Development of an in vitro embryotoxicity test using murine embryonic stem cell cultures

Mouse embryonic stem (ES) cell line D3 was used to establish conditions for a reproducible differentiation of ES cells in culture. ES cells can be maintained in an undifferentiated state by cultivation on a feeder layer of embryonic fibroblasts. ES cells form aggregates in suspension and can spontaneously differentiate into complex organized embryoid bodies (EBs), which in many respects resemble early postimplantation mouse embryos. Under appropriate culture conditions various cell and tissue types will develop in EBs: these include myocardial and skeletal muscle, nerve cells, chondrocytes and blood cells. Retinoic acid (RA) was used as an embryotoxic substance to test the application of ES cell cultures in in vitro embryotoxicity testing. RA (1 x 10(-8)m) induced an increase in skeletal muscle cell differentiation, which followed a characteristic pattern: day 10 is characterized by the first appearance of mononucleated myoblasts; day 12 shows the fusion of myoblasts; on day 13, multinucleated myotubes can be detected, and on day 25 contractile myofibres are present in ES cell cultures. The development of blood islands with red cells enhanced by erythropoietin in EBs has encouraged the hope that, subsequently, more mature stages of erythroid, myeloid and lymphoid cell development could occur in vitro. These data provide further support for the use of ES cells in an in vitro assay for embryotoxicity testing.