Abstract
Cross-species comparisons of hematopoietic systems will elucidate the conservation and diversity among species such as zebrafish, Xenopus, chick and mouse, which are not only of interest but different approaches would contribute to general hematology. To begin to understand their hematopoietic systems, particularly the whole animal-physiology, across non-mammalian vertebrates, we have focused on amphibian hematopoiesis. We tried to clarify the localization of definitive hematopoietic progenitors in adult Xenopus laevis, which is still to be determined. When Xenopus was induced acute hemolytic anemia by intraperitoneal phenylhydrazine (PHZ) administration, immature erythroblasts emerging in the circulation and notable increase in erythropoiesis within the liver were observed. We first screened putative hematopoietic tissues, liver, spleen, bone marrow and kidney, for erythroid progenitors using polyclonal antibodies to putative Xenopus erythropoietin receptor (xlEPOR) that we recently identified. MACS and FACS sorting and analysis revealed the existence of xlEPOR expressing cells in both liver and anemic peripheral blood. These xlEPOR positive cells were hemoglobin-positive with o-dianisidine staining, and had typical blastic morphology with high nucleus-to-cytoplasm ratio. We next developed and established an in vitro colony assay system to identify and score the hematopoietic progenitors retrospectively. The method enabled the identification and quantification of erythroid progenitors. Briefly, cells were prepared from liver, spleen, bone marrow and kidney followed by placing in semi-solid culture medium (α-MEM containing 0.8% methylcellulose, 20% FCS with appropriate hematopoietic stimulators), and cultured at 23°C with 5% CO2. The anemic serum exhibited the apparent erythropoietic stimulating activity toward the formation of remarkable number of colonies derived from anemic peripheral blood cells, resembling typical mammalian hematopoietic colony formation. Most of the colonies consisted of hemoglobin-expressing erythroids after two days culture, indicating that colony-forming units-erythroid (CFU-e) appeared in anemic blood. The normal and anemic liver also contained CFU-e, resulting in the formation of mixed and pure hematopoietic colonies. This also proved to be a useful in vitro assay system for identifying and quantifying various hematopoietic progenitors and activities of related cytokines. Figure shows the number of erythroid colonies derived from PHZ-induced anemic peripheral blood and liver stimulated with anemic serum. We furthermore examined spleen and bone marrow side-by-side, since amphibian hematopoietic system is known to unique as erythropoiesis, granulopoiesis, and thrombopoiesis occur at distinct organs. The results demonstrated the direct evidences of predominant contribution of adult liver to erythropoiesis rather than bone marrow or spleen. A new animal model developed here should provide new insights into the basis of hematopoietic regulations.
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