Functional characterization of primitive and definitive iRBCs. (A) Erythroid expansion of primitive and definitive HPCs in erythroid liquid cultures. (B) Representative histograms of CD36, CD71, and CD235a expression of primitive (top) or definitive erythroblasts during erythroid differentiation. Differentiation days are indicated on the right and negative control is shown in gray. (C) Cytospins showing May-Grünwald Giemsa staining of erythroid cells from primitive and definitive cultures at day 6 and day 12 of differentiation. Scale bars represent 20 μm. (D) Relative abundance of β-like transcripts (β+ε+γ_g+γ_a) at day 12 of erythroid liquid culture (mean ± SD, n = 3-4). (E) HPLC analysis of globin monomers in differentiated erythroblasts, relative quantification of α-like and β-like subunits are shown (primitive and definitive, mean ± SD, n = 3; FL n = 1). (F) HPLC analysis of globin tetramers in differentiated erythroblasts, relative quantification of adult hemoglobin (adult, α₂β₂), HbF (fetal, α₂ γ₂), and embryonic (Gower 1 ζ₂ε₂ and Gower 2, α₂ε₂) globins (mean ± SD). HPLC, high-performance liquid chromatography.