Haematopoietic stem cells (HSCs) are important for human health and clinical therapy. A heterogenous pool of HSCs sustain blood production through life by balancing self-renewal and multilineage differentiation. Aging is associated with a decline in the function of the HSC pool and haematopoietic perturbations. Additionally, aging correlates with clonal haematopoiesis, driven by the accumulation of genetic mutations in this long-lived cell population that can lead to altered cell function and ultimately to leukemic transformation. Alongside their natural role in sustaining haematopoiesis, healthy HSCs are also used clinically for their regenerative capacity in stem cell transplantation, where they reconstitute the entire adult blood system and can cure a range of blood disorders. Within these contexts, the term fitness is regularly used but often poorly defined. In this perspective, we summarise two distinct types of HSC fitness, clonal fitness and stem cell fitness. We go on to introduce mechanisms known to shape each and discuss the therapeutic implications for modulating HSC fitness mechanisms. Teaser Abstract This Perspective discusses two types of hematopoietic stem cell fitness, clonal fitness and stem cell fitness, and summarise mechanisms known to shape each and their therapeutic implications.
Mast cells (MCs) are well known for their roles in immunity, but their influence on hematopoietic stem cell (HSC) regulation remains poorly defined. Here, we identify MC-derived histamine as a suppressor of hematopoiesis. MC-deficient "SASH" mice exhibited increased bone marrow HSC frequency, a transcriptional quiescence signature, and resistance to myeloablative chemotherapy, associated with niche remodeling, including increased HSC-supportive stromal populations and elevated maintenance factor expression, and improved engraftment of wild-type donor cells. Reciprocal transplants showed this phenotype is driven by the recipient niche rather than an HSC-intrinsic property, and MC-derived secretory factors were directly sufficient to suppress HSCs in vitro and in vivo. Pharmacologic H1 receptor blockade with cetirizine, an FDA-approved inverse agonist, phenocopied the SASH model, expanding HSCs and enhancing both early recovery and long-term reconstitution in transplant settings, while exogenous histamine reversed the SASH phenotype. Supporting clinical relevance, electronic health record analysis revealed that antihistamine use was associated with elevated white blood cell counts in humans. These findings establish mast cell-derived histamine as a suppressor of hematopoiesis and suggest that H1R antagonism may offer a tractable strategy to enhance hematopoietic regeneration.Teaser Abstract: Mast cell-derived histamine restrains hematopoietic stem cell activity by shaping the bone marrow niche. Blocking histamine signaling with the FDA-approved antihistamine cetirizine expands the stem and progenitor pool and accelerates hematopoietic recovery after transplant, pointing to a repurposable strategy for boosting blood regeneration in patients.
Infectious complications remain a leading cause of early mortality in patients with acute myeloid leukemia (AML), yet the contribution of qualitative neutrophil abnormalities to infectious risk remains poorly understood. We prospectively evaluated circulating neutrophil phenotypes and oxidative burst reserve in 27 consecutive patients with newly diagnosed AML treated at two tertiary referral centers. Neutrophil subsets were characterized by multiparameter flow cytometry according to CD16/CD62L expression and compared with those of healthy controls (n = 10) and AML patients in complete remission after induction therapy (n = 10). At diagnosis, patients exhibited expansion of activated-like N1 and immature N2B subsets, contraction of the predominant homeostatic N2A population, and impaired oxidative burst reserve (all p < 0.05). Both phenotypic abnormalities and oxidative burst partially recovered after induction therapy. Infection-related mortality occurred in 22.2% of patients. After adjustment for age, the log-transformed N1/N2A ratio remained independently associated with infection-related mortality (adjusted OR 4.90, 95% CI 1.17-20.49; p = 0.029). The ratio also demonstrated moderate discriminatory performance (AUC 0.774, 95% CI 0.585-0.963; p = 0.049), and the optimal cutoff (-2.022) identified patients with significantly inferior infection-related survival (HR 12.83, 95% CI 2.62-62.90; log-rank p = 0.0017). In addition, higher log(N1/N2A) values were associated with lower oxidative burst reserve (Spearman r = -0.45; p = 0.018). These findings suggest that neutrophil phenotypic remodeling reflects innate immune dysfunction in AML and that the log-transformed N1/N2A ratio may serve as a practical biomarker for early identification of patients at increased risk of infection-related mortality.
