Oct4-expressing hESCs isolated by FRET-based FACS demonstrate pluripotency in culture and in vivo. (A–C) Immunocytochemistry demonstrated that FRET-positive hESCs differentiate in culture into cells arising from all 3 embryonic lineages, including cells expressing βIII-tubulin (A; ectoderm), smooth muscle actin (B; mesoderm), and α-fetoprotein (C; endoderm). Scale bars: 100 μm. Immunohistochemical analysis of teratomas showed that FRET-positive hESCs gave rise to tissues derived from the 3 embryonic germ layers, including ectoderm (D, E), mesoderm (F, G), and endoderm (H, I). (D) Bed of eosinophilic neural precursors. (E) Nascent neural tube structure. (F) Pools of cartilage surrounded by loose mesenchyme. (G) Skeletal muscle surrounded by dense mesenchyme. (H) Primitive glandular structure. (I) Glandular intestinal structure. Scale bars: 100 μm. Color images available online at www.liebertonline.com/scd.