Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes Sphere cell transfer to the original feeder-free ESC Cultures resulted in chaotic differentiation in contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors. reversion to the ESC-like state was observed These results indicate that Akt signaling promotes a novel metastable and plunpotent state that is intermediate to those of ESCs and PGCs. (C) 2010 Elsevier Inc All rights reserved.