In vitro teratogenicity testing using a 3D, embryo-like gastruloid system.

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ('replace, reduce and refine') by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D 'gastruloid' model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

Human gastrulation: The embryo and its models.

Technical and ethical limitations create a challenge to study early human development, especially following the first 3 weeks of development after fertilization, when the fundamental aspects of the body plan are established through the process called gastrulation. As a consequence, our current understanding of human development is mostly based on the anatomical and histological studies on Carnegie Collection of human embryos, which were carried out more than half a century ago. Due to the 14-day rule on human embryo research, there have been no experimental studies beyond the fourteenth day of human development. Mutagenesis studies on animal models, mostly in mouse, are often extrapolated to human embryos to understand the transcriptional regulation of human development. However, due to the existence of significant differences in their morphological and molecular features as well as the time scale of their development, it is obvious that complete knowledge of human development can be achieved only by studying the human embryo. These studies require a cellular framework. Here we summarize the cellular, molecular, and temporal aspects associated with human gastrulation and discuss how they relate to existing human PSCs based models of early development.