Experimental Hematology

Background: The ability to repopulate all lineages of blood cells from a single hematopoietic stem cell (HSC) requires a constant balancing between maintenance of quiescence/self-renewal and the need for differentiation and adoption of specific lineage phenotypes. A classical interpretation suggests that during homeostasis, long- and then short-term HSCs differentiate into multipotent progenitors (MPPs), which then lack self-renewal capacity but can still differentiate into all downstream lineages. While much is known about the intrinsic and extrinsic regulators that promote HSC homeostasis, less is known about factors which influence HSC behavior following significant bone marrow (BM) injury, such as sublethal irradiation (SLI). Methods: Conditional knock-out of follistatin-like 1 (FSTL-1) was achieved by breeding mice with floxed Fstl1 sites to mice expressing CreERT2 under control of the ROSA locus, followed by systemic tamoxifen administration. ELISA and RT-PCR quantitated protein and RNA level loss of FSTL-1, respectively. HSC populations were assessed by flow cytometry under multiple conditions. Sublethal irradiation induced BM injury and blocking polyclonal FSTL-1 antibodies were used to recapitulate results seen with genetic ablation. Finally, single cell RNA sequencing defined both cell-specific expression of Fstl1 within the BM compartment, but also identified transcripts and pathways upregulated in the absence of FSTL-1. Results: These studies delineate a novel role for FSTL-1 in regulating HSC biology. In addition to selective expression of Fstl1 within HSCs, genetic deletion of Fstl1 increases Lineage negative;Sca-1+c-Kit+ (LSK) cell number and alters HSC homeostasis. These findings are further amplified in the BM in the context of recovery from sublethal irradiation. Importantly, the post-irradiation increase in BM cellularity seen with Fstl1 deletion is replicated by treatment with anti-FSTL-1 polyclonal antibodies. Finally, single cell RNA sequencing reveals distinct Fstl1 expression in specific LSK subpopulations, including HSCs and proliferating cells, and identifies a novel relationship between decreased FSTL-1 levels and enrichment of canonical metabolic pathways including oxidative phosphorylation, fatty acid metabolism, and glycolysis. Conclusions: Taken together, these results highlight a novel role for FSTL-1 in promoting HSC homeostasis. They also unlock an innovative way to promote HSPC recovery following BM injury, knowledge predicted to be of significant therapeutic value.

In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: {gamma}-globin gene addition, {gamma}-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439* variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

Embryonic hematopoiesis is essential for establishing lifelong blood and immune system function. During development, hematopoietic stem and progenitor cells (HSPCs) acquire intrinsic programs that persist into adulthood and can influence disease susceptibility, yet the molecular signals governing these early-life decisions remain poorly understood. Here, we investigated the role of developmental Transforming Growth Factor-{beta} (TGF-{beta}) signaling in regulating HSPC lineage bias and long-term hematopoietic outcomes. Using the zebrafish model, we found that transient embryonic TGF-{beta} signaling inhibition during the HSPC specification window altered their frequency and migration after emergence from the hemogenic endothelium and movement into the key secondary maturation and expansion niche. Single-cell transcriptomic analysis of embryonic HSPCs revealed repression of migration- and cytoskeleton-associated genes alongside dampened expression of myeloid/macrophage-related genes following ALK5 inhibition. Functionally, early ALK5 blockade reduced macrophage numbers and promoted an M2-like immunosuppressive transcriptional profile. These developmental perturbations produced sustained effects on hematopoietic and immune function into adulthood, including diminished inflammatory gene expression, reduced clonal complexity, and impaired regenerative capacity. Together, our findings identify embryonic TGF-{beta} signaling as a key developmental regulator of HSPC fate and immune programming, with potential implications for immune dysfunction and susceptibility to inflammatory-related disease later in life. PAPER HIGHLIGHTS- Transient developmental signaling perturbations reshape hematopoietic trajectories - Embryogenic TGF-{beta} signaling instructs HSPC lineage priming and macrophage specialization - Early HSPC programming establishes persistent inflammatory states, clonal diversity, and modifies regenerative capacity

