Developmental Toxicity Assay Based on Real-Time Monitoring of Fibroblast Growth Factor Signal Disruption in Human Induced Pluripotent Stem Cells
October 10th, 2025
In this study, we present an in vitro cell assay for identifying teratogenic chemicals. The dynamics of fibroblast growth factor (FGF)/serum response factor (SRF) signaling were measured in human induced pluripotent stem (iPS) reporter cells, and the risk of teratogenicity was evaluated by quantifying chemical disruption of this signaling pathway.
This study explores an alternative to animal testing, that employs culture cell to assess chemical toxicity. The central question is whether disruption in signaling pathway of human iPS cells can serve as a level indicator of developmental toxicity. We found that regardless of the mechanism, developmental toxicant disrupt the signaling pathway, and the temporal sums serves as an indicator.
This iPSC based assay enables comprehensive evaluation of developmental toxicant, with high accuracy and therapeute, and without the use of animals. To begin, seed cells into an ECM coated 96-well plate containing 200 microliters of fresh maintenance medium supplemented with 10 micromolar ROCK inhibitor. Seal the plate with a gas-permeable film.
After 24 hours, replace the medium in each well with 200 microliters of fresh maintenance medium, and reseal the plate. Next, dissolve the test chemical in assay medium to its maximum solubility. Prepare a five-point five-fold serial dilution of the test chemical in assay medium using the previously prepared solution.
Add 11 microliters per well of the prepared chemical solutions. For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO. After 48 hours of treatment, incubate the plates at room temperature for 30 minutes.
Then, add 111 microliters per well of pre-equilibrated ATP-based luminescent cell viability assay reagent at 25 degrees Celsius. For the blank well, add 111 microliters of assay medium and 111 microliters of the reagent. Using a micro-plate shaker, shake the plate at 300 revolutions per minute for two minutes.
Transfer 200 microliters of the mixture from each well to a black plate for luminescence measurement, and incubate the black plate at 25 degrees Celsius for 10 minutes. Then, measure the luminescence intensity. For the determination test, after incubating the cells for 96 hours, replace the medium in each well with 100 microliters of assay medium.
Dissolve the test chemical in assay medium based on the results obtained from the preliminary test. Prepare a nine-point two-fold serial dilution of the test chemical in assay medium using the previously prepared solution. Add 11 microliters per well of the prepared chemical solutions.
For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO. After incubating the cells for 96 hours, replace the medium in each well with 100 microliters of assay medium containing 1%luminescent substrate. Dissolve the test chemical in assay medium at the concentration determined from the previous section.
Prepare an eight-point two-fold serial dilution of the test chemical in assay medium using the previously prepared solution. Two hours after the medium exchange, add 11 microliters per well of the serially diluted chemical solutions. For the vehicle control, add 11 microliters per well of assay medium containing the same concentration of DMSO.
One hour after starting chemical exposure, add 12 microliters per well of assay medium containing basic fibroblast growth factor. Measure luminescence continuously for 48 hours using a real-time luminescence measurement system under 5%carbon dioxide at 37 degrees Celsius. Phase contrast imaging confirmed a confluent cell layer covering nearly the entire surface of the wells, both before and after chemical exposure.
Live and dead staining showed that while the number of propidium iodide stained dead cells increased after chemical exposure, the majority of cells remained viable and were stained with calcein-AM. In the absence of test chemicals, luminescence intensity showed two distinct peaks across all six replicate wells, demonstrating high reproducibility and temporal resolution. Automated real-time monitoring revealed that valproic acid disrupted fibroblast growth factor signaling in a concentration dependent manner, with stronger suppression at higher concentrations.
Manual luminescence measurements captured a general suppression pattern by valproic acid, but lacked the temporal resolution seen in automated detection.
Overview
This study explores an in vitro cell assay designed to identify teratogenic chemicals by assessing the disruption of fibroblast growth factor (FGF)/serum response factor (SRF) signaling in human induced pluripotent stem (iPS) cells. The findings indicate that this assay can serve as a reliable indicator of developmental toxicity without the use of animal testing.
