In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii)

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho^-) cells with that of SP cells by ginbuna transplantation experiments. Rho^- cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho^- cells was lower than that of SP cells, while Rho^- cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho^- cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.