Increasing the time of coculture leads to the production of RBCs with a more mature globin expression program. (A) HPLC chromatograms illustrating globin expression in erythroblasts obtained after 14 days (top panels) or 24 days (bottom panels) of liquid culture of CD34⁺ cells that had been obtained after coculture of hESCs with FH-B-hTERT for 21 (left panels) and 35 days (right panels). Cells in the top panels, which were mostly pro- and basophilic erythroblasts, expressed predominantly α- and γ-globin. Cells in the left panel, which were mostly poly- and orthochromatic erythroblasts, expressed even more α-globin because of the maturation switch. The levels of α- and γ-globins are higher in the 35-day cocultures than in the 21-day cocultures. (B) X-Y plots summarizing the quantification of the HPLC results described in Figure 2 and 4. (C) X-Y plots summarizing the results of a real-time RT PCR analysis on the cells described in Figures 2 and 4. Inset: RT-PCR determination of β-globin expression in 35-day CD34⁺ cells after 14, 21, or 24 days in liquid culture (% β-globin indicates β/β+γ+ϵ). Results show that increasing the length of coculture of hESCs with FH-B-hTERT cells lead to a dramatic switch in the globin produced. Percent α-globin was calculated as 100 × α-globin/(α-globin + ζ-globin). Percent γ-globin was calculated as 100 × γ-globin/(γ-globin + ϵ-globin). Percent ζ-globin was calculated as 100 × ζ-globin/(α-globin + ζ-globin). Percent ϵ-globin was calculated as 100 × ϵ-globin/(γ-globin + ϵ-globin). Error bars represent SD.