Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.
Blood
; 124(24): 3636-45, 2014 Dec 04.
Article
em En
| MEDLINE
| ID: mdl-25339359
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 â¼90%. 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Antígenos de Diferenciação
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Células Precursoras Eritroides
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Regulação da Expressão Gênica
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Eritropoetina
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Eritropoese
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Transcriptoma
Limite:
Female
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Humans
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Male
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article