Effective erythropoiesis from vector-transduced human thalassemia major bone marrow CD34⁺ cells in vitro. Wright-Giemsa–stained cytospins of predominant cell types in erythroid cultures. (A) Cells are mainly pronormoblasts in NBM, TBM, and TBM-BGI at day 4, (B) orthochromic normoblasts in NBM and TBM-BGI, but polychromatophilic normoblasts in TBM, at day 13. (C) Enucleated RBCs produced in NBM and TBM-BGI at day 17, while TBM shows apoptotic cells. (D) Differential interference contrast microscopy of Hoechst 33342–negative cells (enucleated RBCs) sorted by FACS showing normal reticulocyte and discocyte morphology of NBM and TBM-BGI RBC. Occasional RBCs derived from TBM were dysmorphic. (E) Fold expansion of erythroid cultures derived from CD34⁺ cells (n = 4): mock-transduced NBM (⋄), K6I-transduced NBM (▪), mock-transduced TBM (▴), and BGI-transduced TBM (▵). The mean fold expansion at day 13 was NBM 356 ± 22, NBM K6I 360 ± 101, TBM 110 ± 31, TBM-BGI 308 ± 28; and at day 17 was NBM 494 ± 36, NBM K6I 498 ± 119, TBM 108 ± 36, TBM-BGI 458 ± 78 (P < .001 and P < .002 for NBM versus TBM and P = .1 and P = .3 for NBM versus TBM-BGI, at day 13 and day 17, respectively). (F) Representative apoptosis analysis using annexin-V labeling of NBM, TBM, and TBM-BGI erythroid cultures. (G) Quantification of apoptotic (annexin-V⁺) cells at day 7 and day 14 from the 4 experiments. NBM (filled bars), TBM (open bars), and TBM-BGI (hatched bars). Day 7: P < .01 NBM versus TBM; P < .08 NBM versus TBM-BGI; day 14: P < .004 NBM versus TBM; P < .1 NBM versus TBM-BGI.