Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells

Since the differentiation of embryonic stem cells mimics early development, these cells could potentially permit the detection of embryotoxicants which interfere with this process. Although reliable tests based on murine embryonic stem cells exist, no such methods are available for human embryonic stem (hES) cells. Nonetheless, to avoid the false classification of substances due to inter-species differences, human-relevant toxicity tests are needed. We therefore developed an assay based on three human cell types, representing different degrees of developmental maturation, namely, human foreskin fibroblasts, hES cell-derived progenitor cells, and pluripotent hES cells. A set of embryotoxicants for which existing in vivo data were available, namely, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13CRA), valproic acid (VPA) and dimethyl sulphoxide (DMSO), were tested. 5-fluorouracil (5-FU) was used as a positive control, and saccharin as a negative control. Two methods were compared for the assessment of cell viability -- the determination of intracellular ATP content and of resazurin reduction. In addition, the protective capacity of basic fibroblast growth factor (bFGF) against retinoid-induced toxicity was investigated. This novel assay system reliably detected the embryotoxic potentials of the test substances, 5-FU, ATRA, 13-CRA (a substance that displays inter-species differences in its effects) and VPA. This was possible due to the apparent differences in the sensitivities of the human cell types used in the assay system. Thus, our results clearly indicate the advantages and relevance of using hES cells in in vitro developmental toxicity testing.