FIGURE 5.

ES cells maintain the undifferentiated phenotype and pluripotency on the co-immobilized surface. A, expression of three genes that are markers of the undifferentiated state was analyzed by RT-PCR. B, ES cells cultured for 5 days in different conditions were stained with anti-Oct-3/4-specific antibody. On the co-immobilized surface, the expression of Oct-3/4 was observed even though low concentration of LIF-Fc was immobilized. Scale bar indicates 100 μm. C, adaptation of feeder-dependent ES cells onto the co-immobilized surface with E-cad-Fc and LIF-Fc. Jxl1 and R1 cells were seeded on the surface coated with several matrices and cultured for 5 days. The ratio of Oct-3/4 positive cells was calculated by ImageJ software. The data indicate mean ± S.E. of five separate experiments. D, ability to differentiate into multilineage cells was assessed by RT-PCR. Feeder-dependent ES cells were seeded on feeder layer (Feeder) or on the co-immobilized surface (Co-immobilized). After being maintained for 10 passages, cells were cultured to form embryoid bodies. Embryoid bodies were cultured for 11 days, and then expression of marker genes was analyzed by RT-PCR. ES, undifferentiated cells; EB, embryoid bodies. Polr2a was used as an internal control. E, feeder-dependent R1 cells were maintained on the co-immobilized surface of E-cad-Fc and LIF-Fc for 10 passages, and then cells were aggregated with four-cell stage tetraploid CD-1 embryos. The embryos were observed at the stage E9.5.