Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Corrigendum to TBBPA Targets Converging Key Events of Human Oligodendrocyte Development Resulting in Two Novel AOPs.

In this manuscript, which appeared in ALTEX 38, 215-234 (doi:10.14573/altex.2007201), there was an error in Figure 4. The corrected Figure is available at 10.14573/altex.2203151.

The Human Induced Pluripotent Stem Cell Test as an Alternative Method for Embryotoxicity Testing.

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay's applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.

Progenitor cells derived from gene-engineered human induced pluripotent stem cells as synthetic cancer cell alternatives for in vitro pharmacology.

Limitations in genetic stability and recapitulating accurate physiological disease properties challenge the utility of patient-derived (PD) cancer models for reproducible and translational research. A portfolio of isogenic human induced pluripotent stem cells (hiPSCs) with different pan-cancer relevant oncoprotein signatures followed by differentiation into lineage-committed progenitor cells was genetically engineered. Characterization on molecular and biological level validated successful stable genetic alterations in pluripotency state as well as upon differentiation to prove the functionality of our approach. Meanwhile proposing core molecular networks possibly involved in early dysregulation of stem cell homeostasis, the application of our cell systems in comparative substance testing indicates the potential for cancer research such as identification of augmented therapy resistance of stem cells in response to activation of distinct oncogenic signatures.

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery.

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell-based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 µM (5 FRs), 1<10 µM (7 FRs) to the >10 µM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell-based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment.

Academic application of Good Cell Culture Practice for induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSC) are a promising tool for replacing animal-based experiments. To warrant data reproducibility, quality-controlled research material is recommended. While the need for global harmonization of quality standards for stem cell banking centers, commercial providers, pre-clinical and clinical use of cells is well doc­umented, there are no recommendations available for quality control of hiPSC in an academic research environment to date. To fill this gap, we here give an example of a quality-controlled, two-tiered banking process producing a fully characterized master cell bank (MCB) and partially characterized respective working cell banks (WCB). Characteri­zation includes the study of morphology, mycoplasma contamination, cell line identity, karyotype stability, cell antigen expression and viability, gene expression, pluripotency, and post-thaw recovery. Costs of these procedures are cal­culated. We present the results of the proposed testing panel of two hiPSC MCBs and show that both fulfil the defined specifications regarding the above-mentioned characterization assays during and upon banking. In conclusion, we propose a panel of eight assays, which are practical and useful for an academic research laboratory working with hiPSCs. Meeting these proposed specifications ensures the quality of pluripotent stem cells throughout diverse experi­ments at moderate costs.

Stem Cells for Next Level Toxicity Testing in the 21st Century.

The call for a paradigm change in toxicology from the United States National Research Council in 2007 initiates awareness for the invention and use of human-relevant alternative methods for toxicological hazard assessment. Simple 2D in vitro systems may serve as first screening tools, however, recent developments infer the need for more complex, multicellular organotypic models, which are superior in mimicking the complexity of human organs. In this review article most critical organs for toxicity assessment, i.e., skin, brain, thyroid system, lung, heart, liver, kidney, and intestine are discussed with regards to their functions in health and disease. Embracing the manifold modes-of-action how xenobiotic compounds can interfere with physiological organ functions and cause toxicity, the need for translation of such multifaceted organ features into the dish seems obvious. Currently used in vitro methods for toxicological applications and ongoing developments not yet arrived in toxicity testing are discussed, especially highlighting the potential of models based on embryonic stem cells and induced pluripotent stem cells of human origin. Finally, the application of innovative technologies like organs-on-a-chip and genome editing point toward a toxicological paradigm change moves into action.

Neural In Vitro Models for Studying Substances Acting on the Central Nervous System.

Animal models have been greatly contributing to our understanding of physiology, mechanisms of diseases, and toxicity. Yet, their limitations due to, e.g., interspecies variation are reflected in the high number of drug attrition rates, especially in central nervous system (CNS) diseases. Therefore, human-based neural in vitro models for studying safety and efficacy of substances acting on the CNS are needed. Human iPSC-derived cells offer such a platform with the unique advantage of reproducing the "human context" in vitro by preserving the genetic and molecular phenotype of their donors. Guiding the differentiation of hiPSC into cells of the nervous system and combining them in a 2D or 3D format allows to obtain complex models suitable for investigating neurotoxicity or brain-related diseases with patient-derived cells. This chapter will give an overview over stem cell-based human 2D neuronal and mixed neuronal/astrocyte models, in vitro cultures of microglia, as well as CNS disease models and considers new developments in the field, more specifically the use of brain organoids and 3D bioprinted in vitro models for safety and efficacy evaluation.

