Phenotypic and transcriptional analysis of ES-derived erythroid cells. (A) Dissociated D7EB or D14EB cells were double-stained for α-4 integrin and glycophorin-A expression. Only viable glycophorin-A⁺ cells are shown. (B) Dissociated D14EBs and erythroid cells derived from SF expansion of fetal liver cells (FL), dissociated D7EBs, or dissociated D14EBs were triple-stained for glycophorin-A, α-4 integrin, and EP1 expression. Gate was set on viable glycophorin-A⁺ cells, as the example shown on the left. Quadrants of α-4 integrin and EP1 were set according to isotype controls of individual samples. (C) Real-time PCR analysis was performed on erythroid cells derived from the cultures of human adult bone marrow (BM) CD34⁺ cells (n = 2), peripheral blood (PB) mononuclear cells (n = 1), fetal liver (FL) (n = 3), or ES cells (n = 4-5). Duplicates/triplicates were analyzed for each sample. Expression levels of specific transcription factors were normalized to the expression level of β-actin of individual samples. Data from adult BM and PB were pooled together. Data are expressed as fold expression over FL-derived erythroid cells. Error bars indicate SEM. ^* indicates that only data of adult erythroid cells are significantly different from those of both FL- and ES-derived erythroid cells (P < .05).