Derivation and characterization of hESC lines. (A) Schematic diagram to present the procedure to establish a hESC model from poor-quality embryos for study of skewed XCI. The numbers in brackets indicate the number of cell lines in each group. (B) Representative poor-quality embryo culture in modified medium. (C) Colony morphology under an inverted microscope. (D) EB formation at day 7 when plated in suspension dishes. (E-G) Immunostaining analysis of hESCs for anti-SSEA-3 (green), anti-SSEA-4 (green) and anti-TRA-1-60 (green). (H-J) Immunostaining analysis for in vitro differentiation into cell types representative of the three embryonic germ layers, including alpha-fetoprotein (AFP, endoderm), nestin (ectoderm) and smooth muscle actin (SMA, mesoderm). (K-M) Histological examination of in vivo differentiation showing that the teratomas contained tissues of the three embryonic germ layers, including neural-tube like cells (ectoderm), fat cells (mesoderm) and glandular tissue (endoderm). (N, O) Normal karyotypes of 46, XX and 46, XY. Bars in (B-M): 100 μm.