Lineage Specific Differentiation of Mouse ES Cells: Formation and Differentiation of Early Primitive Ectoderm-like (EPL) Cells

Abstract

The purpose of this chapter is to discuss, at a technical level, the formation and use of early primitive ectoderm-like (EPL) cells, including methodologies for further differentiation of EPL cells to the mesodermal or ectodermal lineage and identification of alternative pluripotent and somatic cell populations. There is particular merit in differentiation regimes that recapitulate lineage establishment during normal embryogenesis. These are anticipated to provide a non-transformed model system for the investigation of cell-fate choice, identification, characterization, and production of transient differentiation intermediates, and identification of signaling pathways that regulate cell identity and acquisition of positional information. In conjunction with the extraordinary experimental malleability of the embryonic stem (ES) cell genome, this enables sophisticated analysis of gene function at the cellular level in a system that is not restricted by limitations associated with maintenance of a viable embryo. The most commonly used protocols for ES cell differentiation rely on differentiation within complex cellular aggregates known as “embryoid bodies (EBs).” ES cells share a gene expression, differentiation potential, and cytokine responsiveness with their source cell population, the pluripotent cells of the inner cell mass (ICM). The immediate developmental fate of ICM en route to the formation of the embryo proper, differentiation to primitive ectoderm, can be recapitulated in vitro by the formation of EPL cells. Unlike ES cells, when EPL cells are differentiated in vitro, they do not form visceral endoderm, thus providing a technology for pluripotent cell differentiation that is devoid of extraembryonic signals. This enables control over the formation of somatic lineages from pluripotent cells by manipulation of the differentiation environment and thereby overcomes many of the inherent problems associated with ES cell EBs.