Abstract

Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells.

Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7.

Results:

  1. Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential.

  2. For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them.

  3. When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis.

  4. Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week.

CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character.

Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.

Author notes

Disclosure: No relevant conflicts of interest to declare.