Demonstration of T cell and macrophage progenitors in carp (Cyprinus carpio) kidney hematopoietic tissues. Development of clonal assay system for carp hematopoietic cells

Single hematopoietic cells from carp (Cyprinus carpio) kidney were seeded to each well of 96-well plates and cultured in the presence of a supporting cell layer and conditioned media (CM). The CM were obtained from bulk-cultured carp hematopoietic cells, in which T and macrophage-lineage cells rapidly proliferated as previously reported. After 2–3 weeks, colony formation was found in 0–4 wells of each plate. Three different morphological types of colonies were observed: “type I colonies”, “type II colonies” and “mixed-type colonies”. Type I colony cells were interpreted as composed by macrophage-lineage cells, since they expressed a specific macrophage marker, M-CSFR/csf1r gene, and most of them phagocytosed latex particles. Type II colony cells were interpreted as composed by T lineage cells, since they expressed several T cell marker genes including gata3, lck and TCRβ, but did not engulf latex particles. Mixed-type colonies were interpreted as composed by both macrophages and T lineage cells. They expressed not only the M-CSFR gene but also a T cell marker gene, gata3, but not other T cell markers, such as lck and TCRβ. These results indicated that the mixed-type colonies were developed from immature common progenitors of macrophage and T cell. In contrast, type I and type II colonies were developed from more mature and mono-potent progenitors of macrophage and T cell, respectively.