 In embryonic stem, ES, cell colonies, a small subpopulation that changes cell shape and loses pluripotency, often appears in two-dimensional, 2D, cultures, even in the presence of a stemness factor. This phenomenon can be attributed to the membrane translocation of the syntax in 4, T snare protein. However, in three-dimensional, 3D, aggregates, this effect does not occur due to the suppression of p-ketherin protein expression. The introduction of an expression cassette for p-ketherin practically reproduces the effects induced by extracellular syntax in 4, where the transgene product is clearly detected in 2D ES cells. Furthermore, an expression construct for p-ketherin venous harboring an inframe insertion of the P2A sequence at the joint region gives fluorescent signals only in the cytoplasm of 2D ES cells, demonstrating translational regulation of p-ketherin. These findings suggest that the embryoid body protocol commonly used for ES cell handling may not be suitable for directional differentiation. This article was authored by Yuka Tokeda, Shuji Matsuduchi, Essayu Nozaki, and others. We are article.tv, links in the description below.