TBBPA targets converging key events of human oligodendrocyte development resulting in two novel AOPs

Myelinating oligodendrocytes (OLs) establish saltatory nerve conduction during white matter development. Thus, interference with oligodendrogenesis leads to an adverse outcome on brain performance in the child due to aberrant myelination. An intertwined network of hormonal, transcriptional and biosynthetic processes regulates OL development, thereby simultaneously creating various routes of interference for environmental toxicants. The flame retardant tetrabromobisphenol A (TBBPA) is debated as an endocrine disruptor, especially of the thyroid hormone (TH) system. We identified how TBBPA interferes with the establishment of a population of maturing OLs by two independent modes-of-action (MoA), dependent and independent of TH signaling. Combining the previously published oligodendrocyte maturation assay (NPC6) with large-scale transcriptomics, we describe TBBPA as a TH disruptor, impairing human OL maturation in vitro by dysregulation of oligodendrogenesis-associated genes (i.e., MBP, KLF9 and EGR1). Furthermore, TBBPA disrupts a gene expression network regulating cholesterol homeostasis, reducing OL numbers independently of TH signaling. These two MoA converge in a novel putative adverse outcome pathway (AOP) network on the key event (KE) hypomyelination. Comparative analyses of human and rat neural progenitor cells (NPCs) revealed that human oligodendrogenesis is more sensitive to endocrine disruption by TBBPA. Therefore, ethical, cost-efficient and species-overarching in vitro assays are needed for developmental neurotoxicity hazard assessment. By incorporation of large-scale transcriptomic analyses, we brought the NPC6 assay to a higher readiness level for future applications in a regulatory context. The combination of phenotypic and transcriptomic analyses helps to study MoA to eventually build AOPs for a better understanding of neurodevelopmental toxicity.

Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

Neurotoxicity is mediated by a variety of modes-of-actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC-derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM)) of maturation-supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA-grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC-derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity.

Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells.

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors. Basic Protocol 1: Production and concentration of third-generation lentivirus Support Protocol 1: Cloning of gene of interest into modulation vector Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin-streptomycin Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells Support Protocol 3: Preparation of Matrigel® -coated plates Support Protocol 4: Preparation of mTeSR™1 medium.

Correction to: The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Toll-like receptor agonist imiquimod is metabolized by aryl hydrocarbon receptor-regulated cytochrome P450 enzymes in human keratinocytes and mouse liver.

The Toll-like receptor 7 agonist imiquimod (IMQ) is an approved drug for the topical treatment of various skin diseases that, in addition, is currently tested in multiple clinical trials for the immunotherapy of various types of cancers. As all of these trials include application of IMQ to the skin and evidence exists that exposure to environmental pollutants, i.e., tobacco smoke, affects its therapeutic efficacy, the current study aims to elucidate the cutaneous metabolism of the drug. Treatment of human keratinocytes with 2.5 µM benzo[a]pyrene (BaP), a tobacco smoke constituent and aryl hydrocarbon receptor (AHR) agonist, for 24 h induced cytochrome P450 (CYP) 1A enzyme activity. The addition of IMQ 30 min prior measurement resulted in a dose-dependent inhibition of CYP1A activity, indicating that IMQ is either a substrate or inhibitor of CYP1A isoforms. Incubation of 21 recombinant human CYP enzymes with 0.5 µM IMQ and subsequent LC-MS analyses, in fact, identified CYP1A1 and CYP1A2 as being predominantly responsible for IMQ metabolism. Accordingly, treatment of keratinocytes with BaP accelerated IMQ clearance and the associated formation of monohydroxylated IMQ metabolites. A co-incubation with 5 µM 7-hydroxyflavone, a potent inhibitor of human CYP1A isoforms, abolished basal as well as BaP-induced IMQ metabolism. Further studies with hepatic microsomes from CD-1 as well as solvent- and ß-naphthoflavone-treated CYP1A1/CYP1A2 double knock-out and respective control mice confirmed the critical contribution of CYP1A isoforms to IMQ metabolism. Hence, an exposure to life style-related, dietary, and environmental AHR ligands may affect the pharmacokinetics and, thus, treatment efficacy of IMQ.

