A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism.

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells.

Allogeneic T cell platforms utilizing induced pluripotent stem cell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature T cell receptor (TCR) signaling plays a crucial role in generating T cells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRß chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRß fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce T cell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological T cell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR T cell therapy, offering a promising avenue for advancing adoptive immunotherapies.

TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation.

Hematopoietic stem cells (HSCs) reside in a bone marrow niche in a nondividing state from which they occasionally are aroused to undergo cell division. Yet, the mechanism underlying this unique feature remains largely unknown. We have recently shown that freshly isolated CD34-KSL hematopoietic stem cells (HSCs) in a hibernation state exhibit inhibited lipid raft clustering. Lipid raft clustering induced by cytokines is essential for HSCs to augment cytokine signals to the level enough to re-enter the cell cycle. Here we screened candidate niche signals that inhibit lipid raft clustering, and identified that transforming growth factor-beta (TGF-beta) efficiently inhibits cytokine-mediated lipid raft clustering and induces HSC hibernation ex vivo. Smad2 and Smad3, the signaling molecules directly downstream from and activated by TGF-beta receptors were specifically activated in CD34-KSL HSCs in a hibernation state, but not in cycling CD34+KSL progenitors. These data uncover a critical role for TGF-beta as a candidate niche signal in the control of HSC hibernation and provide TGF-beta as a novel tool for ex vivo modeling of the HSC niche.

TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.

Engineering of human induced pluripotent stem cells via human artificial chromosome vectors for cell therapy and disease modeling.

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.

Sphingosine-1-phosphate receptor 3 potentiates inflammatory programs in normal and leukemia stem cells to promote differentiation.

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.

Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.

57R2A, a newly established monoclonal antibody against mouse GPR56, marks long-term repopulating hematopoietic stem cells.

GPR56 molecule, a G-protein-coupled receptor, was suggested to be expressed in mouse hematopoietic stem cells (HSCs) by gene expression analyses. However, little is known about the cell surface expression of GPR56 protein in mouse HSCs due to the absence of an appropriate monoclonal antibody against GPR56 for flow cytometry analyses. In the present study, we established a novel monoclonal antibody against mouse GPR56 (57R2A) to examine the expression and distribution of GPR56 protein in HSCs. A flow cytometry analysis using 57R2A showed that GPR56 was highly expressed in the CD34-, c-Kit+, Sca-1+, lineage-negative (Lin-) fraction, which are highly enriched with HSCs. The competitive long-term repopulation (LTR) assay showed that LTR cells were included only within the GPR56+ fraction (≤15%) of bone marrow mononuclear cells (BMMNCs), but not within the remaining GPR56- fraction (85%), suggesting that all HSCs express GPR56 protein on their surface. Furthermore, we showed that double staining of BMMNCs with only 57R2A and AMM2 (monoclonal antibody against the HSC marker MPL) enabled enrichment of all LTR cells in the double-positive fraction (0.8% of BMMNCs), within which the LTR potency was consistent with the expression of both GPR56 and MPL. In conclusion, these findings for 57R2A suggest that all HSCs in mouse BMMNCs express GPR56 protein on their surface and that GPR56 is a positive marker for HSCs.

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

A TIM-3/Gal-9 Autocrine Stimulatory Loop Drives Self-Renewal of Human Myeloid Leukemia Stem Cells and Leukemic Progression.

Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and ß-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias.

Molecular identification and characterization of rat Abcc1 cDNA: existence of two splicing variants and species difference in drug-resistance profile.

The human ABCC1 gene, a member of the ATP-binding cassette transporter super-family, plays a critical role in conferring cancer cell resistance to chemotherapeutic drugs. In the present study, we have cloned the full-length cDNA of rat Abcc1 and evaluated its significance in drug resistance. Analysis using the currently available genome database revealed that the rat Abcc1 gene is located on rat chromosome 13 and consists of at least 30 exons. The rat Abcc1 cDNA cloned from the spleen was 4981-bp long, within which two additional splicing variants were discovered. The rat Abcc1 gene is expressed in a wide variety of organs, with the highest expression being observed in the spleen. Human embryonic kidney 293 cells were transfected with the rat Abcc1/pcDNA3.1 vector to stably express rat Abcc1. Overexpression of rat Abcc1 elicited high resistance to etoposide. In contrast to the hitherto known drug-resistance profile of human ABCC1, rat Abcc1 did not significantly confer cellular resistance to anthracyclins or Vinca alkaloids. Our results strongly suggest that there is a significant species difference between human ABCC1 and rat Abcc1 in their contribution to the drug-resistance profile.

Human ATP-binding cassette transporter ABCC10: expression profile and p53-dependent upregulation.

Human ATP-binding cassette (ABC) transporter genes are classified into seven sub-families, where "C" subfamily comprises a total of 13 gene members. The ABCC10 cDNA was cloned in the human full-length cDNA project at the Kazusa DNA Research Institute. However, current information is limited regarding its physiological function and gene expression. In the present study, we have investigated the expression of the ABCC10 gene to gain insight into its biological nature. By quantitative PCR, ABCC10 gene expression is demonstrated to be highest in pancreas among the adult and fetal tissues and tumors presently tested. Decreased expression was observed when resting T- and B-cells were activated. Furthermore, when we examined its expression under apoptotic conditions, we found that ABCC10 mRNA levels remarkably increased in doxorubicin-treated MCF7 cells, whereas its up-regulation was suppressed in p53-dominant-negative MCF7 cells. These results suggest that expression of the ABCC10 gene is regulated in a p53-dependent manner during DNA-damage-related apoptosis.

TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3⁻ AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.

Interleukin-27 directly induces differentiation in hematopoietic stem cells.

Interleukin (IL)-27, one of the most recently discovered IL-6 family cytokines, activates both the signal transducer and activator of transcription (STAT)1 and STAT3, and plays multiple roles in pro- and anti-inflammatory immune responses. IL-27 acts on various types of cells including T, B, and macrophage through the common signal-transducing receptor gp130 and its specific receptor WSX-1, but the effect of IL-27 on hematopoietic stem cells (HSCs) remains unknown. Here, we show that IL-27 together with stem cell factor (SCF) directly acts on HSCs and supports their early differentiation in vitro and in vivo. CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cells, a population highly enriched in mouse HSCs, were found to express both IL-27 receptor subunits. In vitro cultures of CD34(-)KSL cells with IL-27 and SCF resulted in an expansion of progenitors including short-term repopulating cells, while some of their long-term repopulating activity also was maintained. To examine its in vivo effect, transgenic mice expressing IL-27 were generated. These mice exhibited enhanced myelopoiesis and impaired B lymphopoiesis in the bone marrow with extramedullary hematopoiesis in the spleen. Moreover, IL-27 similarly acted on human CD34(+) cells. These results suggest that IL-27 is one of the limited cytokines that play a role in HSC regulation.

Discordant developmental waves of angioblasts and hemangioblasts in the early gastrulating mouse embryo.

Vasculogenesis and hematopoiesis are thought to arise in hemangioblasts, the common progenitors of cells in vessels and in blood. This scheme was challenged by kinetic analysis of vascular endothelial and hematopoietic progenitors in early gastrulating mouse embryos. The OP-9 co-culture system with a combination of cytokines permitted the detection of endothelial progenitors, as well as stroma-dependent hematopoietic progenitors. Endothelial progenitors were detected as early as embryonic day (E) 5.50, after which time their numbers increased. Stroma-dependent hematopoietic progenitors were detected at E6.75, the time point when hemangioblasts reportedly emerge. Colony-forming units in culture (CFU-c), most likely generated from stroma-dependent hematopoietic progenitors via contact with the microenvironment, were detected at E7.50, concomitant with the onset of primitive hematopoiesis in the yolk sac. The presence of nucleated erythrocytes and the expression of an embryonic-type globin in erythroid colonies derived from stroma-dependent hematopoietic progenitors and from CFU-c support the notion that these progenitors coordinately establish primitive hematopoiesis. Using Oct3/4 promoter-driven GFP transgenic mice, early endothelial progenitors, stroma-dependent hematopoietic progenitors, and CFU-c were all shown to express the Oct3/4 transcription factor. Among Oct3/4-positive cells, both endothelial and hematopoietic progenitors were present in the CD31-positive fraction, leaving a subset of endothelial progenitors in the CD31-negative fraction. These data imply that Oct3/4-positive mesoderm gives rise to CD31-negative angioblasts, CD31-positive angiboblasts and CD31-positive hemangioblasts. We propose a distinct developmental pathway in which the angioblast lineage directly diverges from mesoderm prior to and independent of hemangioblast development.

Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche in a noncycling state and enter the cell cycle at long intervals. However, little is known about inter- and intracellular signaling mechanisms underlying this unique property of HSCs. Here, we show that lipid raft clustering is a key event in the regulation of HSC dormancy. Freshly isolated HSCs from the BM niche lack lipid raft clustering, exhibit repression of the AKT-FOXO signaling pathway, and express abundant p57(Kip2) cyclin-dependent kinase inhibitor. Lipid raft clustering induced by cytokines is essential for HSC re-entry into the cell cycle. Conversely, inhibition of lipid raft clustering caused sustained nuclear accumulation of FOXO transcription factors and induced HSC hibernation ex vivo. These data establish a critical role for lipid rafts in regulating the cell cycle, the survival, and the entry into apoptosis of HSCs and uncover a striking similarity in HSC hibernation and Caenorhabditis elegans dauer formation.

Genetic marking of hematopoietic stem and endothelial cells: identification of the Tmtsp gene encoding a novel cell surface protein with the thrombospondin-1 domain.

Using an in silico database search, we identified a novel gene encoding a cell surface molecule with a thrombospondin domain, and designated the gene as transmembrane molecule with thrombospondin module (Tmtsp). Expression profiling of Tmtsp using specific monoclonal antibodies and Venus, a variant of yellow fluorescent protein knock-in mice in the Tmtsp locus, demonstrated its specific expression in hematopoietic and endothelial cells. In lymphohematopoietic cells, Tmtsp was predominantly expressed in hematopoietic stem and progenitor cells, and the level of expression gradually declined as the cells differentiated. Venus expression faithfully traced the expression of Tmtsp, and the level of Venus expression correlated well to the in vitro hematopoietic activity as well as the in vivo bone marrow repopulating capacity. Notably, Venus expression marked the development of definitive hematopoiesis in both the extraembryonic yolk sac and the intraembryonic aorta-gonad-mesonephros (AGM) region and, in combination with CD41, strikingly promoted the enrichment of developing progenitors in the CD41(+)Venus(high) fraction at embryonic day 10.5 (E10.5). In this context, Tmtsp is a novel marker gene for primitive hematopoietic cells and endothelial cells, and Tmtsp(Venus/)(+) mice would serve as a valuable mouse model for the analysis of both embryonic and adult hematopoiesis, as well as for vascular biology.