Formation of a primitive ectoderm like cell population, EPL cells, from EScells in response to biologically derived factors

The primitive ectoderm of the mouse embryo arises from the inner cell mass between 4.75 and 5.25 days post coitum, around the time of implantation, Po sitioned at a pivotal time in development, just prior to formation of the t hree germ layers of the embryo proper, the primitive ectoderm responds dire ctly to the signals generated during gastrulation, We have identified a con ditioned medium, MEDII, which caused the homogeneous conversion of ES cells to a morphologically distinct cell population, termed early primitive ecto derm-like (EPL) cells. EPL cells expressed the pluripotent cell markers Oct 4, SSEA1 and alkaline phosphatase. However, the formation of EPL cells was accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chi maera forming ability? changes in factor responsiveness and modified differ entiation capabilities, all consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post coitum embryo. EPL cell formation could be reversed in the presence of LIF and withdrawal of MEDII, which suggested that EPL cell formation was n ot a terminal differentiation event but reflected the ability of pluripoten t cells to adopt distinct cell states in response to specific factors. Part ial purification of MEDII revealed the presence of two separable biological activities, both of which were required for the induction and maintenance of EPL cells, We show here the first demonstration of uniform differentiation of ES cells in response to biological factors. The formation of primitive ectoderm, bo th in vivo and in vitro, appears to be an obligatory step in the differenti ation of the inner cell mass or ES cells into cell lineages of the embryoni c germ layers, EPL cells potentially represent a model for the development of lineage specific differentiation protocols and analysis of gastrulation at a molecular level. An understanding of the active components of MEDII ma y provide a route for the identification of factors which induce primitive ectoderm formation in vivo.