Implementing a trilineage differentiation in the ReproTracker assay for improved teratogenicity assessment

Area of Science:

• Toxicology
• Developmental Biology
• Stem Cell Biology

Background:

• Teratogenic compounds pose risks during pregnancy, causing birth defects.
• Human-based in vitro assays are crucial for identifying teratogens due to species variation and ethical concerns with animal testing.
• ReproTracker, a human induced pluripotent stem cells (hiPSCs)-based assay, previously assessed hepatocyte and cardiomyocyte differentiation for teratogenicity.

Purpose of the Study:

• To enhance the ReproTracker assay's predictability in identifying potential teratogens.
• To evaluate the performance of an extended hiPSC differentiation assay including neural lineage commitment.

Main Methods:

• Extended the ReproTracker assay by incorporating hiPSC differentiation towards neural rosette-like cells.
• Evaluated the assay's performance using 51 known in vivo teratogens and non-teratogens, including ICH S5 reference compounds.
• Combined morphological profiling with time-dependent biomarker expression analysis.

Main Results:

• The optimized assay identified (neuro)developmental toxicants missed by previous hepatocyte/cardiomyocyte assays.
• Detected compounds selectively downregulated neuroectodermal (PAX6) and neural rosette (NESTIN) markers in a dose-dependent manner.
• Improved assay accuracy from 72.55% to 86.27% and sensitivity from 67.50% to 87.50% with neural differentiation.

Conclusions:

• Trilineage differentiation (hepatocytes, cardiomyocytes, neural cells) significantly expands the range of detectable teratogenic agents.
• The enhanced ReproTracker assay is a valuable tool for early in vitro teratogenicity screening.
• This improved assay aids in identifying potential human teratogens, mitigating risks of birth defects.