Leukemic stem cells (LSCs) play a central role in disease progression, therapeutic resistance, and relapse in acute myeloid leukemia (AML). However, the identification and characterization of LSCs remain challenging because of their low abundance and their close phenotypic resemblance to normal hematopoietic stem and progenitor cells. Although patient-derived xenograft (PDX) models have provided important insights into AML biology and LSC heterogeneity, the relative engraftment potential of distinct CEBPA mutation subtypes and the immunophenotypic identity of LSCs in CEBPA N-terminal mutant AML (CEBPA-N-AML) remain poorly defined. To address these questions, we compared the engraftment characteristics of primary human CEBPA-mutated AML samples representing the major mutational subtypes using the highly permissive NSGS xenograft model. Primary CEBPA-N-AML samples exhibited markedly greater engraftment efficiency and leukemogenic potential than other CEBPA-mutated AML subtypes. Furthermore, we identified a CD366CD73CD123CD117CD371CD247 cell population that is highly enriched for functional LSCs in CEBPA-N-AML, demonstrating enhanced clonogenic activity, leukemia-initiating capacity, and long-term self-renewal. Collectively, our findings demonstrate that the leukemogenic potential of CEBPA-mutated AML is strongly influenced by mutation subtype, with CEBPA-N-AML exhibiting superior leukemia-propagating capacity in vivo. We further define a novel immunophenotypic LSC signature specific to CEBPA-N-AML, providing new insights into LSC heterogeneity in CEBPA-mutated AML and establishing a foundation for the development of LSC-directed therapeutic strategies.

Decoding the bone marrow (BM) hematopoietic progenitor compartment is central to understanding the development of mature immune cells in health and disease. Single-cell multimodal approaches are transforming the analysis of this heterogeneous compartment by simultaneously capturing transcriptomic and proteomic information. Still, integrated studies covering the full spectrum of murine hematopoietic stem and progenitor cells (HSPCs) remain scarce. Here, we report a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) based atlas of 9,281 low-frequency mouse BM HSPCs, profiled using a 123 antibody-derived tag (ADT) panel coupled with genome-wide single-cell transcriptomes. Multimodal analysis resolved 28 progenitor stages across eight blood lineages, each defined by distinctive surface marker and transcription factor (TF) profiles, and provided novel marker combinations to dissect transcriptionally closely related progenitor stages. Classical mouse progenitor surface markers, such as CD117, CD34, and CD115, were detected in accordance with well-established lineage relationships, whereas CD43 and CD48 showed, unexpectedly, broader expression patterns. Neglected surface molecules such as CD27, CD36, CD106, and PIR-A/B are nominated as novel stage-specific markers. ADT versus transcriptome comparison revealed transcription-translation inconsistencies for markers, including CD54 and CD68. A CITE-seq-guided gating strategy translates these population definitions into FACS-compatible panels for prospective isolation and functional interrogation of previously unresolved progenitor stages. Comprehensive TF network analysis delineated lineage- and stage-specific transcriptional programs. This contributes to clarifying unresolved ontogenies and functions, including the lympho-myeloid dual origin of pDCs or rapid neutrophil turnover. This new and comprehensive resource enables exploration of mouse hematopoietic progenitor states driving discoveries in hematology and immunology.

