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. 2009 May 7;113(19):4763-70.
doi: 10.1182/blood-2008-12-197012. Epub 2009 Mar 3.

Preclinical transfusion-dependent humanized mouse model of beta thalassemia major

Yongliang Huo  1 , Sean C McConnell, Thomas M Ryan
Affiliations

Preclinical transfusion-dependent humanized mouse model of beta thalassemia major

Yongliang Huo et al. Blood. .

Abstract

A preclinical humanized mouse model of beta thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult alpha and beta globin genes were replaced with adult human alpha globin genes (alpha2alpha1) and a human fetal to adult hemoglobin (Hb)-switching cassette (gamma(HPFH)deltabeta(0)), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (alpha(2)gamma(2)), with a small amount of human minor adult Hb, HbA2 (alpha(2)delta(2)). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful for the development of new transfusion and iron chelation regimens, the study of iron homeostasis in disease, and testing of cellular and genetic therapies for the correction of thalassemia.

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Figures

Figure 1
Figure 1
CA mice were generated by replacing the adult mouse β globin genes with a human γHPFHδβ0 globin gene cassette in mouse ES cells. (A) Scheme of targeted gene replacement of the adult mouse β globin genes by a human γHPFHδβ0 globin gene cassette in mouse ES cells. The hyg marker gene was deleted by breeding to CRE recombinase transgenic mice. (B) Southern blot confirmation of correct homologous recombination. The 5′ probe hybridizes with a 9.2-kb XbaI fragment from the wild-type allele and a 7.9-kb fragment from the human globin KI allele. The 3′ probe derived from part of the βmin globin gene anneals to a 14.8-kb EcoRI fragment from the βmin globin gene, a 7.3-kb fragment from the βmaj globin gene, and a 10.3-kb fragment from the human globin KI allele.
Figure 2
Figure 2
Hb switching and survival curves of humanized γHPFH δβ0 CA mice. (A) Humanized compound heterozygous γHPFHδβ0/γβA mice complete the switch from high levels of fetal γ globin to adult δ and β globins after birth. Weekly hemolysates from peripheral blood were analyzed by HPLC to quantify the β-like globin chains. The fractional percentage of γ (□), β (◇), and δ (▵) globin chains relative to total β-like chains is plotted over time. Values represent mean ± SEM, n = 8. (B) Survival curves of humanized homozygous γHPFHδβ0 CA (□) and littermate control (◇) mice. The majority of fully humanized γHPFHδβ0 CA mice expire within 3 weeks of age as the human γ globin switches to the human δ and nonfunctional human β0 globin genes. Moribund homozygous CA mice were humanely euthanized before death, and their genotype was confirmed by PCR of tail tip DNA. All humanized littermate control mice (γHPFHδβ0/γβA and γβA/γβA) survived beyond 5 weeks of age.
Figure 3
Figure 3
Erythroid hyperplasia, ineffective erythropoiesis, and hemolysis in humanized γHPFHδβ0 CA mice. (A) Erythroid hyperplasia in the bone marrow and spleen of CA mice was measured by flow cytometry. Bone marrow and spleen cells from age- and sex-matched CA and wild-type mice were stained with fluorescently labeled antibodies to the erythroid antigen Ter119 and transferrin receptor CD71 and fluorescent-labeled annexin V. Dead cells were excluded by 7-aminoactinomycin D (7AAD) staining. The percentages of proerythroblasts (region I: CD71HIGH, Ter119LOW), early erythroblasts (region II: CD71+, Ter119+), late erythroblasts (region III: CD71−, Ter119+), and total Ter119+ erythroid cells (total Ter119+) in the bone marrow and spleen are shown in Table 2. Erythroid populations of humanized HbA mice (α2α1/α2α1 γβA/γβA) and C57BL/6J wild-type mice did not differ significantly (data not shown). (B) Demonstration of ineffective erythropoiesis in CA mice by apoptosis of erythroid progenitors by annexin V–binding assay. Representative histograms are shown of annexin V staining of early (region II) and late (region III) erythroblasts in CA and control mouse bone marrow and spleen cells from panel A. No antibody control samples were stained with all the antibodies in panel A, except annexin V. Annexin V+ cell ratios are quantified in Table 3. Annexin V+ erythroid populations of humanized HbA mice (α2α1/α2α1 γβA/γβA) and C57BL/6J wild-type mice did not differ significantly (data not shown). (C) Bilirubin levels increased significantly in untransfused CA mice compared with age- and sex-matched wild-type control mice, indicating increased hemolysis in CA mice. Bilirubin levels returned to control levels in hypertransfused, but not hypotransfused CA mice. *P < .05; **P < .0001; n = 3 in each group.
Figure 4
Figure 4
Histopathology of blood, spleen, and liver of humanized HbA control and CA mice before and after transfusion. Peripheral blood smears of untransfused CA mice exhibit severe anemia compared with HbA littermate control mice. There are significant numbers of reticulocytes, circulating erythroblasts, and microcytic, hypochromic, and fragmented RBCs in untransfused CA mouse blood. In addition, the normal structure of red pulp and white pulp in the spleen is absent. In the liver of untransfused CA mice, there are extensive clusters of extramedullary hematopoiesis and increased iron staining. In contrast, the hypertransfused CA mouse has a normal peripheral blood smear and greatly improved histology similar to the HbA control, except excess iron is present in the liver. Hypotransfused CA mice are still anemic, have significant numbers of erythroblasts and thalassemic RBCs in the blood, and have a histopathology similar to the untransfused CA mouse. All mice are 8 weeks of age, except for the untransfused CA mouse, which is only 2 weeks old. Scale bars: blood smear, 10 μm; spleen, 100 μm; liver (low power), 50 μm; liver (high power), 10 μm; liver iron, 50 μm.
Figure 5
Figure 5
Hb levels and GFP+ RBC chimerism in peripheral blood of hyper- and hypotransfused CA mice. Humanized CA mice were transfused weekly with packed donor RBCs from GFP+ transgenic mice. Two days after transfusion, mice were bled for determination of Hb level by spectrophotometry (A) and percentage of donor GFP+ RBCs by flow cytometry (B). Values represent mean (± SEM), n = 5 in each group.
Figure 6
Figure 6
Erythropoietic activity and storage iron levels in hypertransfused and hypotransfused CA mice. (A) Erythroid progenitors in the bone marrow of untransfused CA mice, wild-type controls, and transfused CA mice were determined by flow cytometry analysis of Ter119 and CD71 stained bone marrow cells. Dead cells and transfused GFP+ RBCs were excluded from analysis by using the 7AAD− and GFP− populations, respectively. Percentages of Ter119+ erythroid cells are shown. Untransfused and hypotransfused CA mice had increased erythropoietic activity compared with control, whereas hypertransfused CA mice had reduced erythropoietic activity. All mice were 8 weeks of age, except the untransfused CA mice, which were only 2 weeks old. (B) Storage iron concentrations in livers and hearts were quantified in male and female CA mice after 8 weeks of transfusion. Compared with littermate control γβA/γβA mice, the storage iron in the livers of both hyper- and hypotransfused CA mice increased significantly (P ≤ .0001). There was no significant increase in the hearts of either transfused group. P values were calculated by 2-tailed unpaired Student t test, n ≥ 5 in each group.
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