Development of a New Experimental Animal Model of Human Megakaryopoiesis Using Immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) Mice. (2005)

Abstract [Background] We developed novel small molecules with thrombopoietin (TPO) mimetic activities in vitro. These small molecules induced colony formation of megakaryocytes from human bone marrow cells but not murine and cynomolgus monkey cells, indicating strict species specificity. However, the lack of experimental animal models for human megakaryopoiesis hampered evaluation of whether these molecules can increase the number of human platelets in vivo. To solve this problem, we developed a new experimental animal model of human megakaryopoiesis using immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice. [Aims] We developed a new experimental animal model of human megakaryopoiesis using NOG mice to evaluate the in vivo efficacies of the novel human-specific TPO mimetics. We also compared the potentials of cord blood (CB), bone marrow (BM), and peripheral blood (PB)-derived CD34 positive cells at maintaining human megakaryopoiesis in NOG mice. [Methods] After 2.4-Gy X-ray irradiation, 1x105 CD34-positive hematopoietic cells from human CB, BM and PB were intravenously injected into NOG mice. The engraftment of human megakaryocytes and platelets in NOG mice was analyzed by flow cytometry and immunohistochemistry with human-specific antibodies for up to 6 months. [Results] Four weeks after transplantation, we found that around 1% of platelets in these mice were positive for human CD41 antigen (fibrinogen receptor) (n=8). We also confirmed 30–40% of megakaryocytes in the murine bone marrow were positive for human-specific megakaryocyte markers by immunohistochemistry. The percentage of human platelets in the murine blood was maintained at around 2% for 6 months after transplantation with human CD34-positive CB cells. In contrast, the engraftment of human platelets was transient and almost disappeared after 3 months, when CD34-positive cells from human BM and PB were injected into NOG mice. Interestingly, we found around 20 and 40% of murine bone marrow cells were positive for human CD45 three months after transplantation with PB- and BM-derived CD34-positive cells, respectively (n=7). These results indicated that CB cells have higher potencies to maintain human megakaryopoiesis than BM and PB cells in NOG mice. As the percentage of human platelets was much lower than that of human megakaryocytes in NOG mice transplanted with human CB cells, we speculated that the clearance of human platelets, which are larger than murine platelets, might be high or that the murine bone marrow microenvironment lacks factors related to platelet release from human megakaryocytes. We also analyzed the function of human platelets in NOG mice. ADP induced the activation of alpha IIb beta IIIa (fibrinogen receptor) and the expression of CD62P on human platelets in NOG mice in vitro, suggesting that human platelets produced from human CB-derived CD34 positive cells in NOG mice, are functional. [Conclusions] We succeeded to develop a new experimental animal model of human megakaryopoiesis using NOG mice. CB was the most suitable donor for maintaining human matured megakaryocytes and functional platelets in NOG mice. Currently, we are using this model to evaluate in vivo efficacies of novel human-specific TPO mimetics.