Hematopoietic stem cells (HSCs) are essential for the reconstitution of the hematopoietic and immune systems and are widely used in transplantation and emerging cell-based therapies. Cryopreservation is a critical technology enabling long-term storage, banking, and distribution of HSC grafts and immuno-oncology cell products. However, the cryopreservation process exposes stem cells and progenitors to multiple sources of cryoinjury, including intracellular ice formation, osmotic shock, solute effects and ice recrystallization, which can compromise post-thaw viability, recovery and functional potency. For decades, dimethyl sulfoxide (DMSO) has remained the gold-standard cryoprotective agent (CPA) due to its ability to reduce osmotic stress, limit intracellular ice formation and stabilize cellular structures. Despite its widespread use, it is associated with dose- and time-dependent cytotoxicity and adverse infusion-related reactions, motivating efforts to reduce or replace its use. This review summarizes key cryobiological principles underlying HSC preservation, including the importance of optimized CPA exposure, controlled cooling, rapid thawing, and storage below glass transition temperature. We also discuss established and emerging permeating and non-permeating CPAs, including sugars, polymers, and carbohydrate-based ice recrystallization inhibitors (IRI), several of which have shown to improve post-thaw outcomes and engraftment in preclinical models. Finally, we review strategies to mitigate DMSO toxicity, including reduced-DMSO formulations and newly developed "all-in-one" DMSO-free cryosolutions. Collectively, these advances are driving the evolution of safer and more effective cryopreservation strategies for HSC transplantation and next-generation cellular therapeutics.
Acute myeloid leukemia (AML) exhibits heterogeneous outcomes and lacks reliable prognostic markers. As a critical regulator of cell fate, calcium signaling's prognostic value in AML requires investigation. This study aims to construct a calcium-related gene (CRG)-based prognostic model for AML. Differential analysis on RNA-seq data was conducted for AML from TCGA and GEO. Intersecting differentially expressed genes and CRGs yielded AML-associated differentially expressed CRGs (DECRGs). A prognostic model was developed through univariate/multivariate Cox regression and LASSO, and validated in a GEO dataset. Bioinformatics analyses explored the links between risk groups and immune characteristics, genomic mutations, and drug sensitivity. Key genes' effects on cell proliferation, apoptosis, and differentiation were verified in vitro using CCK-8 assay, colony formation assay, and flow cytometry. The 13-DECRG-based model distinguished high- and low-risk patients in both training and validation cohorts, with high-risk patients showing a worse prognosis. The risk score was an independent prognostic factor. Immune analysis revealed a unique immune microenvironment for the high-risk group. CAMK2A overexpression inhibited cell proliferation and colony-forming ability, promoted cell apoptosis, and induced an increased proportion of CD11b- and CD14-positive cells. In vitro experiments indicated CAMK2A-induced suppression of AML cells' malignant phenotype by activating the P53 signaling pathway. An AML CRG-based model with favorable risk stratification performance was constructed. In vitro experiments revealed CAMK2A-induced inhibition of the malignant phenotype via suppressing proliferation, promoting apoptosis, and facilitating myeloid differentiation in AML cells. This study provides novel evidence for understanding CRGs in AML as well as the potential functions of CAMK2A.
Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene-editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) and associated systems (CRISPR-Cas), and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, nongenotoxic conditioning strategies. Here, we reviewed recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide.
Ji and colleagues show that endothelial deletion of PCBP1, a multifunctional nucleic acid-binding protein, reduces intra-arterial hematopoietic cluster formation, and impairs subsequent hematopoietic development. These findings identify PCBP1 as a new regulator of early definitive hematopoiesis although leaving its underlying molecular mechanism unresolved.
