Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized.

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34(-)c-Kit(+)Sca-1(+)Lin(-) (CD34(-)KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)-HSCs were exclusively present in the Endomucin(+)CD34(-)KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45(-)CD41(+)Endomucin(+) fraction at day 10.5 of gestation (E10.5) and in the CD45(+)CD41(+)Endomucin(+) fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) could potentially provide unlimited HSCs for clinical transplantation, a curative treatment for numerous blood diseases. However, to date, bona fide HSC generation has been largely unsuccessful in vitro. We have previously described proof of concept for in vivo HSC generation from PSCs via teratoma formation. However, our first-generation system was complex and the output low. Here, we further optimize this technology and demonstrate the following: (1) simplified HSC generation using transcription factor overexpression; (2) improved HSC output using c-Kit-deficient host mice, and (3) that teratomas can be transplanted and cryopreserved. We demonstrate that overexpression of Gfi1b, c-Fos, and Gata2, previously reported to transdifferentiate fibroblasts into hematopoietic progenitors in vitro, can induce long-term HSC formation in vivo. Our in vivo system provides a useful platform to investigate new strategies and re-evaluate existing strategies to generate HSCs and study HSC development.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.

Hematopoietic stem cells (HSCs) exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation, with age. The HSC niche, which is essential for the maintenance of HSCs, also undergoes marked changes with aging. However, it has been technically challenging to directly evaluate the contribution of niche aging to age-associated HSC alterations without niche-damaging myeloablation in HSC transplantation assays. We herein transplanted an excess of aged HSCs into young mice without preconditioning. Although aged HSCs successfully engrafted in the intact young bone marrow niche, they poorly regenerated downstream progenitors and exhibited persistent myeloid-biased differentiation, resulting in no significant functional rejuvenation. Transcriptome and methylome analyses revealed that the young niche largely restored the transcriptional profile of aged HSCs, but not their DNA methylation profiles. Therefore, the restoration of the young niche is insufficient for rejuvenating HSC functions, highlighting a key role for age-associated cell-intrinsic defects in HSC aging.

Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms.

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.

Leukocyte mono-immunoglobulin (Ig)-like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc-induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1-LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

The negative regulator of p53, MDM2, is frequently overexpressed in acute myeloid leukemia (AML) that retains wild-type TP53 alleles. Targeting of p53-MDM2 interaction to reactivate p53 function is therefore an attractive therapeutic approach for AML. Here we show that an orally active inhibitor of p53-MDM2 interaction, DS-5272, causes dramatic tumor regressions of MLL-AF9-driven AML in vivo with a tolerable toxicity. However, the antileukemia effect of DS-5272 is markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. In relation to this, DS-5272 triggers immune-inflammatory responses in MLL-AF9 cells including upregulation of Hif1α and PD-L1, and inhibition of the Hif1α-PD-L1 axis sensitizes AML cells to p53 activation. We also found that NK cells are important mediators of antileukemia immunity. Our study showed the potent activity of a p53-activating drug against AML, which is further augmented by antitumor immunity.

Reprograming human primary cancer cells remains technically challenging, and leukemia-specific human induced pluripotent stem cell (hiPSC) lines have not yet been generated from tissues of patients with acute leukemia (Muñoz-López et al., 2016). Here, we developed an hiPSC line with a doxycycline-inducible expression system with the fusion gene ETV6-RUNX1 integrated into the genome. This is the most common genetic aberration found in childhood leukemia. This hiPSC line will be a useful model for research on childhood leukemia pathology and risk assessment.

Transcription factor 21 (TCF21) is one of the essential transcription factors in kidney development. To elucidate its influence on glomerular disease, we have investigated TCF21 expression in human and rat kidney tissue, and its urinary concentration. Immunohistological analysis suggested the highest TCF21 expression in nephrotic syndrome along with the urinary protein level. Urinary TCF21 concentration in human showed a positive correlation with its podocyte expression level. In nephrotic rat models, TCF21 expression in podocytes increased along with the severity of nephrotic syndrome. Next, in vitro experiments using Tcf21-expressing murine podocyte cell line, we could observe some Tcf21-dependent effects, related with actin cytoskeleton dysregulation and apoptosis. Our study illustrated TCF21 expression changes in vivo and its in vitro-functional significance injured podocytes.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

We generated dual-antigen receptor (DR) T cells from induced pluripotent stem cells (iPSCs) to mitigate tumor antigen escape. These cells were engineered to express a chimeric antigen receptor (CAR) for the antigen cell surface latent membrane protein 1 (LMP1; LMP1-CAR) and a T cell receptor directed to cell surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSCs derived from LMP2-specific cytotoxic T lymphocytes (CTLs) to generate rejuvenated CTLs (rejTs) active against LMP1 and LMP2, or DRrejTs. All DRrejT-treated mice survived >100 days. Furthermore, DRrejTs rejected follow-up inocula of lymphoma cells, demonstrating that DRrejTs persisted long-term. We also demonstrated that DRrejTs targeting CD19 and LMP2 antigens exhibited a robust tumor suppressive effect and conferred a clear survival advantage. Co-operative antitumor effect and in vivo persistence, with unlimited availability of DRrejT therapy, will provide powerful and sustainable T cell immunotherapy.

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.