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  • Physioxia improves the selectivity of hematopoietic stem cell expansion cultures. Blood advances Igarashi, K. J., Kucinski, I., Chan, Y. Y., Tan, T., Khoo, H. M., Kealy, D., Bhadury, J., Hsu, I., Ho, P. Y., Niizuma, K., Hickey, J. W., Nolan, G., Bridge, K. S., Czechowicz, A., Gottgens, B., Nakauchi, H., Wilkinson, A. C. 2023


    Hematopoietic stem cells (HSCs) are a rare hematopoietic cell type that can entirely reconstitute the blood and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases, but remains a high-risk therapy due to potential side effects including poor graft function and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, we demonstrate that we can improve the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures through the use of physioxic culture conditions. Single-cell transcriptomic analysis confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, as well as highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

    View details for DOI 10.1182/bloodadvances.2023009668

    View details for PubMedID 36809781

  • Bioluminescent Tracking of Human Induced Pluripotent Stem Cells In Vitro and In Vivo. Methods in molecular biology (Clifton, N.J.) Nishimura, T., Niizuma, K., Nakauchi, H. 2022; 2524: 291-297


    The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that arise from using embryonic stem cells. Understanding the in vivo behavior of iPSCs, as well as their derivatives, requires the monitoring of their localization, proliferation, and viability after transplantation. Bioluminescence imaging (BLI) gives investigators a non-invasive and sensitive means for spatio-temporal tracking in vivo. For scientists working within the field of iPSCs, this protocol provides a walk-through on how to conduct in vitro and in vivo experiments with an iPSCs constitutively expressing luciferase.

    View details for DOI 10.1007/978-1-0716-2453-1_22

    View details for PubMedID 35821480

  • Cutting Edge: NKG2D Signaling Enhances NK Cell Responses but Alone Is Insufficient To Drive Expansion during Mouse Cytomegalovirus Infection JOURNAL OF IMMUNOLOGY Nabekura, T., Gotthardt, D., Niizuma, K., Trsan, T., Jenus, T., Jonjic, S., Lanier, L. L. 2017; 199 (5): 1567–71


    NK cells play a critical role in host defense against viruses. In this study, we investigated the role of NKG2D in the expansion of NK cells after mouse CMV (MCMV) infection. Wild-type and NKG2D-deficient (Klrk1-/- ) Ly49H+ NK cells proliferated robustly when infected with MCMV strains engineered to allow expression of NKG2D ligands, which enhanced the response of wild-type NK cells. Naive NK cells exclusively express NKG2D-L, which pairs only with DAP10, whereas NKG2D-S expressed by activated NK cells pairs with DAP10 and DAP12, similar to Ly49H. However, NKG2D alone was unable to drive robust expansion of Ly49H- NK cells when mice were infected with these MCMV strains, likely because NKG2D-S was only transiently expressed postinfection. These findings demonstrate that NKG2D augments Ly49H-dependent proliferation of NK cells; however, NKG2D signaling alone is inadequate for expansion of NK cells, likely due to only transient expression of the NKG2D-DAP12 complex.

    View details for DOI 10.4049/jimmunol.1700799

    View details for Web of Science ID 000408005200006

    View details for PubMedID 28760883

    View details for PubMedCentralID PMC5567695

  • Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family JOURNAL OF BIOLOGICAL CHEMISTRY Niizuma, K., Tahara-Hanaoka, S., Noguchi, E., Shibuya, A. 2015; 290 (36): 22298–308


    Recruitment of circulating monocytes and neutrophils to infection sites is essential for host defense against infections. Here, we identified a previously unannotated gene that encodes an immunoglobulin-like receptor, designated CD300H, which is located in the CD300 gene cluster. CD300H has a short cytoplasmic tail and associates with the signaling adaptor proteins, DAP12 and DAP10. CD300H is expressed on CD16(+) monocytes and myeloid dendritic cells. Ligation of CD300H on CD16(+) monocytes and myeloid dendritic cells with anti-CD300H monoclonal antibody induced the production of neutrophil chemoattractants. Interestingly, CD300H expression varied among healthy subjects, who could be classified into two groups according to "positive" and "negative" expression. Genomic sequence analysis revealed a single-nucleotide substitution (rs905709 (G → A)) at a splice donor site on intron 1 on either one or both alleles. The International HapMap Project database has demonstrated that homozygosity for the A allele of single nucleotide polymorphism (SNP) rs905709 ("negative" expression) is highly frequent in Han Chinese in Beijing, Japanese in Tokyo, and Europeans (A/A genotype frequencies 0.349, 0.167, and 0.138, respectively) but extremely rare in Sub-Saharan African populations. Together, these results suggest that CD300H may play an important role in innate immunity, at least in populations that carry the G/G or G/A genotype of CD300H.

    View details for DOI 10.1074/jbc.M115.643361

    View details for Web of Science ID 000360968500045

    View details for PubMedID 26221034

    View details for PubMedCentralID PMC4571981

  • Toll-like receptor 4 and MAIR-II/CLM-4/LMIR2 immunoreceptor regulate VLA-4-mediated inflammatory monocyte migration NATURE COMMUNICATIONS Totsuka, N., Kim, Y., Kanemaru, K., Niizuma, K., Umemoto, E., Nagai, K., Tahara-Hanaoka, S., Nakahasi-Oda, C., Honda, S., Miyasaka, M., Shibuya, K., Shibuya, A. 2014; 5: 4710


    Inflammatory monocytes play an important role in host defense against infections. However, the regulatory mechanisms of transmigration into infected tissue are not yet completely understood. Here we show that mice deficient in MAIR-II (also called CLM-4 or LMIR2) are more susceptible to caecal ligation and puncture (CLP)-induced peritonitis than wild-type (WT) mice. Adoptive transfer of inflammatory monocytes from WT mice, but not from MAIR-II, TLR4 or MyD88-deficient mice, significantly improves survival of MAIR-II-deficient mice after CLP. Migration of inflammatory monocytes into the peritoneal cavity after CLP, which is dependent on VLA-4, is impaired in above mutant and FcRγ chain-deficient mice. Lipopolysaccharide stimulation induces association of MAIR-II with FcRγ chain and Syk, leading to enhancement of VLA-4-mediated adhesion to VCAM-1. These results indicate that activation of MAIR-II/FcRγ chain by TLR4/MyD88-mediated signalling is essential for the transmigration of inflammatory monocytes from the blood to sites of infection mediated by VLA-4.

    View details for DOI 10.1038/ncomms5710

    View details for Web of Science ID 000341078900008

    View details for PubMedID 25134989

    View details for PubMedCentralID PMC4143930