Derivation and propagation of embryonic stem cells in serum- and feeder-free culture.

The availability of murine embryonic stem (ES) cells has revolutionized the study of mammalian development and disease. We recently developed a culture medium that has enabled us to identify the essential signaling pathways required for maintenance of pluripotency in vitro. Addition of leukemia inhibitory factor and bone morphogenetic protein4 to this medium is sufficient to activate the signal transducer and activator of transcription3 and mammalian homolog of Drosophila mothers against decapentaplegic pathways, respectively. We have successfully derived and propagated ES cells in the absence of feeder cells and serum. This chapter describes a simple protocol for efficient derivation and maintenance of ES cells from embryos of the 129 and C57B1/6 strains of mice.