Regulation of human B lymphopoiesis by the transforming growth factor-beta superfamily in a newly established coculture system using human mesenchymal stem cells as a supportive microenvironment.
OBJECTIVE
To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology.


METHODS
We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development.


RESULTS
Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells.


CONCLUSIONS
hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.
Ichii
Michiko
M
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan.
Oritani
Kenji
K
Yokota
Takafumi
T
Nishida
Makoto
M
Takahashi
Isao
I
Shirogane
Takahiro
T
Ezoe
Sachiko
S
Saitoh
Norimitsu
N
Tanigawa
Rie
R
Kincade
Paul W
PW
Kanakura
Yuzuru
Y
D028341:Activins / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D018952:Antigens, CD34 / Q000187:drug effects / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D018920:Coculture Techniques / Q000379:methods; D004353:Drug Evaluation, Preclinical; D005434:Flow Cytometry; D006801:Humans; D038041:Lymphopoiesis / Q000187:drug effects / Q000276:immunology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D008970:Molecular Weight; D015260:Neprilysin / Q000096:biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology; D012016:Reference Values; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000906:Antibodies; D018952:Antigens, CD34; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C422405:activin A; D028341:Activins; D015260:Neprilysin