Generation of an immortalised erythroid cell line from haematopoietic stem cells of a haemoglobin E/β-thalassemia patient

Abstract

The β-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent β-globin chain synthesis. HbE/β-thalassemia is a subtype of β-thalassemia with extremely high frequency in Asia. Studying molecular defects behind β-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/β-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient's red blood cells. This is the first human cellular model for β-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.

Figure 1

(A) A schematic diagram illustrating steps in the generation of the immortalised erythroid cell line (SiBBE) from stem cells collected from blood of a patient with haemoglobin E/β-thalassemia. (B) Undifferentiated erythroid cells (undif.) on day 114 in expansion medium and differentiated erythroid cells (dif.) on day 2, 4, 6, 8 and 10 in erythroid differentiation medium stained with Leishman reagent and analysed by light microscopy (scale bar = 10 μm). Green arrows = proerythroblasts; Blue arrows = basophilic erythroblasts; Orange arrows = polychromatic erythroblasts; black arrows = orthochromatic erythroblasts; red arrows = reticulocytes. (C) Extrapolated number of erythroid cell types at different time points during erythroid differentiation (mean ± SD, n = 3). (D) Percentage of living cells during erythroid differentiation determined by Trypan blue assay (mean ± SD, n = 3). (E) Expression of HPV16 E6, E7 and gamma globin (HBG) (control) determined by RT-PCR. Full-length gels are presented in Supplementary Fig. 3. (F) Undifferentiated early (day 81) and late (day 144) passage cells in expansion medium (undif.) and at day 10 in differentiation medium (dif. day 10) stained with Leishman reagent and analysed by light microscopy (scale bar = 10 μm). (G) Flow cytometric analysis of cells in (E) incubated with antibodies to glycophorin A (GPA; CD235a) and CD36. (H) Extrapolated cell numbers of early (day 77 to 81; blue) and late (day 136 to 144; orange) passage cells during erythroid differentiation (mean ± SD, n = 3).

Figure 2

(A) Flow cytometric analysis of SiBBE on day 77 in expansion medium showing key erythroid cell marker levels in undifferentiated cells (day 0) and differentiated cells on day 3, 5, 7 and 10 in erythroid differentiation medium. Cells were incubated with antibodies to CD36, glycophorin A, α4 integrin, band3, Rh and RhAG followed by incubation with an IgG1 APC secondary antibody, or with α4 integrin FITC conjugate. The cells were also dual stained by incubation with antibodies to CD36 and GPA or α4 integrin and band 3. (B) Pellets of undifferentiated erythroid cells and differentiated erythroid cells on day 8 in culture. (C) HPLC traces showing HbF, HbA and HbE levels in the patient RBC and the differentiated cell line (SiBBE).

Figure 3

(A) Extrapolated cell numbers of HU treated erythroid cells (blue) and control cells (green) (mean ± SD, n = 3). (B) Flow cytometric analysis of the cell line showing erythroid differentiation of HU treated erythroid cells and control cells. The cells were dual stained with anti-CD36 or anti-α4 integrin and anti-GPA or anti-band 3. (C) Percentage of living cells of HU treated erythroid cells (blue) and control cells (green) determined by Trypan blue assay (mean ± SD, n = 3). (D) Gamma and beta globin expression in HU treated erythroid cells (blue) and control cells (green) analysed by Western blot analysis. Alpha globin was used as control. Western blot is representative of 3 repeats. Density of bands on Western blots were quantified using ImageJ (mean ± SD, p < 0.05 by T-test, n = 3). Full-length blots are presented in Supplementary Fig. 4.