Functional characterization of primitive and definitive RBCs from KLF1 edited iPSC lines. (A) Schematic representation of the experimental layout. (B) Erythroid expansion of primitive or definitive erythroid liquid cultures (mean ± SD, n = 2-5). (C) Percentage of erythroid colonies derived from primitive and definitive HPCs from wild type (WT) and KLF1 edited lines (mean ± SD, n = 2-9, 1-way analysis of variance, Tukey test; ∗∗P < .01 and ∗∗∗P < .001). (D) Expression of KLF1-dependent or independent red cell antigens by flow cytometry in primitive or definitive day 12 RBCs from WT or KLF1^WT/L300P lines. (E) Relative abundance of β-like transcripts (β+ε+γ_g+γ_a) at day 12 of erythroid liquid culture (mean ± SD, n = 3-5, ∗P < .05, paired 2-tailed t test WT vs KLF1^WT/L300P). (F-G) HPLC quantification of globin subunits (F) and tetramers (G) in primitive and definitive RBCs from WT or KLF1^WT/L300P iPSC lines (mean ±SD, n = 2-4, ∗ = P < .05, paired 2-tailed t test WT vs KLF1^WT/L300P). HPLC, high-performance liquid chromatography.