Previous studies reported that the introduction

Previous studies reported that the introduction of KitW41 or KitWv mutations into BALB-RG or NSG mice could render mouse HSCs uncompetitive to donor human HSCs (Cosgun et al., 2014; McIntosh et al., 2015). They also showed that human HSPCs injected at a high dose (5 × 104 to 3 × 105 CD34+ CB cells), without irradiation preconditioning, could outcompete mouse HSCs for engraftment and differentiate into myeloerythroid cells. This is probably because the introduction of loss-of-function Kit mutations might render host HSCs uncompetitive in occupying HSC niches. The BRGSKWv/Wv mouse also reconstituted human hematopoiesis, without irradiation, after a high dose of human HSPC transplantation (data not shown). However, when these mice were irradiated, only a small number (3.5–5 × 103 cells) of human CD34+CD38− CB folate analogue was sufficient to achieve a high level of human cell chimerism of erythroid and megakaryocyte lineage cells. Thus, the introduction of homozygous KitWv mutations into mice with severe combined immunodeficiency, in combination with irradiation preconditioning, allows the robust reconstitution of human erythropoiesis and thrombopoiesis with terminal maturation. Mice with Kit mutations display macrocytic anemia (Nocka et al., 1990) and impairment of megakaryopoiesis and myelopoiesis (Chervenick and Boggs, 1969; Ebbe et al., 1973) as the major hematopoietic phenotypes. These changes might be explained by the decreased affinity between HSPCs and their niches by attenuated KIT signaling. In this context, Kit mutations might help open the putative “myeloid,” “erythroid,” or “megakaryocyte” niches, resulting in the ample reconstitution of human myeloid, erythroid, and megakaryocyte cell lineages in the bone marrow. Indeed, robust human erythropoiesis occurred in the sternal bone marrow of BRGSKWv/Wv mice, where human erythroid cells colonized, forming “islands” independently of mouse erythroid cells. This suggested that these cells migrated and competed for mouse “erythroid” niches in the bone marrow. However, the level of human CD235a+ or CD41+ cell chimerism was lower than that of human CD45+ cell chimerism, even in BRGSKWv/Wv mice. In addition, maturation of human erythroid cells was imperfect, as the number of CD71−CD235a+ mature erythrocytes was limited. Human erythroid reconstitution was not improved further by injecting recombinant human erythropoietin into BRGSKWv/Wv mice (data not shown). Therefore, some other unknown determinants, critical for the faithful regeneration of human hematopoiesis, may still be lacking in the bone marrow microenvironment of BRGSKWv/Wv mice. We did not observe circulating erythrocytes or platelets, even in BRGSKWv/Wv mice with a very high chimerism of human hematopoiesis. In the bone marrow, we detected cells at every stage of human erythroid differentiation, including mature enucleated erythrocytes that synthesized hemoglobin, and mature human megakaryocytes with multiple nuclei. Of note, nearly 50% of platelets in the bone marrow were of human origin. Furthermore, elimination of phagocytic cells by clodronate successfully released a fraction of mature erythrocytes and platelets into the circulation. Thus, the final developmental step, migration from the bone marrow to the circulation and sustainment in the circulation, might not yet be “humanized” even in BRGSKWv/Wv mice harboring NOD-Sirpa. It is possible that the xenogeneic activation of mouse macrophages to engulf human cells does not depend solely on the SIRPA-CD47 interaction, and that other unknown mechanisms of macrophage activation against human cells exist. Clodronate administration, however, was unable to deplete all host macrophages, because a considerable number of residual macrophages were present, at least in the spleen (data not shown). Macrophages reside throughout the body, including around blood vessels in the bone marrow niches (Ehninger and Trumpp, 2011). New strategies to completely inhibit mouse macrophage activity against human cells should be developed to establish a more faithful humanized model with circulating mature human blood cells.