title: Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death.
abstract: The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.
authors.affiliation: Cell Biology and Neuroscience, Istituto Superiore di Sanita', Rome, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
authors.forename: Francesca
authors.initials: F
authors.lastname: Masiello
authors.forename: Valentina
authors.initials: V
authors.lastname: Tirelli
authors.forename: Massimo
authors.initials: M
authors.lastname: Sanchez
authors.forename: Lilian
authors.initials: L
authors.lastname: Varricchio
authors.forename: Carolyn
authors.initials: C
authors.lastname: Whitsett
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Migliaccio
mesh_terms: D000740:Anemia / Q000473:pathology; D017209:Apoptosis / Q000502:physiology; D001343:Autophagy / Q000502:physiology*; D001803:Blood Transfusion / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D004900:Erythroblasts / Q000166:cytology*; D004912:Erythrocytes / Q000166:cytology; D004920:Erythropoiesis / Q000502:physiology*; D005938:Glucocorticoids / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping
chemical_list: D005938:Glucocorticoids; D006133:Growth Substances