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PLoS One. 2013 Jul 8;8(7):e68307. doi: 10.1371/journal.pone.0068307. Print 2013.

A synthetic model of human beta-thalassemia erythropoiesis using CD34+ cells from healthy adult donors.

Author information

1
Molecular Genomics and Therapeutics Section, Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Abstract

Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14-21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.

PMID:
23861885
PMCID:
PMC3704632
DOI:
10.1371/journal.pone.0068307
[Indexed for MEDLINE]
Free PMC Article

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