S. Bremer et al., Effects of embryotoxic chemicals on the in vitro differentiation of genetically engineered embryonic stem cells into cardiac cells, TOX VITRO, 13(4-5), 1999, pp. 645-650
A project has been started using transgenic embryonic stem cells as a toxic
ological endpoint in order to register chemical effects on the development
of embryonic tissues which are known to be sensitive during their different
iation. The green fluorescent protein (GFP) is used as a reporter gene and
is linked to a cardiac specific promotor. This construct is integrated into
the native DNA of undifferentiated embryonic stem cells. The expression of
GFP was switched on after specific activation of the promotor (human-alpha
-actin) which allows a quantification of cardiac cells using the fluorescen
ce activated cell sorter. Kinetic analysis shows a differentiation of 25% o
n cells with activated human-alpha-actin promotor on day 3, increasing to 8
6% on day 7, and decreasing again to 35% on day 11. The known animal terato
gens retinoic acid and 5-fluorouracil were chosen and the measurements were
compared to the IC50 values given by other in vitro endpoints in order to
investigate the potential of this toxicological endpoint. The results show
a higher sensitivity of endpoints which analysed specific effects on a sele
cted target tissue. The exposure of embryonic stem cells to chemicals lead
to the following IC50 values. 1.149 +/- 0.170 ng/ml (cytotoxicity) versus 0
.216 +/- 0.126 ng/ml (GFP expression) after treatment with retinoic acid an
d 54.2 +/- 5.2 ng/ml (cytotoxicity) versus 26.7 +/- 2 ng/ml (GFP expression
) after treatment with 5-fluorouracil. The data shows the necessity to deve
lop specific in vitro methods which take the complexity of embryotoxicology
into account. (C) 1999 Published by Elsevier Science Ltd. All rights reser
ved.