EBs were grown from each iPSC clone in low-attachment plates (Corning) in hES medium without bFGF. After 21 days, total RNA was extracted for expression analyses using an AllPrep DNA/RNA/Protein kit (Qiagen). Germ layer-specific expression of EBs was analyzed using antibodies which recognize β-III-tubulin (R&D Systems, MAB1195), AFP (DAKO, A0008), and vimentin (Dako, M0725).
