Stromal cell-independent differentiation of human cord blood CD34+CD38- lymphohematopoietic progenitors toward B cell lineage.
To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.
Yoshikawa
Y
Y
Hokkaido Red Cross Blood Center, Sapporo, Japan.
Hirayama
F
F
Kanai
M
M
Nakajo
S
S
Ohkawara
J
J
Fujihara
M
M
Yamaguchi
M
M
Sato
N
N
Kasai
M
M
Sekiguchi
S
S
Ikebuchi
K
K
D018952:Antigens, CD34 / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology*; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D005312:Fetal Blood / Q000166:cytology*; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D007231:Infant, Newborn; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology
D018952:Antigens, CD34; D015415:Biomarkers; D007148:Immunoglobulin mu-Chains; D007377:Interleukin-3; D015850:Interleukin-6; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor