A Novel Role for γ‐Secretase in the Formation of Primitive Streak‐like Intermediates from ES Cells in Culture

γ‐Secretase is a membrane‐associated protease with multiple intracellular targets, a number of which have been shown to influence embryonic development and embryonic stem (ES) cell differentiation. This paper describes the use of the γ‐secretase inhibitor N ‐[ N ‐(3,5‐difluorophenacetyl)‐ L ‐alanyl]‐ S ‐phenylglycine t ‐butyl ester (DAPT) to evaluate the role of γ‐secretase in the differentiation of pluripotent stem cells to the germ lineages. The addition of DAPT did not prevent the formation of primitive ectoderm‐like cells from ES cells in culture. In contrast, the addition of DAPT during primitive ectoderm‐like cell differentiation interfered with the ability of both serum and BMP4 to induce a primitive streak‐like intermediate and resulted in the preferential formation of neurectoderm. Similarly, DAPT reduced the formation of primitive streak‐like intermediates from differentiating human ES cells; the culture conditions used resulted in a population enriched in human surface ectoderm. These data suggest that γ‐secretase may form part of the general pathway by which mesoderm is specified within the primitive streak. The addition of an E‐cadherin neutralizing antibody was able to partially reverse the effect of DAPT, suggesting that DAPT may be preventing the formation of primitive streak‐like intermediates and promoting neurectoderm differentiation by stabilizing E‐cadherin and preventing its proteolysis. S TEM C ELLS 2009;27:2941–2951