We differentiated human induced pluripotent stem cells (hiPSCs) to embryonic endoderm and sought to identify a tipping point at which the developing system did not recover from perturbations caused by exposure to a known teratogen, all-trans retinoic acid (ATRA). Differentiating iPSC-derived endoderm was exposed to five concentrations of ATRA between 0.001 and 10 µM at 6h, 96h, or 192h and assessed for forkhead box A2 (FOXA2) protein expression and global gene transcript expression measured by RNA-sequencing. A tipping point of 17±11 nM was identified where patterns of differentially expressed genes supported a shift in the developmental trajectory away from embryonic endoderm in favor of mesoderm and extraembryonic endoderm. Five concentrations of all-trans retinoic acid (ATRA) between 0.001 and 10 µM were compared to time-matched 0.1% DMSO controls at three timepoints (6h, 96h, and 192h) in differentiating endoderm. Two biological replicates were used. Undifferentiated controls (not in DMSO) were also included in duplicate as internal controls for 6h, 96h, and 144h. This dataset is associated with the following publication: Saili, K., T. Antonijevic, T. Zurlinden, I. Shah, C. Deisenroth, and T. Knudsen. Molecular characterization of a toxicological tipping point during human stem cell differentiation. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 91(January 2020): 1-13, (2020).

The data presented here support the application of the Stemina devTOXqP platform for predictive toxicology and further demonstrate its value in ToxCast as a novel resource that can generate testable hypotheses aimed at characterizing potential pathways for teratogenicity and HTS prioritization of environmental chemicals for an exposure-based assessment of developmental hazard. The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Rush, P. Kothiya, R. Judson, K. Houck, E. Hunter, N. Baker, J. Palmer, R. Thomas, and T. Knudsen. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 174(2): 189-209, (2020).

We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 2 replicates of a 384-well plate design. The majority of wells contain U-2 OS cells exposed to 11 test chemicals at 7 different concentrations (two replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (22 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR). This dataset is associated with the following publication: Nyffeler, J., C. Willis, D. Harris, L. Taylor, R. Judson, L. Everett, and J. Harrill. Combining phenotypic profiles and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: retinoic acid as an example.. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, USA, 444: 116032, (2022).

Background: High-content imaging (HCI) allows simultaneous measurement of multiple cellular phenotypic changes and is an important tool for evaluating the biological activity of chemicals. Objectives: Our goal was to analyze dynamic cellular changes using HCI to identify the “tipping point” at which the cells did not show recovery towards a normal phenotypic state. Methods: HCI was used to evaluate the effects of 967 chemicals (in concentrations ranging from 0.4 to 200 μM) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A.x, α-tubulin, histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size, and cell number. A computational model was developed to interpret HCI responses as cell-state trajectories. Results: Analysis of cell-state trajectories showed that 336 chemicals produced tipping points and that HepG2 cells were resilient to the effects of 334 chemicals up to the highest concentration (200 μM) and duration (72 hr) tested. Tipping points were identified as concentration-dependent transitions in system recovery, and the corresponding critical concentrations were generally between 5 and 15 times (25th and 75th percentiles, respectively) lower than the concentration that produced any significant effect on HepG2 cells. The remaining 297 chemicals require more data before they can be placed in either of these categories. Conclusions: These findings show the utility of HCI data for reconstructing cell state trajectories and provide insight into the adaptation and resilience of in vitro cellular systems based on tipping points. Cellular tipping points could be used to define a point of departure for risk-based prioritization of environmental chemicals. This dataset is associated with the following publication: Shah , I., W. Setzer , J. Jack, K. Houck , R. Judson , T. Knudsen , J. Liu, M. Martin , D. Reif, A.M. Richard , R.S. Thomas , K. Crofton , D.J. Dix , and R.J. Kavlock. (Envir. Health Perspect.) Using ToxCast data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA, 1-33, (2015).

