Identifications of HPP-CFC and primitive progenitor-related gene expression within day 6 wild-type and Smad5^−/− EBs.

HPP-CFCs derived from day 6 EBs of wild-type (A) and Smad5^−/− (B) ES cells were able to generate macroscopic colonies in vitro. Macroscopic colonies by HPP-CFCs are shown by white arrows; secondary EBs are shown by white arrowheads. Grid lines on the dishes are spaced 2 mm apart. Note the solid and round morphology of secondary EBs, whereas the HPP-CFCs displayed a looser morphology and often with red hemoglobin. The actual number of HPP-CFC, erythroid mix (CFU-mix), and CFU-GM colonies derived from day 6 EBs are shown in panel C. The results are shown as means ± SEM from 3 representative experiments; the other 2 experiments demonstrated similar results. Significance was determined using the Studentt test. *represents data found to be significantly different from wild-type control values (P < .05); #, data found to be significantly different from Smad5^+/− values (P < .05). The patterns of gene expression were defined using day 6 EBs by RT-PCR (D). The arrows on the top of the photo in panel D represent the 10-fold dilution of samples.