Background: Relapse remains the principal cause of treatment failure in pediatric acute leukemia. Clinically validated minimal residual disease (MRD) assays - multiparameter flow cytometry, immunoglobulin/T-cell-receptor rearrangement sequencing, and targeted next-generation sequencing panels -- are highly sensitive but read out predefined features and do not reconstruct clonal architecture or link a cell's genotype to its functional state. We systematically reviewed what single-cell sequencing has revealed about clonal evolution, chemoresistance, the immune microenvironment, and post-therapeutic immune dynamics in this disease. Methods: This review followed PRISMA 2020. PubMed/MEDLINE, the Cochrane Library, and Google Scholar were searched to September 2026 for primary studies applying single-cell DNA, RNA, chromatin-accessibility, surface-protein, or immune-repertoire sequencing to pediatric acute leukemia samples or pediatric-derived models. Risk of bias in non-randomized studies was assessed with ROBINS-I. Because designs, platforms, and outcomes were heterogeneous, findings were synthesized narratively by biologic theme without meta-analysis, following the SWiM guideline. Results: Of 63 records identified, 10 studies were included (published 2016-2026; 6-159 patients or samples per study; up to ~700,000 cells profiled). Single-cell DNA sequencing traced minor NOTCH1-mutant diagnostic subclones into PTEN- or TP53-mutant relapse clones that bulk sequencing missed. Rare dormant, label-retaining cells and developmental-state-dependent drug sensitivity defined chemoresistant compartments. Remodeling of the bone marrow immune microenvironment identified patients with adverse outcome among those classified as MRD-low, and pre-infusion CAR T-cell type 2 functionality and post-transplant cytotoxic memory CD8+ T-cell subsets were associated with durable remission. Of the seven studies assessed with ROBINS-I, four were at moderate and three at serious overall risk of bias, driven chiefly by small, selected cohorts. Conclusions: Single-cell sequencing yields mechanistic insight that complements, rather than replaces, clinically validated MRD assays, and nominates candidate biomarkers for risk stratification, treatment selection, cell-therapy manufacturing, and post-transplant monitoring. The evidence remains preliminary; adequately powered, prospective, and harmonized studies are required before clinical adoption.

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Abstract Background: The stem cell factor receptor or c-Kit is a type III receptor tyrosine kinase, activated by its ligand Stem cell factor (SCF). Up on activation, c-kit induces signaling pathways that regulates blood cell proliferation, survival, differentiation, and migration. Several studies reported that c-Kit/SCF signaling, contributes to the development and progression of acute myeloid leukemia (AML) in patients. However, the downstream proteins regulated by c-kit activation and their clinical significance in AML remain poorly explored. Methods: Human Acute megakaryoblastic leukemia (Mo7e) cells, were-stimulated with SCF and global protein expression were profiled using two-dimensional gel electrophoresis coupled with MALDI-TOF and LC-MS/MS. Differentially expressed proteins were functionally characterized and validated using patient data from the TCGA-LAML and matched normal data from GTEx, GEO datasets, and quantitative RT-PCR. Their diagnostic and prognostic significance was assessed using ROC, Cox regression, LASSO, Kaplan Meier survival analyses, and a prognostic nomogram model. Results: Proteomic profiling identified 14 differentially expressed proteins in SCF-stimulated Mo7e cells, which are predicted to involved in cytoskeletal organization, protein folding, metabolism, vesicular trafficking, and translational regulation. Transcriptomic analysis of the TCGA-LAML cohort revealed significant dysregulation of CFL1, CCT8, HSP90B1, MDH2, EIF5A, GSN, and TPI1. Integrated ROC, Cox regression, and LASSO analyses identified CFL1, CCT8, and GSN as the most robust prognostic biomarkers associated with poor overall survival in LAML patients. Their expression patterns were validated in independent GEO datasets and by qRT-PCR in SCF stimulated Mo7e cells. Finally, a three-gene nomogram model was developed and validated to predict the overall survival probability of AML patients at 1-, 3-, and 5-year time points. Conclusions: This study identifies CFL1, CCT8, and GSN as key downstream effectors of c-Kit signaling as prognostic biomarkers for AML. These findings provide mechanistic insights into c-Kit-driven leukemogenesis and establish a clinically relevant three-gene signature for AML risk stratification and potential therapeutic targeting.

