DEVELOPMENT OF AN IN-VITRO EMBRYOTOXICITY TEST USING MURINE EMBRYONICSTEM-CELL CULTURES

Mouse embryonic,stem (ES) cell line D3 was used to establish condition s for a reproducible differentiation of ES cells in culture. ES cells r-an be maintained in an undifferentiated state by cultivation on a fe eder layer of embryonic fibroblasts. ES cells form aggregates in suspe nsion and can spontaneously differentiate into complex organized embry oid bodies (EBs), which in many respects resemble early postimplantati on mouse embryos. Under appropriate culture conditions various cell an d tissue types will develop in EBs: these include myocardial and skele tal 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) i nduced an increase in skeletal muscle cell differentiation, which foll owed a characteristic pattern: day 10 is characterized by the first ap pearance of mononucleated myoblasts; day 12 shows the fusion of myobla sts; on day 13, multinucleated myotubes can be detected, and on day 25 contractile myofibres are present in ES cell cultures. The developmen t of blood islands with red cells enhanced by erythropoietin in EBs ha s encouraged the hope that, subsequently, more mature stages of erythr oid, myeloid and lymphoid cell development could occur in vitro. These data provide further support for the use Of ES cells in an in vitro a ssay for embryotoxicity testing.