title: Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.
abstract: Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) and CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.
authors.affiliation: Laboratory of Membrane Biology.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Red Cell Physiology Laboratory, New York Blood Center, New York, NY;
authors.forename: John
authors.initials: J
authors.lastname: Hale
authors.affiliation: Red Cell Physiology Laboratory, New York Blood Center, New York, NY; Pediatric Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY;
authors.forename: Pooja
authors.initials: P
authors.lastname: Bhagia
authors.affiliation: Laboratory of Membrane Biology.
authors.forename: Fumin
authors.initials: F
authors.lastname: Xue
authors.affiliation: Laboratory of Membrane Biology.
authors.forename: Lixiang
authors.initials: L
authors.lastname: Chen
authors.affiliation: Red Cell Physiology Laboratory, New York Blood Center, New York, NY; Pediatric Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY;
authors.forename: Julie
authors.initials: J
authors.lastname: Jaffray
authors.affiliation: Red Cell Physiology Laboratory, New York Blood Center, New York, NY;
authors.forename: Hongxia
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Orthopedic Surgery, The New York Presbyterian Cornell Hospital, New York, NY;
authors.forename: Joseph
authors.initials: J
authors.lastname: Lane
authors.affiliation: Departments of Pediatrics, Pathology and Genetics, Yale University School of Medicine, New Haven, CT;
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Gallagher
authors.affiliation: Red Cell Physiology Laboratory, New York Blood Center, New York, NY;
authors.forename: Narla
authors.initials: N
authors.lastname: Mohandas
authors.affiliation: State Key Laboratory of Medical Genetics and School of Life Science, Central South University, Changsha, Hunan, China; and.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Laboratory of Membrane Biology, College of Life Science, Zhengzhou University, Zhengzhou, Henan, China.
authors.forename: Xiuli
authors.initials: X
authors.lastname: An
mesh_terms: D000943:Antigens, Differentiation / Q000096:biosynthesis*; D002469:Cell Separation / Q000379:methods; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism*; D004920:Erythropoiesis / Q000502:physiology*; D004921:Erythropoietin / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008297:Male; D059467:Transcriptome / Q000502:physiology*
chemical_list: D000943:Antigens, Differentiation; C554270:EPO protein, human; D004921:Erythropoietin