title: Neutralization of autocrine transforming growth factor-beta in human cord blood CD34(+)CD38(-)Lin(-) cells promotes stem-cell-factor-mediated erythropoietin-independent early erythroid progenitor development and reduces terminal differentiation.
abstract: Transforming growth factor (TGF)-beta1 exerts autocrine and paracrine effects on hematopoiesis. Here, we have attempted to evaluate the effect of endogenous TGF-beta1 on early erythroid development from primitive human hematopoietic stem cells (HSCs) and to assess the effects of TGF-beta1 on different phases of erythropoiesis. Cord blood CD34(+)CD38(-) lineage-marker-negative (Lin(-)) cells were cultured in serum-free conditions using various combinations of stem cell factor (SCF), erythropoietin (Epo), and TGF-beta-neutralizing antibody. Generation of erythroid progenitors was assessed using colony assay and flow cytometry. Terminal erythroid differentiation was examined when SCF/Epo-stimulated cells were recultured in the presence of Epo with and without TGF-beta1. Anti-TGF-beta augmented the proliferation of CD34(+)CD38(-)Lin(-) cells (day 21) in SCF-stimulated (6.4-fold +/- 1.5-fold) and SCF/Epo-stimulated (2.9-fold +/- 1.2-fold) cultures. Cells stimulated by SCF/Epo underwent similar levels of erythroid differentiation with and without anti-TGF-beta. While SCF alone stimulated the production of tryptase-positive mast cells, cells stimulated by SCF/anti-TGF-beta were predominantly erythroid (CD36(+)CD14(-) and glycophorin A positive). A distinct expansion of erythroid progenitors (CD34(+)CD36(+)CD14(-)) with the potential to form erythroid colonies was seen, revealing early Epo-independent erythroid development. In contrast, the kinetics of erythroid progenitor generation from primitive HSCs indicate that TGF-beta1 is not inhibitory in late erythropoiesis, but it accelerated the conversion of large BFU-E into colony-forming units-erythroid. Finally, TGF-beta1 accelerated Epo-induced terminal erythroid differentiation and resulted in a greater level of enucleation (22% +/- 6% versus 7% +/- 3%) in serum-free conditions. Serum addition stimulated enucleation (54% +/- 18%), which was lower (26% +/- 14%) with anti-TGF-beta, suggesting that optimal erythroid enucleation is Epo dependent, requiring serum factors including TGF-beta1.
authors.affiliation: Leukocyte Biology Section, Basic Research Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702-1201, USA.
authors.forename: Salem
authors.initials: S
authors.lastname: Akel
authors.forename: Cari
authors.initials: C
authors.lastname: Petrow-Sadowski
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Laughlin
authors.forename: Francis W
authors.initials: FW
authors.lastname: Ruscetti
mesh_terms: D036541:ADP-ribosyl Cyclase / Q000032:analysis* / Q000276:immunology; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D018952:Antigens, CD34 / Q000032:analysis* / Q000276:immunology; D000943:Antigens, Differentiation / Q000032:analysis; D019898:Autocrine Communication; D002454:Cell Differentiation; D019070:Cell Lineage; D015672:Erythroid Precursor Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D004920:Erythropoiesis; D004921:Erythropoietin / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology* / Q000378:metabolism; D006410:Hematopoiesis; D016298:Hematopoietic Cell Growth Factors / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D009500:Neutralization Tests; D019089:Stem Cell Factor / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D053773:Transforming Growth Factor beta1
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D016298:Hematopoietic Cell Growth Factors; D008562:Membrane Glycoproteins; D019089:Stem Cell Factor; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D004921:Erythropoietin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1