Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin- 3

Abstract

The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony- forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL- 3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (= 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor- dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.

Ovarian fibrosarcoma producing multiple cytokines

“…Human mast cell proliferation and differentiation are regulated by cytokines such as IL-3, IL-4, IL-6 and stem cell factor. 19,20 In this study, by RT-PCR we demonstrated expression of IL-6 as well as stem cell factor, but failed to demonstrate IL-3 and IL-4. The production of these factors may promote proliferation and maturation of mast cell precursors in bone marrow.…”

Role of Thrombopoietin in Mast Cell Differentiation

“…A further contribution to the identification of the extrinsic pathway has been provided by the definition of culture conditions necessary to observe mast cell differentiation in vitro. [13][14][15][16] The extrinsic regulatory pathway includes the growth factors stem cell factor (SCF) and interleukin-3 (IL-3). 15,16 In fact, marrow (and spleen) hematopoietic progenitors generate, within 14 days of culture stimulated with optimal concentrations of SCF and IL-3, cells with the phenotypical (c-Kit high CD34 low Fc⑀RI pos cells with small alcian blue neg cytoplasmic granules), 17 and functional (reconstitute connective and mucosal mast cells when transplanted into mast cell-deficient animals) 18 profile of mast cell precursors (see also FIG.…”

“…Instrumental to the clarification of these pathways has been the analyses of the phenotype of mice harboring specific gene mutations. A further contribution to the identification of the extrinsic pathway has been provided by the definition of culture conditions necessary to observe mast cell differentiation in vitro 13–16 …”

The role of mast cells in atopy: what can we learn from canine models? A thorough review of the biology of mast cells in canine and human systems

“…[46][47][48][49][50][51][52] In addition, studies of human and animal mast cell cultures have made significant contributions to our understanding of the pathogenesis of atopic diseases. [53][54][55][56][57] Although the guinea pig, 58,59 the monkey 60 and the sheep 61,62 have also provided valuable information, their use is limited for a variety of reasons.…”