Characterization of the erythroid cells under normoxia and hypoxia. (A) CD analysis of various markers (CD45, CD71, and CD235a) for all populations under normoxia (21% O2). The cells are cultured as described in Fig. 1. Note that the CD profiles are similar for all groups under normoxia. (B) CD analysis of various markers (CD45, CD71, and CD235a) for all populations under hypoxia (5% O2). The cells were cultured as described in Fig. 1. During week 3, we see significant differences in the proportion of CD235a between CMS and the controls (sea level and non-CMS). (A and B) Each experiment was done in three replicates, and experiments were repeated at least three times. (C) BFU-e assay under hypoxia (5% O2). The y axis represents the number of BFU-e colonies. The experiment was done in three replicates and repeated twice. (D) Hemoglobin (Hb) analyses and function of erythroid cells with high-performance liquid chromatography profiles of erythroid cells at weeks 6 and 8. Note a shift in hemoglobin from fetal to adult A0. The O2-binding curve also shows a transition of P50 from 14 mmHg (week 6) to 22 mmHg (week 8). Shown is one representative image of data points at weeks 6 and 8. The experiment was repeated at least five times. A.U., arbitrary units; Sat, saturation. (E) Western blot of RBCs (erythroblasts). Note the presence of BAND-3 and GLUT1 in all lanes (normalized to actin). Retic, reticulocytes used as controls. (F) Globin expression by quantitative PCR for sea level and globin switching in time in culture. Similar trends were observed during maturation for all groups. n = 3 and error bars represent SD. Globin percentage is calculated as described by Qiu et al. (2008). The experiment was repeated at least three times. *, P < 0.05.
