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

Exposure to teratogenic compounds during pregnancy can lead to significant
birth defects. Given the considerable variation in drug responses across
species, along with the financial and ethical challenges associated with
animal testing, the development of advanced human-based in vitro assays is
imperative for effectively identifying potential human teratogens.
Previously, we developed a human induced pluripotent stem cell
(hiPSCs)-based biomarker assay, ReproTracker, that follows the
differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay
combines morphological profiling with the assessment of time-dependent
expression patterns of cell-specific biomarkers to detect developmental
toxicity responses. To further increase the predictability of the assay in
identifying potential teratogens, we added differentiation of hiPSCs
towards neural rosette-like cells. We evaluated the performance of the
extended assay with a set of 51 well-known in vivo teratogens and
non-teratogens, including the compounds listed in the ICH S5 reference
list. The optimized assay correctly identified (neuro)developmental
toxicants that were not detected in the hepatocyte and cardiomyocyte
differentiation assays. These compounds selectively downregulated gene and
protein expression of the neuroectodermal marker PAX6 and/or neural
rosette marker NESTIN in a concentration-dependent manner and disrupted
the differentiation of hiPSCs towards neural rosette-like cells. Overall,
based on the current dataset, the addition of neural commitment improved
the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to
87.50%), when compared to the previously described assay. In summary,
trilineage differentiation expanded the spectrum of teratogenic agents
detectable by ReproTracker, making the assay an invaluable tool for early
in vitro teratogenicity screening.