Embryotoxicity screening using embryonic stem cells in vitro: Correlation to in vivo teratogenicity

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can b e induced to differentiate in culture into a variety of cell types, includi ng cardiac muscle cells. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in viv o studies in reproductive toxicology about toxic effects of chemicals. ES c ells of the mouse cell line D3 are investigated for their preserved capabil ity to differentiate following drug exposure, and both ES cells and differe ntiated fibroblast cells of the mouse cell line 3T3 are comparatively analy zed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embry otoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1)t he inhibition of differentiation of ES cells into cardiomyocytes after 10 d ays of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 c ells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-di methylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibit ion concentrations for differentiation (ID50) and cytotoxicity (IC(50)D3 an d IC(50)3T3) are calculated from concentration-response curves. Applying li near analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the Ig(lC(50)D3), the Ig(IC(50)3T3) and the relative distance between IC(50)3T3 and ID50, that i mproved the separation of the three classes of embryotoxicity compared to t he prediction model that was originally proposed after test development. Un like the original PM, the improved PM incorporates as one variable the rela tive distance between IC(50)3T3 and ID50, instead of the ratio ID50/IC(50)D 3 that was used previously. Copyright (C) 1999 S. Karger AG, Basel.