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.

The currently accepted methods for neurotoxicity (NT) testing rely on animal studies. However, high costs and low testing throughput hinder their application for large numbers of chemicals. To overcome these limitations, in vitro methods are currently being developed based on human-induced pluripotent stem cells (hiPSC) that allow higher testing throughput at lower costs. We applied six different protocols to generate 3D BrainSphere models for acute NT evaluation. These include three different media for 2D neural induction and two media for subsequent 3D differentiation resulting in self-organized, organotypic neuron/astrocyte microtissues. All induction protocols yielded nearly 100% NESTIN-positive hiPSC-derived neural progenitor cells (hiNPCs), though with different gene expression profiles concerning regional patterning. Moreover, gene expression and immunocytochemistry analyses revealed that the choice of media determines neural differentiation patterns. On the functional level, BrainSpheres exhibited different levels of electrical activity on microelectrode arrays (MEA). Spike sorting allowed BrainSphere functional characterization with the mixed cultures consisting of GABAergic, glutamatergic, dopaminergic, serotonergic, and cholinergic neurons. A test method for acute NT testing, the human multi-neurotransmitter receptor (hMNR) assay, was proposed to apply such MEA-based spike sorting. These models are promising tools not only in toxicology but also for drug development and disease modeling.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumour in adulthood. Despite strong research efforts current treatment options have a limited impact on glioma stem-like cells (GSCs) which contribute to GBM formation, progression and chemoresistance. Invasive growth of GSCs is in part associated with epithelial–mesenchymal-like transition (EMT), a mechanism associated with CD73 in several cancers. Here, we show that CD73 regulates the EMT activator SNAIL1 and further investigate the role of enzymatic and non-enzymatic CD73 activity in GBM progression. Reduction of CD73 protein resulted in significant suppression of GSC viability, proliferation and clonogenicity, whereas CD73 enzymatic activity exhibited negative effects only on GSC invasion involving impaired downstream adenosine (ADO) signalling. Furthermore, application of phosphodiesterase inhibitor pentoxifylline, a potent immunomodulator, effectively inhibited ZEB1 and CD73 expression and significantly decreased viability, clonogenicity, and invasion of GSC in vitro cultures. Given the involvement of adenosine and A₃ adenosine receptor in GSC invasion, we investigated the effect of the pharmacological inhibition of A₃AR on GSC maintenance. Direct A₃AR inhibition promoted apoptotic cell death and impaired the clonogenicity of GSC cultures. Taken together, our data indicate that CD73 is an exciting novel target in GBM therapy. Moreover, pharmacological interference, resulting in disturbed ADO signalling, provides new opportunities to innovate GBM therapy.

Despite growing awareness of endocrine disrupting chemicals (EDCs), knowledge gaps remain regarding their effects on human brain development. EDC risk assessment focuses primarily on EATS modalities (estrogens, androgens, thyroid hormones, and steroidogenesis), overlooking the broader range of hormone receptors expressed in the developing brain. This limits the evaluation of chemicals for their potential to cause endocrine disruption-mediated developmental neurotoxicity (ED-DNT). The Neurosphere Assay, an in vitro test method for developmental neurotoxicity (DNT) evaluation, is an integral component of the DNT in vitro testing battery, which has been used to screen a broad domain of environmental chemicals. Here, we define the endocrine-related applicability domain of the Neurosphere Assay by assessing the impact and specificity of 14 hormone receptors on seven key neurodevelopmental processes (KNDPs), neural progenitor cell (NPC) proliferation, migration of radial glia, neurons, and oligodendrocytes, neurite outgrowth, and differentiation of neurons and oligodendrocytes. Comparative analyses in human and rat NPCs of both sexes revealed species- and sex-specific responses. Mechanistic insights were obtained through RNA sequencing and agonist/antagonist co-exposures. Most receptor agonists modulated KNDPs at concentrations in the range of physiologically relevant hormone concentrations. Phenotypic effects induced by glucocorticoid receptor (GR), liver X receptor (LXR), peroxisome proliferator-activated receptor beta/delta (PPARβδ), retinoic acid receptor (RAR) and retinoid X receptor (RXR) activation were counteracted by receptor antagonists, confirming specificity. Transcriptomics highlighted receptor crosstalk and the involvement of conserved developmental pathways (e.g. Notch and Wnt). Species comparisons identified limited concordance in hormone receptor-regulated KNDPs between human and rat NPCs. This study presents novel findings on cellular and molecular hormone actions in human fetal NPCs, highlights major species differences, and illustrates the Neurosphere Assay's relevance for detecting endocrine MoAs, supporting its application in human-based ED-DNT risk assessment.

Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (¹H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/Glu^High GSCs were found to be resistant to glutamine deprivation, whereas Gln/Glu^Low GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.

Plasticizers like di-(2-ethylhexyl) phthalate (DEHP) are commonly used in medical devices to enhance plastic flexibility. DEHP is classified as a CMR1b substance due to its adverse effects on reproduction and fertility, and it has been linked to neurodevelopmental disorders such as ADHD, autism spectrum disorder, and learning disabilities. While DEHP is scheduled for phase-out by 2030, data on the developmental neurotoxicity (DNT) of alternative plasticizers remain scarce. We evaluated the DNT potential of DEHP and three alternative plasticizers: di-(2-ethylhexyl) terephthalate (DEHT), di-(2-ethylhexyl) adipate (DEHA), and tris-(2-ethylhexyl) trimellitate (TOTM), aiming to identify safer substitutes, particularly for neonates in neonatal intensive care units (NICUs). The human Neurosphere Assay was used to assess plasticizer effects on seven key neurodevelopmental processes: neural progenitor cell (NPC) proliferation, migration of radial glia, neurons, and oligodendrocytes, neurite outgrowth, and differentiation of neurons and oligodendrocytes. Concentration-response analyses provided benchmark concentrations (BMCs) and lowest observed adverse effect concentrations (LOAECs). Gene expression profiling provided mechanistic insights, and toxicity was ranked using the most sensitive endpoint (MSE) and ToxPi Tool. DEHP and TOTM showed the highest DNT potential, with NPC proliferation as the MSE. DEHT impacted oligodendrocyte differentiation, while no BMC was determined for DEHA within the tested concentrations. Considering an exposure scenario in NICUs, the estimated neonatal DEHP plasma levels exceeded the LOAEC for NPC proliferation, raising concerns for DNT. Overall, DEHA emerged as the least hazardous alternative for neurodevelopment, highlighting the value of combined human-relevant in vitro phenomics and human biomonitoring for DNT hazard evaluation.