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Blood. 2014 Dec 4;124(24):3636-45. doi: 10.1182/blood-2014-07-588806. Epub 2014 Oct 22.

Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.

Author information

1
Laboratory of Membrane Biology.
2
Red Cell Physiology Laboratory, New York Blood Center, New York, NY;
3
Red Cell Physiology Laboratory, New York Blood Center, New York, NY; Pediatric Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY;
4
Department of Orthopedic Surgery, The New York Presbyterian Cornell Hospital, New York, NY;
5
Departments of Pediatrics, Pathology and Genetics, Yale University School of Medicine, New Haven, CT;
6
State Key Laboratory of Medical Genetics and School of Life Science, Central South University, Changsha, Hunan, China; and.
7
Laboratory of Membrane Biology, College of Life Science, Zhengzhou University, Zhengzhou, Henan, China.

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.

PMID:
25339359
PMCID:
PMC4256913
DOI:
10.1182/blood-2014-07-588806
[Indexed for MEDLINE]
Free PMC Article
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