Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse.

The transplantation of human mesenchymal stromal/stem cells (hMSCs) has potential as a curative and permanent therapy for congenital skeletal diseases. However, the self-renewal and differentiation capacities of hMSCs markedly vary. Therefore, cell proliferation and trilineage differentiation capacities were tested in vitro to characterize hMSCs before their clinical use. However, it remains unclear whether the ability of hMSCs in vitro accurately predicts that in living animals. The xenograft model is an alternative method for validating clinical MSCs. Nevertheless, the protocol still needs refinement, and it has yet to be established whether hMSCs, which are expanded in culture for clinical use, retain the ability to engraft and differentiate into adipogenic, osteogenic, and chondrogenic lineage cells in transplantation settings. In the present study, to establish a robust xenograft model of MSCs, we examined the delivery routes of hMSCs and the immunological state of recipients. The intra-arterial injection of hMSCs into X-ray-irradiated (IR) NOG, a severely immunodeficient mouse, achieved the highest engraftment but failed to sustain long-term engraftment. We demonstrated that graft cells localized to a collagenase-released fraction (CR), in which endogenous colony-forming cells reside. We also showed that Pdgfrα+Sca1+ MSCs (PαS), which reside in the CR fraction, resisted IR. These results show that our protocol enables hMSCs to fulfill a high level of engraftment in mouse bone marrow in the short term. In contrast, long-term reconstitution was restricted, at least partially, because of IR-resistant endogenous MSCs.

PBMC-engrafted humanized mice models for evaluating immune-related and anticancer drug delivery systems.

Immune-related drug delivery systems (DDSs) in humanized mouse models are at the forefront of cancer research and serve as bridges between preclinical studies and clinical applications. These systems offer unique platforms for exploring new therapies and understanding their interactions with human cells and the immune system. Here, we focus on a DDS and a peripheral blood mononuclear cell (PBMC)-engrafted humanized mouse model that we recently developed, and consider some of the key components, challenges, and applications to advance these systems towards better cancer treatment on the basis of a better understanding of the immune response. Our DDS is unique and has a dual function, an anticancer effect and a capacity to fine-tune the immune reaction. The PBL-NOG-hIL-4-Tg mouse system is superior to other available humanized mouse systems for the development of such multifunctional DDSs because it supports the rapid reconstruction of an individual donor's immunity and avoids the onset of graft-versus-host disease.

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

Ileal Crohn's Disease Exhibits Reduced Activity of Phospholipase C-ß3-Dependent Wnt/ß-Catenin Signaling Pathway.

Crohn's disease is a chronic, debilitating, inflammatory bowel disease. Here, we report a critical role of phospholipase C-ß3 (PLC-ß3) in intestinal homeostasis. In PLC-ß3-deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-ß3 deficiency in multiple non-hematopoietic cell types. PLC-ß3 deficiency resulted in reduced Wnt/ß-catenin signaling, which is essential for homeostasis and the regeneration of the intestinal epithelium. PLC-ß3 regulated the Wnt/ß-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and, potentially, protein-protein interaction levels. PLC-ß3-deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/ß-catenin signaling. Reduced expression of PLC-ß3 and its signature genes was found in biopsies of patients with ileal Crohn's disease. PLC-ß regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that a reduction in PLC-ß3-mediated Wnt/ß-catenin signaling contributes to the pathogenesis of ileal Crohn's disease.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. 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. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice.

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Liposome-encapsulated progesterone efficiently suppresses B-lineage cell proliferation.

Progesterone suppresses several ancient pathways in a concentration-dependent manner. Based on these characteristics, progesterone is considered a candidate anticancer drug. However, the concentration of progesterone used for therapy should be higher than the physiological concentration, which makes it difficult to develop progesterone-based anticancer drugs. We previously developed liposome-encapsulated progesterone (Lipo-P4) with enhanced anticancer effects, which strongly suppressed triple-negative breast cancer cell proliferation in humanized mice. In this study, we aimed to clarify whether Lipo-P4 effectively suppresses the proliferation of B-lineage cancer cells. We selected six B-cell lymphoma and two myeloma cell lines, and analyzed their surface markers using flow cytometry. Next, we prepared liposome-encapsulated progesterone and examined its effect on cell proliferation in these B-lineage cancer cells, three ovarian clear cell carcinoma cell lines, two prostate carcinoma cell lines, and one triple-negative breast cancer adenocarcinoma cell line. Lipo-P4 suppressed the proliferation of all cancer cell lines. All B-lineage cell lines, except for the HT line, were more susceptible than the other cell types, regardless of the expression of differentiation markers. Empty liposomes did not suppress cell proliferation. These results suggest that progesterone encapsulated in liposomes efficiently inhibits the proliferation of B-lineage cells and may become an anticancer drug candidate for B-lineage cancers.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

Exophiala dermatitidis coinfection with nontuberculous mycobacteria: A case report and literature review.

