Delta-4 Notch ligand promotes erythroid differentiation of human umbilical cord blood CD34+ cells.
OBJECTIVE
Important roles of Notch signaling have been demonstrated in hematopoiesis. In many cases, activation of the Notch pathway leads to the inhibition of differentiation of immature precursors, suggesting a potential role in self-renewal promotion. However, the function of Notch and Notch ligands is not so straightforward because it is considerably dependent on cytokine context. In this study, we analyzed effects of one Notch ligand, Delta-4, whose function is less clear than others, such as Delta-1 and Jagged-1 and -2.


METHODS
CD34(+) cells isolated from human umbilical cord blood were cocultured with a Delta-4-expressing murine stromal cell line, SC9-19, and induced to erythroid differentiation by adding stem cell factor and erythropoietin. To examine the involvement of Delta-4, we utilized stromal cell subclones expressing Delta-4 protein at higher or lower level than parental SC9-19 by plasmid transfection. Erythroid maturation was examined by surface phenotype (CD34 and glycophorin A) and cytospin morphology. Recombinant human Delta-4 protein was prepared to analyze direct effects of Delta-4.


RESULTS
Under erythroid lineage-inducing conditions, we found that the increase in Delta-4 expression of SC9-19 promoted erythroid differentiation whereas the decrease in Delta-4 expression inhibited it. Morphologic examination as well as colony formation analysis supported this observation. Moreover, the experiment using recombinant Delta-4 provided direct evidence of the Delta-4 activity found in coculture system.


CONCLUSIONS
By modifying Delta-4 expression of the stromal cells and using the recombinant protein, we demonstrated that Delta-4 had a differentiation promoting activity for human primitive hematopoietic cells into erythroid lineage.
Sugimoto
Akira
A
Hayashibara Biochemical Laboratories, Inc. Okayama, Japan. asugimoto@hayashibara.co.jp
Yamamoto
Mayuko
M
Suzuki
Motoyuki
M
Inoue
Toshiya
T
Nakamura
Shuji
S
Motoda
Ryuichi
R
Yamasaki
Fumiyuki
F
Orita
Kunzo
K
D000818:Animals; D018952:Antigens, CD34; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000502:physiology*; D004920:Erythropoiesis / Q000502:physiology*; D005260:Female; D005312:Fetal Blood / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression; D006021:Glycophorins / Q000096:biosynthesis; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D000072101:Jagged-2 Protein; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction / Q000502:physiology; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology; D014162:Transfection / Q000379:methods
D018952:Antigens, CD34; D002135:Calcium-Binding Proteins; D006021:Glycophorins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C000606274:Jag1 protein, mouse; C490503:Jag2 protein, mouse; D000072100:Jagged-1 Protein; D000072101:Jagged-2 Protein; D008565:Membrane Proteins; D011994:Recombinant Proteins; D000072099:Serrate-Jagged Proteins; C064045:delta protein