Figure 5. Single cell culture of hPS cells in the hESF9a_2i medium.

(A) Cell growth of dissociated single H9 hES cells cultured in each indicated condition for three passages. Cells were reseeded at the cell density of 1×10⁶ cells/well every 5 days. When the cells were passages, cell numbers were counted. Cell growth in the hESF9a_2i medium was significantly different (P<0.05) from hESF9 (FGF-2), FGF-2 + activin A, FGF-2 + activin A + U0126. Cell growth in hESF9a + GFX was significantly different (P<0.05) from hESF9 (FGF-2), FGF-2 + activin A, FGF-2 + activin A + U0126, and FGF2 + U0126. The data are represented as means ± SE (n = 3). (B) Gene expression in the hPS cells cultured in each indicated condition for three passages. The gene expression levels of NANOG, OCT3/4, FOXA2, T in the cells were measured by real-time RT-PCR. On the y axis, the gene expression level in the cells cultured with FGF-2 in a experiment was taken as 1.0. The data are represented as means ± SE (n = 3). *P<0.05. (C) Phase-contrast image of hPS cells grown on FN in hESF9a_2i medium for 3 passages. The cells were dissociated into single cells for passage, and reseeded at a ratio of 1∶3 - 1∶5 every five days. Scale bars, 200 µm. (D) OCT3/4 expression in hPS cells grown on FN in hESF9a_2i. The cells grown in hESF9a_2i as described above in Figure 5C were reseeded on a 6-well-plate and cultured for 5 days. The cells stained with anti-OCT3/4 antibody were visualized with Alexa Fluor 488 (upper panels). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 200 µm. Whole cell images in whole plate were captured and OCT3/4 expression profiles were analyzed by Image Analyzer (lower panels). Antigen histogram (red); control histogram (green); Y axis is cell numbers and X axis is fluorescence intensity for anti-OCT3/4 antibody.