In Vitro Study of Ineffective Erythropoiesis in Thalassemia: Diverse Intrinsic Pathophysiological Features of Erythroid Cells Derived from Various Thalassemia Syndromes

Abstract

Defective hemoglobin production and ineffective erythropoiesis contribute to the pathophysiology of thalassemia syndromes. Previous studies in the field of erythropoiesis mainly focused on the severe forms of thalassemia, such as β-thalassemia major, while mechanisms underlying the pathogenesis of other thalassemia syndromes remain largely unexplored. The current study aimed to investigate the intrinsic pathophysiological properties of erythroid cells derived from the most common forms of thalassemia diseases, including α-thalassemia (hemoglobin H and hemoglobin H-Constant Spring diseases) and β-thalassemia (homozygous β⁰-thalassemia and β⁰-thalassemia/hemoglobin E diseases), under an identical in vitro erythroid culture system. Cell proliferation capacity, differentiation velocity, cell death, as well as globin synthesis and the expression levels of erythropoiesis modifying factors were determined. Accelerated expansion was found in erythroblast cells derived from all types of thalassemia, with the highest degree in β⁰-thalassemia/hemoglobin E. Likewise, all types of thalassemia showed limited erythroid cell differentiation, but each of them manifested varying degrees of erythroid maturation arrest corresponding with the clinical severity. Robust induction of HSP70 transcripts, an erythroid maturation-related factor, was found in both α- and β-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous β⁰-thalassemia erythroblasts and associated with the up-regulation of pro-apoptotic (Caspase 9, BAD, and MTCH1) genes and down-regulation of the anti-apoptotic BCL-XL gene.

Keywords: apoptosis; cell death; erythropoiesis; globin; hemoglobin; thalassemia.

Figure 1

Defective globin production in thalassemic erythroid cells derived from in vitro culture. (a) Amount of α- and β-globin transcripts in cells day 10 of culture. (b) Chromatograms showing hemoglobin content in cells day 14 of culture. An error bar represents mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. N: normal, H: HbH disease, HCS: HbH-Constant Spring disease, BE: β⁰-thalassemia/HbE disease, BB: homozygous β⁰-thalassemia disease.

Figure 2

Elevated thalassemic erythroid cell expansion during in vitro erythropoiesis. (a) Cell number at the different time points of culture (day 4–14) of normal (N), HbH disease (H), HbH-CS disease (HCS), β⁰-thalassemia/HbE (BE), and homozygous β⁰-thalassemia (BB). *** p ≤ 0.001, **** p ≤ 0.0001 as compared to normal. (b) Dye dilution assay in erythroid cells day 6 of culture. The fluorescent intensity was captured by flow cytometer at day 6–14 of culture.

Figure 3

Delayed thalassemic erythroid cell differentiation during in vitro erythropoiesis. (a) Representative flow cytometry density dot plots of normal and thalassemic erythroid cells maturation during in vitro erythropoiesis. Red dots: CD71 high/GPA negative cells, blue dots: CD71 high/GPA positive cells, purple dots: CD71 moderate/GPA positive cells, pink dots: CD71 low/GPA positive cells. (b) Wright–Giemsa staining of cells harvested from day 14 culture. (c) Levels of HSP70 mRNA in erythroid cells days 8 and 10 of culture. Results from quantitative RT-PCR have been depicted from day 8 (white bar) and day 10 (black bar) of culture as relative fold change with the mean and SD value from independent subjects of each thalassemia group compared to the normal group. N: normal, H: HbH disease, HCS: HbH-Constant Spring disease, BE: β⁰-thalassemia/HbE disease, BB: homozygous β⁰-thalassemia disease.

Figure 4

Increased apoptotic cells in the homozygous β⁰-thalassemia erythroid cells during in vitro culture. (a) Representative flow cytometry density dot plots of erythroblasts derived from normal control (N), HbH disease(H), HbH-CS disease (HCS), β⁰-thalassemia/HbE disease (BE), and homozygous β⁰-thalassemia disease (BB) on days 10, 12, and 14 of cell culture. (b) Percentage of erythroid cells undergoing apoptosis (Annexin V positive and PI positive population) at days 12 (white bar) and 14 (black bar) of culture. (c) Quantitative RT-PCR analysis of transcript levels of apoptotic related genes in days 10 (black bar) and 12 (gray bar) of culture. The results were calculated as a fold change to the mean of transcript levels of cells from normal control day 10 of culture. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.