BackgroundThe Philadelphia negative myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), are characterized by myeloid cell proliferation, thrombosis and inflammation. Suspicion of MPN arises from increased blood count in one or more lineages; however, knowledge on the MPN plasma proteome including biomarkers measurable in blood, are lacking. Comparing the plasma proteome of MPN patients to subjects with elevated blood counts but without MPN diagnosis, may provide an increased understanding of the MPN disease biology as well as diagnostic biomarkers measurable in blood. Patients and methodsWe performed plasma proteome profiling in 87 patients referred to the Department of Hematology due to elevated blood counts. Of these, 55 were diagnosed with MPN and 32 did not fulfill MPN diagnostic criteria and thus constituted the non-MPN control group. ResultsThe frequency of thrombosis was equal between the groups. We found 189 differentially expressed proteins between MPN and non-MPN, enriched for Hemostasis and Platelet activation proteins. Using Lasso multiple regression, we identified SORT1, GP1BA, PSPN, MMP1 and BAG6 separating MPN from non-MPN individuals, and TFRC and SEMA7A specific for MPN subtype PV. Interestingly, TFRC alone had a diagnostic accuracy for identifying PV of 89.3%, and when combined with serum erythropoietin it increased to 99.3%. Only two proteins, IL-6 and GH1, were increased in JAK2 mutant MPN compared to JAK2 wildtype MPN. The same trend was seen for JAK2 mutant ET compared to JAK2 wildtype ET, indicating that IL-6 is induced by JAK STAT activation. However, IL-6 levels did not differ between MPN and non-MPN patients. Discussion/conclusionIn conclusion, hemostasis and platelet activation are inherent to MPN disease whereas little difference was found in proinflammatory cytokines between MPN and non-MPN groups. We demonstrate novel potential blood biomarkers for MPN and MPN subtypes, in particular TFRC for identifying PV patients.

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

Background: Epigenetic deregulation is a defining feature of leukemia, and DNA methylation and chromatin accessibility profiles encode the cell of origin, lineage and clinical behaviour of the disease. Artificial intelligence (AI) and machine learning (ML) can extract predictive patterns from these high-dimensional data, but the published models have not been systematically appraised. We systematically reviewed AI models built on epigenomic data in leukemia for diagnosis, prognosis, relapse and treatment-response prediction, and measurable residual disease (MRD) detection. Methods: This review followed PRISMA 2020 and the SWiM guideline. PubMed was searched on 7 October 2026 with three structured queries, supplemented by a broader PubMed query, citation searching and web searching. Eligible studies were peer-reviewed primary studies that applied an ML or deep-learning algorithm to DNA methylation, chromatin-accessibility or histone-modification data from human leukemia samples and reported a quantitative diagnostic, prognostic, response or MRD outcome. Risk of bias and applicability were assessed with PROBAST, informed by PROBAST+AI. Owing to heterogeneity, results were synthesized narratively and in structured tables, without meta-analysis. Results: Of 68 unique database records and 32 reports from other sources, 30 studies (2010-2026; 22 since 2020) were included: 16 in acute myeloid leukemia, 4 in acute lymphoblastic leukemia, 2 spanning acute leukemia lineages, 3 in juvenile myelomonocytic leukemia and 5 in chronic lymphocytic leukemia; 10 were pediatric. DNA methylation was the input in 28 studies and chromatin accessibility in 2; no eligible study modelled histone marks. Tree ensembles (n = 9) and support vector machines (n = 5) were the most frequent algorithms. Diagnostic classifiers reported accuracy, concordance or F1 scores of 0.87-1.00, and two classifiers were applied to nanopore sequencing data, one returning acute leukemia subtype calls within about 2 hours. Prognostic epigenomic scores were associated with survival (reported hazard ratios 2.36-10.82), but discrimination fell on transport to independent cohorts (C-index 0.677 internally vs 0.529 in one external cohort). Fifteen studies validated models on an external epigenomic cohort, one prospectively. Overall risk of bias was high in 21 studies, unclear in 8 and low in 1, driven by the analysis domain (small samples, absent calibration, possible data leakage). Calibration was reported in 3 studies and code was available for 11. Conclusions: Epigenomic AI models classify leukemia subtypes with high reported accuracy and can deliver same-day diagnoses, and methylation-based scores add prognostic information. However, the evidence is dominated by retrospective, high-risk-of-bias development studies with limited external validation and almost no prospective or impact evaluation. Prospective, multi-center validation with calibrated, transparently reported models is required before clinical adoption.

Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, affect millions of individuals worldwide who have limited access to curative therapies. Autologous hematopoietic stem cell transplant following ex vivo CRISPR editing of the BCL11A erythroid enhancer reactivates fetal hemoglobin (HbF) and achieves an effective cure, but the resource constraints of clinically approved procedures for editing by electroporation (EP) severely limit widespread implementation. We directly compared the functional outcomes of EP delivery of Cas9 ribonucleoprotein with lipid nanoparticle (LNP) delivery of Cas9 mRNA in primary human HSPCs obtained from healthy HbAA donors and from patients with SCD. While higher editing rates are achieved with EP, LNP-treated HSPCs exhibited greater viability and cell yields that persisted throughout a multi-stage in vitro erythroid differentiation protocol. By day 20, the yield of mature red blood cells (CD71lowCD235ahigh) was lowest in the EP cohorts. Across treatment groups, we observed HbF induction proportional to indel frequency. LNP editing of SCD patient-derived HSPCs as low as 25% modified alleles still caused HbF production and reduced the propensity for sickling of in vitro differentiated RBCs. These findings highlight the critical trade-offs among manufacturing ease, delivery-associated toxicity, and functional performance across two modalities of therapeutic genome editing for hemoglobinopathies.

Haematopoietic stem cells (HSCs) have long been used in the clinic for bone marrow transplantation applications, critical for the survival of an increasingly wide range of patients with haematological, oncological and immunological pathologies. Despite this, little is known about the mechanisms through which relatively few stem cells are able to regenerate the entire haematopoietic tissue of transplant recipients. To gain insights on this biological process, we used intravital microscopy of calvarium bone marrow, collecting tissue-wide images and a total of 850 hours of tracks of engrafting haematopoietic stem and progenitor cells from 24 hours to 8 days following injection in lethally irradiated recipients. Analysis of the data revealed that regenerating HSCs and their immediate progeny are highly dynamic, migrating through the parenchyma at both the microscopic and near-macroscopic scales, i.e. within and in-and-out of fields of view. HSC-derived cell clusters, expanded locally between day 2 and day 4 post-transplant, leading to patches of densely populated bone marrow by day 8. Single cell level analysis highlighted increasingly heterogenous cellular behaviours over time. Track clustering based on migration and niche interaction parameters combined with post-tracking whole mount immunostaining revealed that persistence in the vicinity of nestin-GFP perivascular cells and relatively slow movement correlated with stemness, while more heterogeneous niche interactions coupled with slowest or faster migration correlated with differentiation, validated through flow cytometry analysis and functional studies. The findings presented here shed light on the fundamental principles driving haematopoietic regeneration in transplantation settings.

Acute myeloid leukemia (AML) is an aggressive blood cancer driven by genetic and epigenetic alterations that disrupt normal hematopoiesis. Among these, the histone H3-K27M mutation, originally identified in pediatric high-grade gliomas, reshapes gene repression programs by reducing global H3-K27 trimethylation. Although rare, H3-K27M mutations have been detected in preleukemic hematopoietic stem cells (HSCs) of AML patients, suggesting its role in early leukemogenesis and identifying it as a promising therapeutic target. Here, we investigated how H3-K27M alters hematopoiesis using a longitudinal xenotransplantation mouse model of human hematopoietic stem and progenitor cells carrying either wild-type H3 or H3-K27M. To quantify hematopoietic dynamics, we developed a collection of mathematical models representing alternative lineage hierarchies and fit them to our longitudinal experimental data. Using information criteria, we identified the model that best depicts blood dynamics in each condition. Our results show that H3-K27M promotes HSC proliferation and differentiation and erythroid commitment while reducing multipotent progenitor self-renewal potential and lymphoid commitment, ultimately increasing blood cell numbers despite impairing erythroid maturation. Mathematical modelling inferred the progenitor-level alterations, which were not readily evident from the blood cell count alone. H3-K27M also induced distinct kinetics in human HSC1 and HSC2 subpopulations that were not observed in wild-type H3 controls. Together, our results provide the first quantitative framework to reveal how H3-K27M influences the hematopoietic hierarchy roadmap and alters blood cell dynamics in preleukemia.