This study aimed to generate lentiviral vectors carrying an array of AP-1 motifs driving luciferase gene expression as reporters of mitogen-activated protein kinase (MAPK) activity. We created a series of vectors based on LeGO-iG that were used to generate stably transduced leukemia cell lines. A vector termed LEGO-AP1×6-GM55 containing an array of 6 AP-1 sites linked to the minimal CSF2 promoter was sufficient to support high levels of MAPK-inducible luciferase activity in leukemic cell lines that was suppressed by MAPK inhibitors. The inclusion of a putative chromatin priming element encompassing RUNX and ETS motifs increased the activity of these vectors. The additional inclusion of the full-length mouse CSF2 promoter, or the human DUSP5 promoter further increased the MAPK-dependent activity of these vectors in leukemic cells. These vectors support moderate levels of constitutive activity in cells carrying mutations that activate the RAS/RAF/MEK MAPK signaling pathway, and high-level activity after direct activation of MAPK signaling. They also respond to T-cell receptor activation via MAPK and Ca2+ signaling pathways. This resource will now make it easier to track receptor or oncogene-inducible MAPK activity in cultured cells, and potentially in tumors, close to real-time.
Hematopoietic stem and progenitor cells (HSPCs) are characterized by the ability to proliferate, differentiate into multiple lineages, self-renew and repopulate the hematopoietic system. Development of functional assays enabling qualitative and quantitative measurements of these different features are cornerstones of the field. In this review, we summarize key approaches, from early transplantation and spleen colony-forming unit assays to in vitro clonogenic systems, long-term culture assays, xenotransplantation models, and single-cell platforms with historical perspectives and evaluation of their utilities as well as limitations. We emphasize the principles of functional definition of stem and progenitor cells and the discoveries of clonal origin and functional heterogeneity within phenotypically defined HSPC populations.
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a key component of the innate immune machinery, that detects the presence of cytosolic dsDNA and initiates type I interferon-driven inflammation, resulting in downstream immune responses. In cancer, aberrant activation or suppression of this signaling pathway connects genomic instability with immune surveillance, causing context-dependent outcomes that can either augment antitumor immunity or promote tumor progression. This paradox can be exemplified using acute myeloid leukemia (AML), a genetically and clinically heterogeneous hematologic malignancy with poor long-term survival, as a model disease. Emerging evidence indicates that cGAS-STING signaling plays multifaceted roles in AML pathogenesis, influencing leukemic cell survival, stemness, inflammatory signaling, and interactions with the immune microenvironment. Genetic and epigenetic alterations common in AML engage the cGAS-STING axis to drive chronic inflammatory states, clonal expansion, and leukemic transformation, whereas acute or therapeutic activation of STING can improve antileukemic immunity. Pharmacologic STING activation promotes dendritic cell maturation, type I interferon production, and cytotoxic T cell and natural killer (NK) cell responses, and can synergize with hypomethylating agents, poly(ADP-ribose) polymerase (PARP) inhibitors, immune checkpoint blockade, and nanoparticle-based delivery systems. This review highlights current understanding of cGAS-STING biology, its contentious roles in cancer, and its specific functions in AML, highlighting therapeutic opportunities, caveats and challenges. We discuss how the delicate balance between acute and chronic STING signaling in the setting of underlying genetic regulation and immune landscape can play critical role in determining clinical outcome, positioning cGAS-STING as a promising therapeutic target and biomarker of sterile inflammation in AML.
Early achievement of deep remission improves patients' outcome in chronic myeloid leukemia (CML) treatment, highlighting the need for predictive indicators before therapy initiation. This study aimed to investigate whether baseline hematologic profiles are associated with molecular response in CML. Using hierarchical clustering of complete blood count (CBC) data at diagnosis, patients were stratified into two clusters. Patients in Cluster 1 had higher BCR::ABL1IS mRNA levels at 3 and 6 months post-treatment and lower rates of major molecular response compared with cluster 2. Cluster 1 also showed increased granulocyte and immature white blood cell counts and decreased erythroid parameters. Flow cytometric analysis of bone marrow mononuclear cells revealed that cluster 1 had a significant increase in hematopoietic stem cell fractions and a higher ratio of granulocyte-macrophage progenitors to megakaryocyte-erythroid progenitors compared with cluster 2. These findings suggest that differences in bone marrow progenitor cell differentiation affect peripheral blood profiles. Artificial intelligence-driven ghost cytometry (GC) comprehensively captured these differences, distinguishing poor responders, with diagnostic GC scores correlating with BCR::ABL1IS mRNA levels. The study indicates that multivariate analysis of CBC and GC captures biological features associated with early molecular response in CML.
