Fibroblast‐dependent growth of mouse mast cells in vitro: Duplication of mast cell depletion in mutant mice of w/w^v genotype

Abstract

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.

W mutant mice with mild or severe developmental defects contain distinct point mutations in the kinase domain of the c-kit receptor.

“…An alternate mode of mast cell growth has been described (Fujita et al 1988b) that has no requirement for exogenous growth factors but is dependent on contact with embryonic fibroblasts. W/W v mast cell cultures show normal proliferation in suspension culture but are deficient in fibroblast-dependent growth, a result of a defect in transition from G1 to S phase of the cell cycle (Fujita et al 1988a).…”

Distinct role for c-kit receptor tyrosine kinase and SgIGSF adhesion molecule in attachment of mast cells to fibroblasts

“…12 When cultured mast cells (CMCs) were cocultured on a monolayer of NIH/3T3 fibroblasts, CMCs derived from wild-type ( þ / þ ) mice proliferated without any specific growth factors. 13,14 However, when þ / þ CMCs were prevented from being in contact with NIH/3T3 fibroblasts, they gradually disappeared from the coculture. 13 In this coculture, adhesion of CMCs to NIH/3T3 fibroblasts appeared to be important for CMCs to proliferate, probably because the adhesion allowed KIT on CMCs to bind to membrane-bound SCF expressing on NIH/3T3 fibroblasts.…”

“…13,14 However, when þ / þ CMCs were prevented from being in contact with NIH/3T3 fibroblasts, they gradually disappeared from the coculture. 13 In this coculture, adhesion of CMCs to NIH/3T3 fibroblasts appeared to be important for CMCs to proliferate, probably because the adhesion allowed KIT on CMCs to bind to membrane-bound SCF expressing on NIH/3T3 fibroblasts.…”

In vitro duplication and in vivo cure of mast-cell deficiency of Sl/Sld mutant mice by cloned 3T3 fibroblasts.

“…Therefore, those cultured for >4 wk were used in these experiments. The NIH 3T3 fibroblast cell line derived from a Swiss mouse embryo (14) or other fibroblast cell lines were cocultured with mast cells in the absence of PWM-SCM as described (11,12). Briefly, 105 cultured mast cells suspended in 2 ml of medium containing 5% fetal calf serum but not PWM-SCM were added to a confluent culture Abbreviations: IL, interleukin; PWM-SCM, pokeweed mitogenstimulated spleen cell-conditioned medium; BrdUrd, 5-bromodeoxyuridine.…”