Malik and Arumagan Figure 1.
Malik and Arumagan Figure 1. Correction of β-thalassemia major in vitro and in vivo, in immune deficient mice. . / (A) Morphology of cultured CD34+ cells at day 10 of erythroid differentiation showing formation of hemoglobinized orthochromatic normoblasts in normal bone marrow (NBM). Normoblasts derived from thalassemia bone marrow CD34+ cells (TBM) fail to hemoglobinize and arrest at the polychromatophil normoblast stage of erythropoiesis, and undergo apoptosis thereafter. In contrast, normoblasts derived from TBM CD34+ cells transduced with the BGI vector (TBM-BGI) hemoglobinize and mature like those from NBM. (B) High level gene transfer and β-globin production in individual BFUe from thalassemia major bone marrow CD34+ cells transduced with the BGI vector. Individual BFUe were picked at 2 weeks and labeled with anti-HbA antibody and analyzed by FACS. The mean fluorescence intensity (MFI) of HbA expression from 20 individual BFUe colonies from NBM, TBM and TBM-BGI from one experiment is shown (left panel). Normal levels of β-globin production were seen in TBM-BGI erythroid liquid cultures, as shown by reverse phase HPLC analysis (right panel). (C) Reversal of ineffective erythropoiesis and expression of human β-globin in β2Mnull NOD-SCID mice. FACS analysis of β2Mnull NOD-SCID mouse bone marrow and blood was performed 12–16 weeks following transplant with NBM, TBM and TBM-BGI CD34+ cells. Representative dot-plots showing human CD45 (column 1) and Annexin-V staining (column 2), demonstrate sustained human cell engraftment in all mice, but presence of apoptotic cells only in TBM xenografts. Peripheral blood of the mice transplanted with NBM, TBM and TBM-BGI analyzed for glycophorin A (column 3) and HbA (column 4) expression shows presence of circulating human erythroid cells. All samples in columns 1–4 were gated on the basis of appropriate isotype controls and events falling within this gate are shown in dark gray. All events labeled with the respective human antibodies are shown in red. For intracellular HbA staining, human ζ-globin was used as the negative control and events falling within this gate are shown in dark gray. HbA labeled events falling outside this gate are shown in red.

Correction of β-thalassemia major in vitro and in vivo, in immune deficient mice.

(A) Morphology of cultured CD34+ cells at day 10 of erythroid differentiation showing formation of hemoglobinized orthochromatic normoblasts in normal bone marrow (NBM). Normoblasts derived from thalassemia bone marrow CD34+ cells (TBM) fail to hemoglobinize and arrest at the polychromatophil normoblast stage of erythropoiesis, and undergo apoptosis thereafter. In contrast, normoblasts derived from TBM CD34+ cells transduced with the BGI vector (TBM-BGI) hemoglobinize and mature like those from NBM. (B) High level gene transfer and β-globin production in individual BFUe from thalassemia major bone marrow CD34+ cells transduced with the BGI vector. Individual BFUe were picked at 2 weeks and labeled with anti-HbA antibody and analyzed by FACS. The mean fluorescence intensity (MFI) of HbA expression from 20 individual BFUe colonies from NBM, TBM and TBM-BGI from one experiment is shown (left panel). Normal levels of β-globin production were seen in TBM-BGI erythroid liquid cultures, as shown by reverse phase HPLC analysis (right panel). (C) Reversal of ineffective erythropoiesis and expression of human β-globin in β2Mnull NOD-SCID mice. FACS analysis of β2Mnull NOD-SCID mouse bone marrow and blood was performed 12–16 weeks following transplant with NBM, TBM and TBM-BGI CD34+ cells. Representative dot-plots showing human CD45 (column 1) and Annexin-V staining (column 2), demonstrate sustained human cell engraftment in all mice, but presence of apoptotic cells only in TBM xenografts. Peripheral blood of the mice transplanted with NBM, TBM and TBM-BGI analyzed for glycophorin A (column 3) and HbA (column 4) expression shows presence of circulating human erythroid cells. All samples in columns 1–4 were gated on the basis of appropriate isotype controls and events falling within this gate are shown in dark gray. All events labeled with the respective human antibodies are shown in red. For intracellular HbA staining, human ζ-globin was used as the negative control and events falling within this gate are shown in dark gray. HbA labeled events falling outside this gate are shown in red.

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