Six years ago, a 60-year-old man presented to our hospital with a cough and sputum. Upon suspicion of nontuberculous mycobacterial (NTM) infection, he was followed up at our hospital. Because the abnormal shadows in the bilateral lung fields deteriorated slightly over 6 years, bronchoscopy was performed. Exophiala dermatitidis and Mycobacterium intracellulare were detected in the bronchial lavage fluid. The patient underwent follow-up examinations without drug administration. Currently, the patient's condition remains stable. E. dermatitidis is regulatory found in the lungs of patients with cystic fibrosis, but only rarely is it found in respiratory samples from patients without cystic fibrosis. However, NTM complications have been reported more frequently in recent years. Due to the increasing number of NTM patients, E. dermatitidis pulmonary infections may also increase. Additional research is required to develop strategies for treating this infection.

Construction of the systemic anticancer immune environment in tumour-bearing humanized mouse by using liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated progesterone.

Immune checkpoint inhibitors highlight the importance of anticancer immunity. However, their clinical utility and safety are limited by the low response rates and adverse effects. We focused on progesterone (P4), a hormone produced by the placenta during pregnancy, because it has multiple biological activities related to anticancer and immune regulation effects. P4 has a reversible immune regulatory function distinct from that of the stress hormone cortisol, which may drive irreversible immune suppression that promotes T cell exhaustion and apoptosis in patients with cancer. Because the anticancer effect of P4 is induced at higher than physiological concentrations, we aimed to develop a new anticancer drug by encapsulating P4 in liposomes. In this study, we prepared liposome-encapsulated anti-programmed death ligand 1 (PD-L1) antibody-conjugated P4 (Lipo-anti-PD-L1-P4) and evaluated the effects on the growth of MDA-MB-231 cells, a PD-L1-expressing triple-negative breast cancer cell line, in vitro and in NOG-hIL-4-Tg mice transplanted with human peripheral blood mononuclear cells (humanized mice). Lipo-anti-PD-L1-P4 at physiological concentrations reduced T cell exhaustion and proliferation of MDA-MB-231 in vitro. Humanized mice bearing MDA-MB-231 cells expressing PD-L1 showed suppressed tumor growth and peripheral tissue inflammation. The proportion of B cells and CD4+ T cells decreased, whereas the proportion of CD8+ T cells increased in Lipo-anti-PD-L1-P4-administrated mice spleens and tumor-infiltrated lymphocytes. Our results suggested that Lipo-anti-PD-L1-P4 establishes a systemic anticancer immune environment with minimal toxicity. Thus, the use of P4 as an anticancer drug may represent a new strategy for cancer treatment.

UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease.

UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.

Establishment of a human microbiome- and immune system-reconstituted dual-humanized mouse model.

Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγnull (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34+ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3+ T cells than the SPF humanized mice. Additionally, we found that the human CD3+ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

High-progesterone environment preserves T cell competency by evading glucocorticoid effects on immune regulation.

Progesterone (P4) and glucocorticoid (GC) play crucial roles in the immunoregulation of a mother to accept and maintain a semi-allogenic fetus. P4 concentration increases during pregnancy and becomes much higher in the placenta than in the other peripheral tissues, wherein the concentration of cortisol (COR), the most abundant GC and a strong immunosuppressor, remains uniform throughout the rest of the body. Here, we evaluated the effect of a high-P4 environment on pregnant immunity by comparing it with COR. Naïve T cell proportion increased transiently in peripheral blood of pregnant women just after delivery and decreased after one month. T cells stimulated with superantigen toxic-shock-syndrome-1 (TSST-1) in the presence of P4 stayed in the naïve state and did not increase, irrespective of the presence of COR, and reactive T cells could not survive. Treatment of T cells with P4 without T cell receptor (TCR) stimulation transiently suppressed T cell activation and proliferation, whereas the levels remain unaltered if P4 was not given before stimulation. Comparison of the engraftment and response against specific antigens using hu-PBL-NOG-hIL-4-Tg mice showed that P4-pretreated lymphocytes preserved CD62L expression and engrafted effectively in the spleen. Moreover, they produced antigen-specific antibodies, whereas COR-pretreated lymphocytes did not. These results suggest that a high-P4 environment suppresses T cell activation and induces T cell migration into lymphoid tissues, where they maintain the ability to produce anti-pathogen antibodies, whereas COR does not preserve T cell function. The mechanism may be pivotal in maintaining non-fetus-specific T cell function in pregnancy.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Antinuclear antibodies produced in HLA-DR transgenic humanized mice developed chronic graft-versus-host disease.

