Both human and mouse c-kit ligand induced differentiation of human mast cells in a long-term culture ofthe mononuclear cells of umbilical cord blood. Growth factor activity for human mast cells present in conditioned medium of BALB/3T3 fibroblasts was due to mouse c-kit ligand. Recombinant c-kit ligand induced differentiation and proliferation of mast cell progenitors in early stages of culture. However, apparent selective growth of mast cells by c-kit ligand in cord blood cell cultures is mainly due to the effect of the cytokine to selectively maintain survival of immature mast cells. Electron microscopic analysis indicated that human mast cells developed by c-kit ligand were similar to human mast cells in the lung and gut mucosa, while those developed in coculture of cord blood cells with Swiss albino/3T3 fibroblasts were similar to skin mast cells. This conclusion was supported by the fact that the majority of mast cells developed by c-kit ligand contained only tryptase in their granules, whereas those developed in the cocultures contained both tryptase and chymase. It was also found that mast cells developed by c-kit ligand were immature even after culture for 14 weeks. Nevertheless, these cells express FceRI, and could be sensitized with human IgE for anti-IgE-induced release of histamine, prostaglandin D2, and leukotriene C4.Several murine cytokines, such as interleukin (IL)-3, IL4, IL-9, and IL-10 promote differentiation and proliferation of mouse mast cells (1-4). In contrast, reproducible growth of human mature mast cells had not been achieved until 1989, when we succeeded in developing morphologically and functionally mature human mast cells in a long-term coculture of mononuclear cells of cord blood with Swiss albino/3T3 fibroblasts (5). Subsequent studies revealed that the culture supernatant of 3T3 fibroblasts contained growth factors that promote differentiation of human mast cells (6). Human mast cell growth-promoting activity could be enriched by fractionation of the culture supernatant with ammonium sulfate precipitation and by ion-exchange chromatography. The molecular size of the factor was estimated by gel filtration to be between 70 and 100 kDa (7).While our studies were in progress, a cytokine, c-kit ligand, was characterized and molecular cloning of the cytokine has been accomplished by several groups of investigators (8)(9)(10)(11)(12). The present experiments show that both human and murine c-kit ligand induce differentiation of cord blood cells to human mast cells in culture and provide evidence that human mast cell growth-promoting activity in culture supernatants of 3T3 fibroblasts is associated with murine c-kit ligand. MATERIALS AND METHODSCell Cutures and Microscopic Examination. Mononuclear cells were obtained from heparinized umbilical cord blood (5) and suspended in RPMI 1640 medium (GIBCO) supplemented with 10%o fetal bovine serum (FBS; GIBCO), 50 ,M 2-mercaptoethanol, 2 mM L-glutamine, 100 units of penicillin per ml, 50 ,ug of streptomycin per ml, and 25 ,ug of gentami...

Effects of recombinant human interleukins on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical-cord blood and bone marrow. The results showed that interleukin 5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with interleukin 5, essentially all nonadherent cells in both bone marrow and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with interleukin 4 resulted in the selective growth of OKT3' lymphocytes. However, OKT3' cells did not develop if the bone marrow cells were depleted of OKT3'/OKT11+ cells prior to the culture, indicating that interleukin 4 induced the proliferation of a subpopulation of resting T cells present in cord blood and bone marrow cell preparations. In suspension cultures of bone marrow cells and cord blood cells grown in the presence of interleukin 3, basophilic, eosinophilic, and neutrophilic myelocytes and macrophages developed within 2 weeks. By 3 weeks, however, the majority of nonadherent cells became eosinophilic myelocytes. In contrast to mouse bone marrow cell cultures, neither interleukin 3 nor a combination of interleukins 3 and 4 induced the differentiation of mast cells in human bone marrow or cord blood cell cultures.

Nucleated cells of human umbilical cord blood were cocultured with mouse skin-derived 3T3 fibroblasts. After 7-8 weeks in culture, when the number of the other hematopoietic cells declined, metachromatic granulecontaining mononuclear cells appeared in the cultures, and the number of the cells increased up to 12 weeks. After 11-14 weeks in culture, the metachromatic mononuclear cells comprised a substantial portion of the cultured cells. These cells contained 1.8-2 jg of histamine per 106 cells and bore receptors for IgE. All of the cells contained tryptase in their granules. Electron microscopic analysis showed that these cells were mature human mast cells, clearly different from the basophilic granulocytes or eosinophils that arise in a variety of circumstances in cord blood cell cultures. Most of the cultured mast cells expressed some granules with regular crystalline arrays and contained both tryptase and chymase, and thus resembled human skin mast cells.In the murine system, recombinant interleukin 3 (IL-3) promotes the proliferation of mouse mast cell-line cells (1) and facilitates the formation of mast cell colonies in semisolid cultures of mouse bone marrow (BM) cells (2). Suspension culture of murine BM cells with recombinant IL-3 results in the development of a pure mast cell population (3). These IL-3-dependent, BM-derived mast cells appear to be analogous to mast cells in the gastrointestinal mucosa and different from mast cells in connective tissue (4). However, LeviSchaffer et al. (5) as well as Dayton et al. (6) demonstrated that coculture of the BM-derived, IL-3-dependent mast cells with 3T3 fibroblasts in the presence of IL-3-containing conditioned medium resulted in a phenotype change to those resembling connective-tissue mast cells.In contrast to the murine mast cell system, requirements for the differentiation and proliferation of human mast cells are unknown. In the past 10 years, numerous attempts were made to develop human basophils and mast cells in vitro. Several groups, including ours, demonstrated in vitro development of human basophils from progenitors in BM, umbilical cord blood, and fetal liver (7-10). However, reproducible success in in vitro development of mature human mast cells has not been achieved. Recombinant human IL-3 promoted the differentiation of human basophils from precursors in BM and cord blood cells, but neither IL-3 nor IL-4 induced the differentiation of human mast cells (11-13). We anticipated that a feeder layer or cytokines from non-T cells might facilitate the differentiation of human mast cells in vitro. In the present experiments, nucleated cells from cord blood were cocultured with various human and murine fibroblasts. Prolonged coculture of cord blood cells with mouse 3T3 fibroblasts resulted in the development of mature human mast cells in vitro. MATERIALS AND METHODSCell Cultures. Total nucleated cells were recovered from heparinized umbilical cord blood by differential centrifugation in 73-75% Percoll (Pharmacia) at 300 x g for 20 min at room te...