The formation of the blastocyst during mammalian development involves the segregation of two populations of cells with unequal potential: pluripotent cells of the inner cell mass (ICM) and multipotent cells of the trophectoderm (TE). ICM cells maintain the capacity to give rise to all cells represented in the organism, while TE cells, which represent the first lineage to emerge during development, are capable of differentiating into trophoblast lineages of the placenta. The ICM and TE are both essential for development. The ICM is genetically programmed to generate all cells of the embryo proper, while the TE forms extraembryonic trophoblast lineages and is required for implantation of the embryo and maternal-fetal exchange of nutrients and waste. Embryonic stem (ES) cells, which can be derived from the ICM of blastocysts in the presence of external signals such as LIF, can self-renewal indefinitely, and because they can differentiate into all cells of the organism, ES cells are a widely used in vitro model to study genetics and development. Trophoblast stem (TS) cells can be derived from the TE of blastocyst stage embryos in the presence of FGF4, and like ES cells, TS cells are also able to self-renew indefinitely. Because TS cells can differentiate into epithelial lineages of the trophoblast, TS cells are an ideal in vitro model to study the biology of the trophoblast. In this chapter, we describe protocols for simultaneous derivation of ES cells and TS cells from mouse blastocysts and culture conditions that promote self-renewal of hybrid ESC/TSC colonies. These protocols are sufficient for efficient derivation of hybrid ESC/TSC colonies.
Keywords: Blastocyst; Derivation; Differentiation; Embryonic stem cells; Inner cell mass; Multipotent; Pluripotent; Trophectoderm; Trophoblast stem cells.