title: A synthetic model of human beta-thalassemia erythropoiesis using CD34+ cells from healthy adult donors.
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.
authors.affiliation: Molecular Genomics and Therapeutics Section, Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Y Terry
authors.initials: YT
authors.lastname: Lee
authors.forename: Ki Soon
authors.initials: KS
authors.lastname: Kim
authors.forename: Colleen
authors.initials: C
authors.lastname: Byrnes
authors.forename: Jaira F
authors.initials: JF
authors.lastname: de Vasconcellos
authors.forename: Seung-Jae
authors.initials: SJ
authors.lastname: Noh
authors.forename: Antoinette
authors.initials: A
authors.lastname: Rabel
authors.forename: Emily R
authors.initials: ER
authors.lastname: Meier
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Miller
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D001782:Blood Donors; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D004920:Erythropoiesis; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006262:Health; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D017086:beta-Thalassemia / Q000235:genetics / Q000473:pathology*
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D006454:Hemoglobins; D012333:RNA, Messenger