In vitro erythroid differentiation from purified human CD34⁺ peripheral blood hematopoietic cells. (A) Wright-Giemsa–stained cytospin samples during in vitro differentiation. (B) Flow cytometric characterization of cells during differentiation. Scatter plots show CD45/CD34 staining and CD71 (transferrin)/CD235 (glycophorin A) at start and completion of differentiation. Graph shows the changing patterns of CD34 and CD235a cell surface expression during in vitro differentiation. (C) Expansion of cells during in vitro differentiation. (D) Relative levels of γ- and β-globin mRNA during in vitro differentiation as determined by quantitative RT-PCR. Values are normalized to the geometric mean of β-actin and G3PD mRNAs as described in “Materials and methods; Quantitative RT-PCR and Hb HPLC.” (E) Patterns of steady-state γ- and β-globin mRNA during differentiation. Values are expressed as the proportion of maximal expression to highlight the time course of expression. (F) Hb HPLC performed on cellular extracts at the end of the culture period.