensive toxicogenomics and -proteomics databases and complement current databases that rely on data obtained from animal experiments. More recently, induced pluripotent stem (iPS) cells with ES cell-like properties have been obtained through reprogramming of somatic cells, thus enabling the generation of disease-specific cell lines. We review the potential of combining ES cells and ES cell-derived somatic cells with "omics" technologies for in vitro toxicology with a particular emphasis on the development of toxicogenomics and toxicoproteomics signatures. A separate section describes the potential of iPS cells for toxicology.