Fibroblast origin shapes tissue homeostasis, epidermal differentiation, and drug uptake.

Preclinical studies frequently lack predictive value for human conditions. Human cell-based disease models that reflect patient heterogeneity may reduce the high failure rates of preclinical research. Herein, we investigated the impact of primary cell age and body region on skin homeostasis, epidermal differentiation, and drug uptake. Fibroblasts derived from the breast skin of female 20- to 30-year-olds or 60- to 70-year-olds and fibroblasts from juvenile foreskin (<10 years old) were compared in cell monolayers and in reconstructed human skin (RHS). RHS containing aged fibroblasts differed from its juvenile and adult counterparts, especially in terms of the dermal extracellular matrix composition and interleukin-6 levels. The site from which the fibroblasts were derived appeared to alter fibroblast-keratinocyte crosstalk by affecting, among other things, the levels of granulocyte-macrophage colony-stimulating factor. Consequently, the epidermal expression of filaggrin and e-cadherin was increased in RHS containing breast skin fibroblasts, as were lipid levels in the stratum corneum. In conclusion, the region of the body from which fibroblasts are derived appears to affect the epidermal differentiation of RHS, while the age of the fibroblast donors determines the expression of proteins involved in wound healing. Emulating patient heterogeneity in preclinical studies might improve the treatment of age-related skin conditions.

The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis.

Ultraviolet B (UVB) radiation induces mutagenic DNA photoproducts, in particular cyclobutane pyrimidine dimers (CPDs), in epidermal keratinocytes (KC). To prevent skin carcinogenesis, these DNA photoproducts must be removed by nucleotide excision repair (NER) or apoptosis. Here we report that the UVB-sensitive transcription factor aryl hydrocarbon receptor (AHR) attenuates the clearance of UVB-induced CPDs in human HaCaT KC and skin from SKH-1 hairless mice. Subsequent RNA interference and inhibitor studies in KC revealed that AHR specifically suppresses global genome but not transcription-coupled NER. In further experiments, we found that the accelerated repair of CPDs in AHR-compromised KC depended on a modulation of the p27 tumor suppressor protein. Accordingly, p27 protein levels were increased in AHR-silenced KC and skin biopsies from AHR-/- mice, and critical for the improvement of NER. Besides increasing NER activity, AHR inhibition was accompanied by an enhanced occurrence of DNA double-strand breaks triggering KC apoptosis at later time points after irradiation. The UVB-activated AHR thus acts as a negative regulator of both early defense systems against carcinogenesis, NER and apoptosis, implying that it exhibits tumorigenic functions in UVB-exposed skin. In fact, AHR-/- mice developed 50% less UVB-induced cutaneous squamous cell carcinomas in a chronic photocarcinogenesis study than their AHR+/+ littermates. Taken together, our data reveal that AHR influences DNA damage-dependent responses in UVB-irradiated KC and critically contributes to skin photocarcinogenesis in mice.

Development of the Concept for Stem Cell-Based Developmental Neurotoxicity Evaluation.

Human brain development consists of a series of complex spatiotemporal processes that if disturbed by chemical exposure causes irreversible impairments of the nervous system. To evaluate a chemical disturbance in an alternative assay, the concept evolved that the complex procedure of brain development can be disassembled into several neurodevelopmental endpoints which can be represented by a combination of different alternative assays. In this review article, we provide a scientific rationale for the neurodevelopmental endpoints that are currently chosen to establish assays with human stem/and progenitor cells. Assays covering these major neurodevelopmental endpoints are thought to assemble as building blocks of a DNT testing battery.

Current Availability of Stem Cell-Based In Vitro Methods for Developmental Neurotoxicity (DNT) Testing.

There is evidence that chemical exposure during development can cause irreversible impairments of the human developing nervous system. Therefore, testing compounds for their developmentally neurotoxic potential has high priority for different stakeholders: academia, industry, and regulatory bodies. Due to the resource-intensity of current developmental neurotoxicity (DNT) in vivo guidelines, alternative methods that are scientifically valid and have a high predictivity for humans are especially desired by regulators. Here, we review availability of stem-/progenitor cell-based in vitro methods for DNT evaluation that is based on the concept of neurodevelopmental process assessment. These test methods are assembled into a DNT in vitro testing battery. Gaps in this testing battery addressing research needs are also pointed out.