Key Study Components
Area of Science
- Neuroscience
- Toxicology
- Stem Cell Research
Background
- Traditional animal testing methods for assessing chemical toxicity are being replaced by in vitro approaches.
- Human iPS cells provide a relevant model for studying developmental toxicity.
- Disruption of specific signaling pathways can indicate potential teratogenic effects.
- High accuracy and therapeutic relevance are crucial for evaluating developmental toxicants.
Purpose of Study
- To develop an assay that evaluates the risk of teratogenicity in chemicals.
- To measure the dynamics of FGF/SRF signaling in response to chemical exposure.
- To provide an alternative to animal testing for assessing developmental toxicity.
Methods Used
- Culture human iPS cells in ECM-coated plates with ROCK inhibitor.
- Perform serial dilutions of test chemicals and assess their effects on cell viability using luminescence measurements.
- Monitor FGF signaling disruption over time using real-time luminescence detection.
- Utilize phase contrast imaging and live/dead staining to evaluate cell health post-exposure.
Main Results
- Valproic acid was shown to disrupt FGF signaling in a concentration-dependent manner.
- Automated monitoring provided higher temporal resolution compared to manual measurements.
- Most cells remained viable despite exposure to developmental toxicants.
- The assay demonstrated high reproducibility and accuracy in detecting chemical effects.
Conclusions
- The iPSC-based assay is a promising tool for evaluating developmental toxicants.
- Disruption of FGF/SRF signaling serves as a reliable indicator of teratogenicity.
- This method supports the reduction of animal testing in toxicological assessments.
Frequently Asked Questions
Executive Industry Relevance
Developmental toxicity remains a critical risk in early drug discovery, with traditional animal models often failing to predict human-specific teratogenicity. This human iPSC-based FGF signaling disruption assay offers a predictive, reproducible, and ethically aligned alternative for developmental toxicity screening. Integrating real-time quantitative readouts, it enables earlier and more confident portfolio triage, reducing late-stage attrition due to unforeseen developmental risks.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of developmental toxicity mechanisms in a human-relevant system.
- Supports functional target validation by linking chemical exposure to pathway disruption.
- Improves predictive confidence for go/no-go decisions in early-stage portfolios.
Screening & Assay Development
- Provides a standardized, reproducible platform for high-throughput compound evaluation.
- Delivers quantitative, time-resolved luminescence outputs for robust assay comparability.
- Facilitates assay scalability and platform reuse across diverse chemical libraries.
Translational & Preclinical Research
- Aligns developmental toxicity assessment with human biology, reducing interspecies translation gaps.
- Enables continuity from discovery through preclinical safety evaluation.
- Supports risk-adjusted advancement by providing mechanistic de-risking data.
Pipeline & Workflow Integration
This assay fits at the intersection of early discovery and preclinical safety, bridging target validation and lead identification with translationally relevant toxicity data.
- Discovery Biology: Quantifies FGF pathway disruption as a surrogate for developmental toxicity risk.
- Screening: Offers reproducible, high-content outputs suitable for compound triage.
- Analytics: Provides real-time, quantitative luminescence data for statistical comparison of test conditions.
- Translational Research: Reduces reliance on animal models by leveraging human iPSC biology.
- Enterprise Reuse: Establishes a reusable, scalable platform for ongoing developmental toxicity assessment.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in developmental toxicity risk.
- Operational Value: Enhances standardization, reproducibility, and throughput for toxicity screening.
- Strategic Value: Enables earlier, data-driven portfolio decisions and reduces late-stage biological risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of candidate molecules.
Implementation Considerations
- Requires expertise in human iPSC culture and luminescence-based analytics.
- Needs access to real-time luminescence measurement instrumentation for optimal temporal resolution.
- Demands cross-team standardization of assay protocols and data analysis workflows.
- May require adaptation for different chemical classes or model systems.
- Temporal resolution and throughput depend on instrumentation and workflow integration.