BACKGROUND: Chronic graft versus host disease (GVHD) has been reported in humanized mice after the implantation of human hematopoietic stem cells (hu-HSC). As such, humanized mice have been applied to a mouse model of chronic GVHD; however, B-cell activation and autoantibody production did not occur, and the clinical features of chronic GVHD were not sufficiently reproduced. The purpose of this study was to establish an improved humanized mouse model with chronic GVHD using HLA-DR transgenic NOD/Shi-scid, IL-2RγKO (NOG) mice. METHODS: CD34-positive cells were isolated from blood extracted from HLA-DRB1∗0405-positive umbilical cords using magnetic cell isolation. Then these were transplanted into NOG-Iab KO, HLA-DR 0405 Tg mice aged 8-16 weeks. GVHD symptoms were observed 26 weeks after transplantation. Histological findings of the skin, lung, liver, and spleen were compared with those of non-humanized mice. Antinuclear antibodies (ANA) were measured by indirect immunofluorescence using sera isolated 26 weeks after transplantation. RESULTS: Although GVHD symptoms were not observed in humanized (hu-HSC) NOG-Iab KO, HLA-DR 0405 Tg mice during the observation period, histological findings of human T-cell infiltration were observed in the skin, liver, and lung, suggesting that GVDH was present; human tingible body macrophages or clusters of BCL-6-positive human B-cells were observed in the spleen. Furthermore, human IgG ANA with peripheral or homogeneous staining patterns were also detected in the sera. CONCLUSION: Hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice differed from conventional models in terms of B-cell activation and ANA production. This study is the first to report on B-cell activation and autoantibody production in humanized mice with chronic GVHD, suggesting that hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice could be applied to a new humanized mouse model of chronic GVHD.

Development of a novel humanized mouse model for improved evaluation of in vivo anti-cancer effects of anti-PD-1 antibody.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer in the clinic. Further discovery of novel drugs or therapeutic protocols that enhance efficacy requires reliable animal models that recapitulate human immune responses to ICI treatment in vivo. In this study, we utilized an immunodeficient NOG mouse substrain deficient for mouse FcγR genes, NOG-FcγR-/- mice, to evaluate the anti-cancer effects of nivolumab, an anti-programmed cell death-1 (PD-1) antibody. After reconstitution of human immune systems by human hematopoietic stem cell transplantation (huNOG-FcγR-/- mice), four different programmed death-ligand 1 (PD-L1)-positive human cancer cell lines were tested. Among them, the growth of three cell lines was strongly suppressed by nivolumab in huNOG-FcγR-/- mice, but not in conventional huNOG mice. Accordingly, immunohistochemistry demonstrated the enhanced infiltration of human T cells into tumor parenchyma in only nivolumab-treated huNOG-FcγR-/- mice. Consistently, the number of human T cells was increased in the spleen in huNOG-FcγR-/- mice by nivolumab but not in huNOG mice. Furthermore, human PD-L1 expression was strongly induced in the spleen of huNOG-FcγR-/- mice. Collectively, our results suggest that the anti-cancer effects of anti-PD-1 antibodies can be detected more clearly in NOG-FcγR-/- mice than in NOG mice.

Generation of Novel Human Red Blood Cell-Bearing Humanized Mouse Models Based on C3-Deficient NOG Mice.

Despite recent advances in immunodeficient mouse models bearing human red blood cells (hRBCs), the elimination of circulating hRBCs by residual innate immune systems remains a significant challenge. In this study, we evaluated the role of mouse complement C3 in the elimination of circulating hRBCs by developing a novel NOG substrain harboring a truncated version of the murine C3 gene (NOG-C3ΔMG2-3). Genetic C3 deletion prolonged the survival of transfused hRBCs in the circulation. Chemical depletion and functional impairment of mouse macrophages, using clodronate liposomes (Clo-lip) or gadolinium chloride (GdCl3), respectively, further extended the survival of hRBCs in NOG-C3ΔMG2-3 mice. Low GdCl3 toxicity allowed the establishment of hRBC-bearing mice, in which hRBCs survived for more than 4 weeks with transfusion once a week. In addition, erythropoiesis of human hematopoietic stem cells (hHSCs) was possible in NOG-C3ΔMG2-3/human GM-CSF-IL-3 transgenic mice with Clo-lip treatment. These findings indicate that mouse models harboring hRBCs can be achieved using NOG-C3ΔMG2-3 mice, which could facilitate studies of human diseases associated with RBCs.