Formation of mast-cell colonies in methylcellulose by mouse skin cells and development of mucosal-like mast cells from the cloned cells in the gastric mucosa of W/Wv mice.

Connective tissue-type mast cells, other than those located in the serosal cavity, are fixed in the tissues. For study of the differentiation processes of mast cells in connective tissue, an in vitro method for producing mast-cell colonies is required. The authors enzymatically dispersed the cells from the skin of either neonatal or adult mice and plated them in methylcellulose containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). More than 97% of the colonies that developed consisted of mast cells alone. The clonal nature of the mast-cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when a mixture of skin cells from C57BL/6-bgJ/bgJ and C57BL/6-+/+ mice was plated, most of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. In spite of depletion of T-cell-derived factors, concentrations of mast cells and mast cell colony-forming units (CFU-Mast) in the skin of nude athymic mice are normal. However, PWM-SCM was necessary for in vitro development of mast-cell colonies from the skin of nude mice. The concentration of CFU-Mast in the skin of genetically mast-cell-deficient WBB6F1-W/Wv mice was negligible when compared with the value observed in the skin of control WBB6F1-+/+ mice. Individual mast-cell colonies derived from the skin of neonatal WBB6F1-+/+ mice were lifted from the methylcellulose, and cells from each colony were injected into the wall of the glandular stomach and the skin of WBB6F1-W/Wv mice. Most of the mast cell that appeared at the skin injection sites of the WBB6F1-W/Wv mice stained with berberine sulfate, indicating that they contained heparin. In contrast, the mast cells that appeared in the stomach mucosa of the recipient WBB6F1-W/Wv mice did not stain. This suggests that CFU-Mast located in the skin have not been committed to the connective tissue type. The present method may be useful for investigation of the mechanisms of mast-cell differentiation in connective tissue other than the serosal cavity.