The use of serum-free culture conditions in the embryonic stem cell test: defined culture conditions for cardiac stem cell differentiation.
The Embryonic Stem Cell Test (EST) is an well established and standardized in vitro method to predict the embryotoxic potential of chemicals and is based on the capacity of murine embryonic stem (ES) cells to differentiate under specific culture conditions into beating cardiomyocytes.An efficient differentiation requires the use of cell culture medium supplemented with 15% fetal calf serum (FCS). Unfortunately, FCS has a great lot to lot variability with regard to its composition. Therefore selected batches of FCS have to be pre-tested if they support differentiation and proliferation of ES cells before the EST can be performed. In order to optimize the culture conditions in the EST our aim was to establish serum-free culture conditions. Chemically defined serum-free culture conditions would provide several advantages: (1) improved protocol transfer to other laboratories (2) improved reproducibility of the differentiation assay, (3) no interference of undefined serum component with the test substance (reproducible bioavailability) and (4) application of the EST in high/medium-throughput screening systems.Chemically-defined serum-free media supplemented with several growth and differentiation factors known to be important for cardiogenesis in vivo have been tested. Our results demonstrate that under these conditions cardiac embryonic stem cell differentiation can be achieved in vitro in a highly standardized and reproducible manner. In addition, we were able to demonstrate the applicability of the serum-free EST to test chemicals with different embryotoxic potentials.