Detection of Thalidomide Embryotoxicity by In Vitro Embryotoxicity Testing Based on Human iPS Cells

Abstract

Abstract. The mouse embryonic stem cell test (mEST) is used to assess the embryotoxicity of drug candidates by evaluating the effects on the cardiac differentiation of stem cells. However, thalidomide embryotoxicity has not yet been reported using the mEST. To detect the effects of thalidomide, we used human induced pluripotent stem cells (hiPSCs) instead of mouse embryonic stem cells, and assessed three endpoints: the inhibition of cardiac differentiation, the cytotoxicity to hiPSCs, and the cytotoxicity to human dermal fibroblasts, according to the mEST. From these data (IC 50 values), the embryotoxicity was classified into one of three different classes based on the mEST and our criteria. Valproate was used as a positive control and ascorbic acid was used as a negative control, and their effects were assessed. Similar to valproate, thalidomide was classified as a Class 2 agent, with weak embryotoxicity, by the mEST criteria, and was classified as Category 3 embryotoxic based on our criteria. Ascorbic acid was classified as a Class 1 / Category 1, non-embryotoxic agent, based on both criteria. Thalidomide embryotoxicity was detected in the embryonic stem cell test based on hiPSCs. This test system is thus considered to have a much greater predictive ability than the mEST.

A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells

“…Therefore, simple assays to achieve similar findings are desired, and many in vitro developmental toxicity assays have been developed, such as the mouse embryonic stem cell test (EST) (Annett et al, 2016;Seiler and Spielmann, 2011;Scholz et al, 1999;Genschow et al, 2002Genschow et al, , 2004, zebrafish test (Truong et al, 2011) and devTOX TM test (Palmer et al, 2013) using human embryonic stem cells or human induced pluripotent stem (iPS) cells. We also reported the preliminary method of in vitro iPS cells testing (iPST) (Aikawa et al, 2014) that is a modified version of the EST and a promising animal-free approach.…”

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

“…We sought here to develop an in vitro teratogenicity assay that recapitulates the early stem cell specification events upstream of limb bud development. The gold standard in vitro assay for teratogenicity testing has been the mEST, which measures the influence of putative teratogens on the spontaneous cardiac differentiation of mouse embryoid bodies 20,[51][52][53] . The mEST presents several limitations, namely the subjectivity of analysis, and was characterized as 53-79% accurate for predicting teratogenicity depending on the chemical reference set used 16,17 .…”

“…The mEST presents several limitations, namely the subjectivity of analysis, and was characterized as 53-79% accurate for predicting teratogenicity depending on the chemical reference set used 16,17 . Thalidomide is teratogenic in humans, NHPs, and rabbits but does not induce teratogenicity in mice 1,7 , and indeed the mEST has been appropriately insensitive to the effects of thalidomide, though it is sensitive to the effects of other known mouse and human teratogens including atRA 20 . Recently, an hPSC-based teratogenicity assay of definitive endoderm differentiation was established and characterized as 94% predictive of visceral malformations in preclinical species 23 .…”

“…Human pluripotent stem cells have proven useful in investigating the teratogenicity of thalidomide in vitro. Studies on the effect of thalidomide on human pluripotent stem cells (hPSCs) characterized cytotoxicity in undifferentiated hPSCs 19,20 and hPSC-derived mesoderm 21 and functional effects on hPSC-derived mesendoderm 22 and cardiac mesoderm 20 at concentrations above 100 µM. However, these studies demonstrated effects of thalidomide on PSCs or PSC-derived mesoderm in vitro at concentrations >5-fold in excess of the thalidomide clinical Cmax 7 , which raises concern regarding the relevance of these effects to thalidomide teratogenicity.…”

Comparative study of the zebrafish embryonic toxicity test and mouse embryonic stem cell test to screen developmental toxicity of human pharmaceutical drugs

“…For example, we could not detect the teratogenicity of thalidomide in the mEST. Recently, Aikawa et al (2014) demonstrated the embryonic toxicity of thalidomide (weak teratogenicity) using human induced pluripotent stem cells. Further modification of the protocol might be needed to achieve better predictions in the mEST.…”