Hematopoietic stem cells (HSCs) give rise to all blood cell lineages and possess long-term self-renewal potential. HSCs undergo symmetric division for their expansion and asymmetric division to generate one HSCs and one progenitor cells which contribute to production of mature blood cells. The midbody is a structure which is formed in the center of the intercellular bridge during cytokinesis. However, the midbody is either asymmetrically inherited by one daughter cell or symmetrically released after cell division, whether these distinct patterns of midbody inheritance influence HSC fate remain poorly understood. In this study, we designed a fusion protein hmKO2 and MgcRacGAP which is a component of midbody. We then traced the midbody inheritance during cell division and the future cell fates of HSC daughters after division by time-lapse imaging. As a result, we found that the midbody release correlated with the delay of the time to the next division but not to the lineage potential of HSCs, indicating the possibility that midbody remnant plays some roles in cell cycle progression. HighlightHematopoietic stem cells exhibit a low frequency of midbody inheritance. Midbody inheritance does not affect the lineage potential of daughter cells. Midbody loss is associated with delayed entry into the next cell cycle.

Invariant natural killer T (iNKT) cells can limit graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), but their scarcity in peripheral blood limitstherapeutic development. Current clinical-grade human iNKT expansion protocols mainly rely on IL-2, require prior iNKT-cell sorting, last 6-8 weeks, and predominantly expand CD4+ iNKT cells, whereas human CD4- iNKT cells are more strongly associated with GVHD control in patients and uniquely regulate antigen-presenting cells and T-cell activation. We developed a scalable culture system to preferentially expand human CD4- iNKT cells directly from total peripheral blood mononuclear cells (PBMCs) using alpha galactosylceramide (-GalCer) and optimized cytokine conditions. IL-15 was the most effective cytokine. The optimized 14-day protocol generated a mean of 3.8x107 iNKT cells from 2x107 PBMCs, including 74% CD4- iNKT cells. Single-cell transcriptomic profiling identified eight major iNKT subsets, differentiation trajectories during expansion, and distinct IL-2- versus IL-15-associated transcriptional programs. IL-15-expanded iNKT cells induced apoptosis of monocyte-derived dendritic and leukemic cells in vitro, controlled xeno-GVHD, and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models. This platform enables reproducible production of human CD4- iNKT cells at clinically relevant scale and position IL-15-expanded iNKT cells as a compelling immunotherapy candidate for allo-HSCT.

Commitment events to specific blood lineages arise from single hematopoietic stem cells (HSCs) and are influenced by stress, inflammation and disease. However, the understanding of how such events are regulated in human haematopoiesis is limited by the lack of tractable in vitro models. In this study, we introduce a novel Early Progenitor Differentiation (EPD) assay to study the initial lineage commitment of human 49+ HSCs, in a system faithfully recapitulating cell states observed in vivo. Combining single cell -omics approaches and single cell functional assays, we show that IL-33 acts directly on human 49f+ HSCs activating the MAPK pathway to enhance their commitment towards Megakaryocytic-Erythroid-Mast cell Progenitors and subsequently megakaryopoiesis. This occurs without affecting HSC self-renewal via accelerated establishment of chromatin programmes associated with Erythroid and Megakaryocyte and mast cells lineages. Our findings demonstrate the utility of the EPD model to identify molecular regulators of human HSC differentiation and uncover a new role of IL-33 in haematopoiesis.

