Figure 3

Derivation and characterization of mouse embryonic stem cells on human foreskin fibroblast without exogenous leukemia inhibitory factor. (A) Derivation of C57H1.2 mouse embryonic stem cells (ESCs) on human foreskin fibroblast (HFF) without exogenous leukemia inhibitory factor (LIF). (a) Outgrowth formed post inner cell mass (ICM) attachment. (b) Manually split ICM clumps. (c) Typical mouse ESC colonies. Scale bars: (a), (b) 100 μm; (c) 1,000 μm. (B) RT-PCR analysis of the expression levels of the pluripotency-associated markers (Oct4, Nanog and Sox2) in newly derived mouse ESC lines on HFF without exogenous LIF. NC, water was used as the negative control. (C) Immunostaining of C57H1.2 mouse ESCs with antibodies against Oct4, Sox2 and Nanog. Scale bars: 25 μm. (D) Detection of the methylation status at the endogenous Oct4 promoter of C57H1.2 mouse ESCs by a combined bisulfite restriction analysis. (E) Immunofluorescence staining of the attached embryonic bodies formed by C57H1.2 mouse ESCs with antibodies against Gata4, Foxa2, Flk1, Vimentin and Tuj1. Scale bars: 250 μm. (F) Teratomas formed by C57H1.2 mouse ESCs were sectioned and stained with H & E. Typical (a, b) smooth muscles, (c, e) intestinal epithelium, (d) fat and (f) neural-like tissues were found. Scale bars: 50 μm. (G) Chimeric mice generated by injection of C57H1.2 mouse ESCs into blastocysts of the ICR mouse. Black colors indicate origination of C57H1.2 mouse ESCs. (H) Immunofluorescence staining of the genital ridge sections from a chimeric embryo (male) at E15.5 with antibodies against EGFP and Oct4. C57H1.2 ESCs were labeled with EGFP. Arrow, an EGFP and Oct4 double-positive cell. Scale bars: 50 μm.