Establishment of a sticky, large, oval-shaped thrombocyte cell line from tree frog as an ancestor of mammalian megakaryocytes.

Maintenance of blood vessels is important for homeostasis. Many types of cells and cytokines are involved in angiogenesis and blood vessel repair. In mammals, platelets, which are produced from megakaryocytes, play a major role in hemostasis. Other vertebrates have no platelets in their bloodstream. In these animals, thrombocytes aggregate to form a thrombus. Therefore, I established a frog hematopoietic cell line to elucidate the mechanism of hematopoiesis in this species. The frog-derived thrombocytic cell line was established from a long-term bone marrow culture of Hyla japonica and was designated as a frog-derived unique hematopoietic non-adherent (FUHEN) cell line. The FUHEN cells had unique characteristics in that they proliferated in suspension culture without adherence to the culture flask, and the shapes of the FUHEN cells changed drastically to become very large ovals with growth. These cells reached more than 40 µm in length and had multi-lobed nuclei. The FUHEN cells expressed CD41, a specific surface marker of thrombocytes. These results indicated that the FUHEN cells were thrombocytes. Deprivation of divalent ions quickly induced adherence of the cells to the petri dish. This characteristic may be important for hemostasis. Furthermore, some of the FUHEN cells survived at 16 °C for 1 month and re-established proliferation when the cells were moved to 28 °C. Taken together, this new thrombocytic frog cell line, as an ancestor of mammalian megakaryocytes, could provide useful material to study the functions of thrombocytes and the hemostasis mechanism of amphibians.