When embryonic stem cells are allowed to aggregate, the outer layer of the 
aggregated spheres (referred to as embryoid bodies) differentiates into 
primitive endoderm. This initial specification of cell lineage facilitates 
further differentiation of the inner mass of the embryoid bodies. These 
processes are considered to recapitulate early embryonic development from the 
blastocyst stage to the egg-cylinder stage. Formation of the primitive endoderm 
layer in the embryoid bodies was induced solely by aggregation of embryonic stem 
cells, in the presence of leukemia inhibitory factor/STAT3 and serum/BMP4, which 
were considered to be sufficient for embryonic stem cell self-renewal. 
Interestingly, cell aggregation by itself induced Nanog repression at the outer 
layer, which was essential for aggregation-induced primitive endoderm formation. 
These data illustrate aggregation-based cell-fate specification during early 
embryonic development, when downregulation of Nanog plays a crucial role.