LONG-TERM GENERATION OF HUMAN MAST-CELLS IN SERUM-FREE CULTURES OF CD34(-BLOOD CELLS STIMULATED WITH STEM-CELL FACTOR AND INTERLEUKIN-3() CORD)

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 gro wth factor produced by murine fibroblasts and required to observe diff erentiation of human mast cells is attributable in part to stem cell f actor (SCF). However, other factors are likely involved. We have previ ously shown that the combination of sep and interleukin-3 (IL-3) effic iently sustains proliferation and differentiation of colony-forming ce lls (CFCs) from pre-CFC enriched from human umbilical cord blood by CD 34(+) selection. With periodic medium changes and the addition of fres h growth factors, five consecutive cultures of different cord blood sa mples gave rise to differentiated cells and CFCs for more than 2 month s. Although differentiated cells continued to be generated far more th an 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue p ositive, are karyotypically normal, are CD33(+), CD34(-), CD45(+), c-k it(-), and c-fms(-), and die in the absence of either SCF or IL-3. The se cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentrat ion of no less than 10(4) cells/mL. At refeedings, the cultures contai n 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 d ie 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 I L-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lin es. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia. (C) 1994 by The American Society of Hematology.