hEBs (formed under -ROCK/-spin condition) expressed proteins characteristic of all the three developmental germ layers.

At 20 days in suspension culture, the hEBs were positive for Alpha Fetoprotein (AFP, endoderm-specific), SOX1 (ectoderm-specific), and brachiury (mesoderm-specific) for both the (a) BG01V/hOG and (b) H9 cell lines. Cell nuclei were stained in blue with DAPI.