Development of the neurovascular unit (NVU) is a complex, multistage process that requires orchestrated cell signaling mechanisms across several cell types and ultimately results in the formation of the blood-brain barrier. Typical high-throughput screening (HTS) assays investigate single biochemical or single cell responses following chemical insult. As the NVU comprises multiple cell types interacting at various stages of development, a methodology for combining high-throughput results across pertinent cell-based assays is needed to investigate potential chemical-induced disruption to the development of this complex cell system. To this end, we developed a novel method for screening putative NVU disruptors across diverse assay platforms to predict chemical perturbation of the developing NVU. Here, HTS assay results measuring chemical-induced perturbations to cellular key events across angiogenic and neurogenic outcomes were combined to create a cell-based prioritization of NVU hazard. Using activity from each biological outcome, chemicals were grouped into similar modes of action and used to train a logistic regression literature model. This model utilizes the chemical-specific pairwise mutual information score for PubMed MeSH annotations to represent how often a chemical was shown to produce a specific outcome in the published literature space. Taken together, this study presents a methodology to investigate NVU developmental hazard using cell-based HTS assays and literature evidence to prioritize screening of putative NVU disruptors. The results from these screening efforts demonstrate how chemicals that represent a range of putative vascular disrupting compound (pVDC) scores based on angiogenic endpoints can also produce effects on neurogenic outcomes such as neurite outgrowth, neuroprogenitor/neural crest migration, representing an additional method for understanding the range of possible modes of action for disruption of the developing NVU. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Baker, T. Toimela, T. Heinonen, and T. Knudsen. A cross-platform approach to characterize and screen potential neurovascular unit toxicants. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 96(September 2020): 300-315, (2020).

Background Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes. Results We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAFII135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAFII135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAFII135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARγ2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAFII135 delays proteolysis of the RARγ2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAFII135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant. Conclusions These observations indicate that appropriately timed proteolysis of TBP and TAFII135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.

Biomarker Image Cytometry. The cell-level frequency of NK2 Homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein staining were quantitatively evaluated by high-content imaging across huThyrEC cell line variants (1-4) in two medium formulations (huThyrEC and h7H) for verification of thyroid follicular epithelial cell enrichment. Data are the % positive expression frequency (mean ± SD) of two replicates. This dataset is associated with the following publication: Hopperstad, K., T. Truschel, T. Wahlicht, W. Stewart, A. Eicher, T. May, and C. Deisenroth. Characterization of Novel Human Immortalized Thyroid Follicular Epithelial Cell Lines. Applied In Vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, USA, 7(2): 39-49, (2021).

The AOP concept was used to translate ToxCast high-throughput screening data into a ToxPi matrix of 1058 chemicals x 24 features ordered by predicted potential to disrupt angiogenesis. This dataset is associated with the following publication: Saili, K., J. Franzosa, N. Baker, R. Ellis-Hutchings, R. Settivari, E. Carney, R. Spencer, T. Zurlinden, N. Kleinstreuer, S. Li, M. Xia, and T. Knudsen. Systems Modeling of Developmental Vascular Toxicity. Current Opinion in Toxicology. Elsevier BV, AMSTERDAM, NETHERLANDS, 15(1): 55-63, (2019).

Organogenesis in the embryo involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the cellular architecture and phenotype of many tissues and organs, and the three-dimensional (3D) architecture of organoids presents an opportunity to study developmental human toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro models to study cell and tissue morphogenesis. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as twelve cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the twelve putative cleft palate teratogens studied here significantly disrupted in vitro fusion, including theophylline, triamcinolone, and valproic acid. Tributyltin chloride and all-trans retinoic acid (ATRA) were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid survival and morphogenesis in a scalable format amenable to toxicology screening. This dataset is associated with the following publication: Belair, D., C. Wolf, S. Moorefield, C. Wood, C. Becker, and B. Abbott. A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 394-408, (2018).

This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. This dataset is not publicly accessible because: no EPA data; all the data generated by external organizations; EPA coauthors. It can be accessed through the following means: Data generated by external organizations. Format: N/A. This dataset is associated with the following publication: Ellis-Hutchings, R., R. Settivari, A. McCoy, N. Kleinstreuer, J. Franzosa, T. Knudsen, and E. Carney. (REPRODUCTIVE TOXICOLOGY) EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 70: 82-96, (2017).