Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Proteomics and disease network associations evaluation of environmentally relevant Bisphenol A concentrations in a human 3D neural stem cell model.

Bisphenol A (BPA) exposure is associated with a plethora of neurodevelopmental abnormalities and brain disorders. Previous studies have demonstrated BPA-induced perturbations to critical neural stem cell (NSC) characteristics, such as proliferation and differentiation, although the underlying molecular mechanisms remain under debate. The present study evaluated the effects of a repeated-dose exposure of environmentally relevant BPA concentrations during the in vitro 3D neural induction of human induced pluripotent stem cells (hiPSCs), emulating a chronic exposure scenario. Firstly, we demonstrated that our model is suitable for NSC differentiation during the early stages of embryonic brain development. Our morphological image analysis showed that BPA exposure at 0.01, 0.1 and 1 µM decreased the average spheroid size by day 21 (D21) of the neural induction, while no effect on cell viability was detected. No alteration to the rate of the neural induction was observed based on the expression of key neural lineage and neuroectodermal transcripts. Quantitative proteomics at D21 revealed several differentially abundant proteins across all BPA-treated groups with important functions in NSC proliferation and maintenance (e.g., FABP7, GPC4, GAP43, Wnt-8B, TPPP3). Additionally, a network analysis demonstrated alterations to the glycolytic pathway, potentially implicating BPA-induced changes to glycolytic signalling in NSC proliferation impairments, as well as the pathophysiology of brain disorders including intellectual disability, autism spectrum disorders, and amyotrophic lateral sclerosis (ALS). This study enhances the current understanding of BPA-related NSC aberrations based mostly on acute, often high dose exposures of rodent in vivo and in vitro models and human GWAS data in a novel human 3D cell-based model with real-life scenario relevant prolonged and low-level exposures, offering further mechanistic insights into the ramifications of BPA exposure on the developing human brain and consequently, later life neurological disorders.

Low-dose Bisphenol A exposure alters the functionality and cellular environment in a human cardiomyocyte model.

Early embryonic development represents a sensitive time-window during which the foetus might be vulnerable to the exposure of environmental contaminants, potentially leading to heart diseases also later in life. Bisphenol A (BPA), a synthetic chemical widely used in plastics manufacturing, has been associated with heart developmental defects, even in low concentrations. This study aims to investigate the effects of environmentally relevant doses of BPA on developing cardiomyocytes using a human induced pluripotent stem cell (hiPSC)-derived model. Firstly, a 2D in vitro differentiation system to obtain cardiomyocytes from hiPSCs (hiPSC-CMs) have been established and characterised to provide a suitable model for the early stages of cardiac development. Then, the effects of a repeated BPA exposure, starting from the undifferentiated stage throughout the differentiation process, were evaluated. The chemical significantly decreased the beat rate of hiPSC-CMs, extending the contraction and relaxation time in a dose-dependent manner. Quantitative proteomics analysis revealed a high abundance of basement membrane (BM) components (e.g., COL4A1, COL4A2, LAMC1, NID2) and a significant increase in TNNC1 and SERBP1 proteins in hiPSC-CMs treated with BPA. Network analysis of proteomics data supported altered extracellular matrix remodelling and provided a disease-gene association with well-known pathological conditions of the heart. Furthermore, upon hypoxia-reoxygenation challenge, hiPSC-CMs treated with BPA showed higher rate of apoptotic events. Taken together, our results revealed that a long-term treatment, even with low doses of BPA, interferes with hiPSC-CMs functionality and alters the surrounding cellular environment, providing new insights about diseases that might arise upon the toxin exposure. Our study contributes to the current understanding of BPA effects on developing human foetal cardiomyocytes, in correlation with human clinical observations and animal studies, and it provides a suitable model for New Approach Methodologies (NAMs) for environmental chemical hazard and risk assessment.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Epigenetic Mechanisms of ART-Related Imprinting Disorders: Lessons From iPSC and Mouse Models.

