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Figure 1. Characterization of EBNA1-expressing H9 hES cells. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of EBNA1 expression in EBNA1 hES cells. Lanes 1–6: six different stably EBNA1-expressing hES cell clones. Lane 7: untransfected hES cells. (B): Western blot analysis of the EBNA1 protein in EBNA1-expressing hES cell clones (lanes 1–6) and untransfected hES cells (lane 7) as in (A). (C): Staining for cell surface markers in EBNA1-expressing hES cells (a–e) and EBNA1 expression (f). Alkaline phosphatase (a) in EBNA1 hES cell clone was stained by fast red (red). SSEA-3 (b), SSEA-4 (c), TRA-1-60 (d), TRA-1-80 (e), and EBNA1 (f) were detected by immunofluorescence. (D): RT-PCR analysis of OCT4 and Nanog expression in untransfected hES cells (U) and EBNA1 hES cells (E). (E): Normal telomerase activity in EBNA1 hES cells. Inactivated MEF was used as a feeder layer; 293: adenovirus-transformed kidney epithelial cell line, used as a positive control. (F): EBNA1 hES cells differentiated into EB-like spheres by suspension culture. (G): Sections of a teratoma formed by EBNA1 hES cells in severe combined immunodeficient-beige mice. a, retina-like structures; b, rosettes of neural epithelium; c, cartilage; d, bone; e, striated muscle; f, pseudostratified ciliated columnar epithelium structures. Bars = 100 µm. (H): Human CG-ß was detected by immunofluorescence (green) in the syncytial cells from EBNA1 hES cells with nuclei stained by Hoechst 33342 (blue). Bars = 25 µm. Abbreviations: E, EBNA1-transfected hES cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, mouse embryonic fibroblast; U, untransfected hES cells.
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