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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1237-7) contains supplementary material, which is available to authorized users.

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates

“…Interestingly, a cell line derived from blood cells of the hibernating (hypoxic conditions) tree frog recently demonstrated that thrombocytes develop polyploidy and megakaryocyte-like features. 181 This finding supports the notion that the hypoxic conditions of the Triassic period contributed to the evolution of megakaryocytes.…”

Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration

“…In contrast, the blood of non‐mammals, including the fish and amphibian species widely used for studies of organ regeneration, contains no platelets, but rather larger nucleated thrombocytes. These cells usually derive from hematopoietic tissues other than bone marrow but act much like platelets after an injury, undergoing degranulation with blood coagulation and releasing bioactive factors which convert extravasated plasma into serum, setting the stage for inflammation and tissue repair (Sugimoto, ).…”

Amphibian thrombocyte‐derived extracellular vesicles, including microRNAs, induce angiogenesis‐related genes in endothelial cells

“…Higher levels of cd41 expression were detected in the bone marrow, spleen and peripheral blood of treefrogs than in the lung, liver, kidney, muscle and heart (Figure 1c). As FUHEN cells were established from long‐term bone marrow culture (LTBMC) of treefrogs (Sugimoto, 2015), high expression of cd41 in treefrog bone marrow was reasonable. Human bone marrow is also reported to be the major production tissue for Mks (Pallotta et al., 2009).…”

“…Maintenance of the FUHEN cell line was described previously (Sugimoto, 2015). Briefly, 5 × 10 5 cells were seeded in 8 ml of alpha‐MEM supplemented with 10% HS in a culture flask (true line, TR6000, 25 cm 2 ).…”

“…Recently, we established an amphibian‐derived thrombocyte cell line named FUHEN from Treefrog ( Hyla japonica ; Sugimoto, 2015). FUHEN cells have polyploid nuclei and express a homologue of Xenopus cd41 , which is a specific marker of megakaryocytes and platelets.…”