Mouse embryonic stem cell derivation, and mouse and human embryonic stem cell culture and differentiation as embryoid bodies

Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long-term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene-targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs) has provided a convenient system for studying early mouse developmental processes, including several aspects of extraembryonic lineage and axis formation associated with the pre- and peri-gastrulating mouse embryo. Relatively little is known regarding the corresponding development of the early human embryo due to limitations associated with the acquisition of relevant tissue material for study. The transfer of methods such as EB formation to human systems should, by association, facilitate a more advanced understanding of similar processes associated with early human development. This unit describes protocols for isolating mouse embryonic stem cells and methods for propagating, freezing, and producing EBs from both mouse and human embryonic stem cells.