Background aimsThe development of next-generation therapies for sickle cell disease (SCD) and beta thalassemia (beta thal), including fetal globin-inducing small molecules and gene therapy approaches, depends on patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) for discovery and preclinical validation, but commercial vendors stock only healthy donor material and disease-specific banks hold limited inventories. Recent US Food and Drug Administration and National Institutes of Health guidance favoring human cell-based methods over animal testing underscores the value of authentic patient cells. Methods: Over 14 months we recovered, purified, and biobanked CD34+ HSPCs from clinical apheresis product waste and mobilized peripheral blood (PB) otherwise discarded after clinical procedures, using immunomagnetic selection adapted for hemoglobinopathy specimens; a microfluidic technology was evaluated separately. We quantified yield and purity for bead-selected material and cell number and viability for the microfluidic pilot; engraftment was tested in NBSGW mice. Results: Immunomagnetic selection recovered a median of 4.71 x 106 CD34+ cells from just 1 to 2 mL of apheresis product waste, comparable to the 6.0 x 106 cells from a 10 to 40 fold larger volume of PB waste, with similar purity across sources and diagnoses. Because apheresis product waste is far more concentrated, it reaches equivalent yields without the density-gradient steps required for PB waste, approximately halving processing time. Recovered cells engrafted NBSGW mice, confirming preserved repopulating capacity. The microfluidic pilot (two patients, 11 specimens) recovered 2.17 x 106 CD34+ cells per specimen at greater than 90% viability and purity. Conclusions: A center with existing apheresis infrastructure can reproducibly recover, bank, and distribute research-grade patient CD34+ HSPCs, addressing a recognized gap in the hemoglobinopathy pipeline. HighlightsO_LIClinical apheresis waste is used to generate a single-center biobank of high-quality, research-grade CD34+ HSPCs from patients with sickle cell disease and beta-thalassemia. C_LIO_LIConcentrated apheresis waste matches large-volume PB waste in CD34+ yield and purity. C_LIO_LIMicrofluidic enrichment recovers CD34+ cells at >90% viability and purity across 2 patients. C_LIO_LIRecovered CD34+ HSPCs engraft mice and form erythroid cells, preserving function. C_LI

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor prognosis and an unmet need for more effective treatment options, particularly after relapse. One potential approach to address this need, which has been used successfully in solid tumors, is adoptive transfer of T cells engineered to express specific T cell receptors (TCRs). Particularly attractive are TCRs that can target epitopes derived from oncogenes on which cancer cells depend while sparing normal cells that do not harbor the mutation. One promising oncogenic target in AML is mutated nucleophosmin 1 (mtNPM1), present in up to 30% of AML cases. Most NPM1 mutations result in a common frameshifted sequence that generates peptides presented by several class I human leukocyte antigens (HLAs), such as HLA-A*11:01. In this study, we vaccinated HLA-A*11:01-transgenic mice with the mtNPM1 peptide AVEEVSLRK and isolated six functional TCRs from mtNPM1-reactive lymphocytes. When engineered into human T cells, two of these TCRs consistently mediated robust and specific lysis of an AML cell line and primary AML cells co-expressing HLA-A*11:01 and mtNPM1. Neither TCR recognized related human peptides, HLA-A*11:01-expressing normal peripheral blood mononuclear cells or stem/progenitor cells, or cells expressing diverse allogeneic HLAs, suggesting a low risk of cross-reactivity and alloreactivity. In an AML xenograft model, both TCRs controlled AML in hematopoietic organs, although extramedullary tumors emerged during treatment, potentially reflecting the aggressive behavior of the model that may not be representative of AML in patients. Together, these data suggest that the two TCRs represent strong candidates for further preclinical development in adoptive T cell therapy for NPM1-mutated, HLA-A*11:01-positive AML. These results also support the use of HLA-transgenic mice as a platform for generating TCRs targeting oncogene-derived epitopes in hematologic malignancies.