The rising frequency of ART-conceived births is accompanied by the need for an improved understanding of the implications of ART on gametes and embryos. Increasing evidence from mouse models and human epidemiological data suggests that ART procedures may play a role in the pathophysiology of certain imprinting disorders (IDs), including Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Prader-Willi syndrome, and Angelman syndrome. The underlying molecular basis of this association, however, requires further elucidation. In this review, we discuss the epigenetic and imprinting alterations of in vivo mouse models and human iPSC models of ART. Mouse models have demonstrated aberrant regulation of imprinted genes involved with ART-related IDs. In the past decade, iPSC technology has provided a platform for patient-specific cellular models of culture-associated perturbed imprinting. However, despite ongoing efforts, a deeper understanding of the susceptibility of iPSCs to epigenetic perturbation is required if they are to be reliably used for modelling ART-associated IDs. Comparing the patterns of susceptibility of imprinted genes in mouse models and IPSCs in culture improves the current understanding of the underlying mechanisms of ART-linked IDs with implications for our understanding of the influence of environmental factors such as culture and hormone treatments on epigenetically important regions of the genome such as imprints.

Environmental Alterations during Embryonic Development: Studying the Impact of Stressors on Pluripotent Stem Cell-Derived Cardiomyocytes.

Non-communicable diseases (NCDs) sauch as diabetes, obesity and cardiovascular diseases are rising rapidly in all countries world-wide. Environmental maternal factors (e.g., diet, oxidative stress, drugs and many others), maternal illnesses and other stressors can predispose the newborn to develop diseases during different stages of life. The connection between environmental factors and NCDs was formulated by David Barker and colleagues as the Developmental Origins of Health and Disease (DOHaD) hypothesis. In this review, we describe the DOHaD concept and the effects of several environmental stressors on the health of the progeny, providing both animal and human evidence. We focus on cardiovascular diseases which represent the leading cause of death worldwide. The purpose of this review is to discuss how in vitro studies with pluripotent stem cells (PSCs), such as embryonic and induced pluripotent stem cells (ESC, iPSC), can underpin the research on non-genetic heart conditions. The PSCs could provide a tool to recapitulate aspects of embryonic development "in a dish", studying the effects of environmental exposure during cardiomyocyte (CM) differentiation and maturation, establishing a link to molecular mechanism and epigenetics.

Maternal One-Carbon Metabolism during the Periconceptional Period and Human Foetal Brain Growth: A Systematic Review.

The maternal environment during the periconceptional period influences foetal growth and development, in part, via epigenetic mechanisms moderated by one-carbon metabolic pathways. During embryonic development, one-carbon metabolism is involved in brain development and neural programming. Derangements in one-carbon metabolism increase (i) the short-term risk of embryonic neural tube-related defects and (ii) long-term childhood behaviour, cognition, and autism spectrum disorders. Here we investigate the association between maternal one-carbon metabolism and foetal and neonatal brain growth and development. Database searching resulted in 26 articles eligible for inclusion. Maternal vitamin B6, vitamin B12, homocysteine, and choline were not associated with foetal and/or neonatal head growth. First-trimester maternal plasma folate within the normal range (>17 nmol/L) associated with increased foetal head size and head growth, and high erythrocyte folate (1538-1813 nmol/L) with increased cerebellar growth, whereas folate deficiency (<7 nmol/L) associated with a reduced foetal brain volume. Preconceptional folic acid supplement use and specific dietary patterns (associated with increased B vitamins and low homocysteine) increased foetal head size. Although early pregnancy maternal folate appears to be the most independent predictor of foetal brain growth, there is insufficient data to confirm the link between maternal folate and offspring risks for neurodevelopmental diseases.

Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres are Suitable for Neurotoxicity Screening.

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

Ca2+ mobilization-dependent reduction of the endoplasmic reticulum lumen is due to influx of cytosolic glutathione.

BACKGROUND: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca2+ store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca2+ from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. RESULTS: We found that Ca2+ mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca2+ efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca2+-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca2+ mobilization. While the action of puromycin was ascribable to Ca2+ leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. CONCLUSIONS: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca2+ mobilization. We postulate the existence of a Ca2+- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.

Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-ß1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-ß1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-ß1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-ß1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1.

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2.

In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2(thyd)) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H2O2, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.

Spatial and temporal analysis of NADPH oxidase-generated hydrogen peroxide signals by novel fluorescent reporter proteins.

AIMS: Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2. RESULTS: We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci. INNOVATION: Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells. CONCLUSION: Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.