Toward better assessments of developmental toxicity using stem cell-based in vitro embryogenesis models

Abstract

In the past few decades, pluripotent stem cells have been explored as nonanimal alternatives to assess the developmental toxicity of chemicals. To date, numerous versions of stem cell-based assays have been reported that are allegedly effective. Nonetheless, none of the assays has become the gold standard in developmental toxicity assessment. Why? This article discusses several issues in the hope of facilitating the refinement of stem cell assays and their acceptance as the cornerstone in predictive developmental toxicology. Each stem cell assay is built on a limited representation of embryogenesis, so that multiple assays are needed to detect the diverse effects of various chemicals. To validate and compare the strengths and weaknesses of individual assays, standardized lists of reference chemicals should be established. Reference lists should consist of exposures defined by toxicokinetic data, namely maternal plasma concentrations that cause embryonic death or malformations, and also by the effects on the molecular machineries that control embryogenesis. Although not entirely replacing human or animal tests, carefully selected stem cell assays should serve as practical and ethical alternatives to proactively identify chemical exposures that disturb embryogenesis. To achieve this goal, unprecedented levels of coordination and conviction are required among research and regulatory communities.

Keywords: Daston list; adverse outcome pathway; embryo; embryotoxicity; gastruloid; risk assessment; teratogen.

FIGURE 1

Schematic diagram depicting the origins of pluripotent stem cells: one from the epiblast of normal embryos and the other from somatic cells that are reprogrammed with the Yamanaka factors. In vivo, the epiblast gives rise to the entire fetal body. In vitro, pluripotent stem cells can be induced to differentiate into various cell types, or to form 3D cell aggregates, known as gastruloids, which undergo axial patterning and elongation morphogenesis. In vitro development of pluripotent stem cells is explored as a model of embryogenesis to assess the developmental toxicity of various chemicals

FIGURE 2

Developmental toxicity assay using gastruloids of mouse P19C5 stem cells. (a) Basic characteristics of the P19C5 gastruloid‐based assay. Right images show the morphological changes of 3D cell aggregates of P19C5 stem cells over the course of 4 days of hanging drop culture. Aggregates have been removed from hanging drops for photography. Scale bar = 500 μm. (b) Exposure‐based validation of the P19C5 gastruloid assay using the Daston list (Daston et al., 2014). Summarized results from the previous studies (Li & Marikawa, ; Warkus & Marikawa, 2017) are shown. For adverse effects, circles represent significant morphological impact (i.e., reduced size or altered shape in Day 4 aggregates), and crosses represent no significant morphological impact. (c) Mechanism‐based validation of the P19C5 gastruloid assay. Summarized results from the previous studies (Li & Marikawa, , ; Warkus, Yuen, Lau, & Marikawa, ; Warkus & Marikawa, 2018) are shown. RA (retinoic acid), HH (hedgehog), PCP (planar cell polarity), HDAC (histone deacetylase). Right images show Wnt signaling‐inhibited Day 4 aggregates (treated with IWP2 at 8 μM) and the corresponding control (untreated) aggregates. Note that axial elongation is markedly diminished in IWP2‐treated aggregates. Scale bar = 500 μm

FIGURE 3

Comparisons of three hypothetical assays that are validated against 10 hypothetical reference (developmentally toxic) exposures. See the text for details