Development of NOG-W41-based humanized mouse model for studying In Vivo human hematopoiesis

Abstract Humanized mouse models engrafted with human CD34⁺ hematopoietic stem cells (HSCs) are essential tools for studying human hematopoiesis, immune cell development, and drug efficacy and safety evaluation in vivo. However, previous humanized mouse models, for example NOG, NSG, and BRG mice often limited engraftment efficiency, poor differentiation, and the need for preconditioning, such as irradiation. To overcome these limitations, we developed a next-generation humanized mouse model based on the NOG-W41 strain, which carries a mutation in the tyrosine kinase domain of the c-Kit receptor. This mutation enhances human hematopoietic engraftment without requiring preconditioning and enables long-term maintenance of HSCs in the bone marrow. Using the NOG-W41 strain, we created several strains introducing additional gene modification to support the development of specific human blood cell lineages. NOG-W41-hIL-3/hGM-CSF Tg (NOGW-EXL) and NOGW-EXL hIL-5 Tg mice enable enhanced differentiation of human myeloid cells such as monocytes/macrophages, mast cells, and eosinophils. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Furthermore, we established NOGW-Mpl KO (NOGWM) mice in which mouse Mpl gene, encodes the thrombopoietin receptor, is depleted. These mice supported high levels of human platelet production, over 50% of circulating platelets in peripheral blood, even when a relatively small number of CD34⁺ cells were transplanted.Overall, NOG-W41-based humanized mice represent a powerful and versatile platform for in vivo studies of human hematopoiesis. Their high engraftment efficiency, ability to support multiple blood lineages, and simplified transplantation procedures make them promising tools for both basic research and translational applications, including disease modeling, immunotherapy development, and drug testing in humanized systems.