Thalidomide is a well-known immunomodulator that has been recently studied for its effects in increasing HbF levels and decreasing the clinical severity of non-transfusion-dependent thalassemia (NTDT) and transfusion-dependent thalassemia (TDT) with promising results regarding the reduction of transfusion requirements. Recent studies demonstrated the effectiveness of thalidomide in reducing transfusion requirements in TDT and increasing hemoglobin levels in NTDT. This manuscript presents a case of NTDT pediatric patient with concomitant autoimmune hemolytic anemia (AIHA) having significant clinical response to thalidomide. The manuscript also provides a focused literature review on thalidomide use in this population.
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 HSC and one progenitor cell 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.
Clonal hematopoiesis (CH), an age-related expansion of somatically mutated hematopoietic clones, is associated with increased risk of severe infections including coronavirus disease (COVID)-19, yet the underlying mechanisms remain unclear. Here, we investigated the impact of Dnmt3a deficiency in a murine model of influenza A virus (IAV) pneumonia. Dnmt3a-deficient mice exhibited increased pulmonary viral burden and reduced neutrophil accumulation in IAV-infected lungs despite comparable circulating neutrophil numbers. Functional analyses of neutrophils showed impaired chemotactic migration in vitro, whereas maturation, antimicrobial enzyme content, and metabolic capacity were unchanged. Transcriptomic profiling revealed downregulation of pathways involved in chemotaxis, cytokine signaling, and cellular activation, including reduced expression of Cxcr1. Supporting the translational relevance of these findings, proteomic analysis of plasma from individuals with germline DNMT3A mutations (Tatton-Brown-Rahman syndrome) revealed alterations in proteins associated with cell migration and cytoskeletal dynamics. Collectively, our findings demonstrate that Dnmt3a loss compromises innate immune defense by impairing neutrophil migration in a cell-intrinsic manner, leading to ineffective pathogen clearance. This work provides mechanistic insight into how CH-associated mutations contribute to age-associated susceptibility to infection and highlights altered leukocyte trafficking as a potential therapeutic target in aging populations with CH.
Hematopoietic stem cell (HSC) aging is often described as a gradual loss of stem cell fitness that culminates in impaired blood production, immune dysfunction, and increased susceptibility to hematologic disease. However, recent work suggests that this view is too simple. Rather than a uniform decline, aging appears to remodel the HSC compartment into metabolically and functionally distinct states, including maladaptive trajectories as well as surprisingly resilient subsets. In this review, we argue that HSC aging is best understood through the interplay of mitochondrial regulation, metabolic uncoupling, and niche-derived stress, with particular emphasis on how recent findings revise several longstanding assumptions in the field (Box 1).
Somatic mutations involving the canonical exon 10 hotspots of the thrombopoietin receptor gene (MPL) are established drivers in myeloproliferative neoplasms (MPNs), whereas the significance of rare, atypical variants remains unclear. We investigated the relevance of three uncommon MPL variants affecting residues Y591 and R592. Among a multicenter cohort of myeloid neoplasms undergoing next-generation sequencing (NGS), eight individuals harboring MPL p.Y591D, p.Y591H, or p.R592Q variants were identified. Clinical data were integrated with a comprehensive literature review, in silico pathogenicity prediction, and structural bioinformatics modeling of the MPL-JAK2 complex. Clinically, these variants were observed across heterogeneous myeloid disorders and consistently co-occurred with canonical driver mutations in MPN cases. Certain patients with Y591 substitutions seem to exhibit aggressive disease phenotypes and/or suboptimal responses to Janus Kinase (JAK) inhibitors, whereas R592Q cases showed variable outcomes influenced by comutational profiles. Computational predictors yielded discordant pathogenicity assessments. Structural modeling indicated that these substitutions induce minor local rearrangement and do not significantly disrupt the overall MPL-JAK2 complex assembly. However, the Y591 substitutions may lead to loss of critical regulatory motifs by removing a key phosphorylation-dependent docking site and disrupting the YXXφ motif, potentially leading to receptor hypersensitivity to thrombopoietin. Regarding pathogenicity classification, the p.R592Q substitution should be considered as a variant of uncertain significance (VUS), whereas the Y591 alterations may be categorized either as VUS or as "likely oncogenic" depending on the chosen framework. Crucially, current evidence indicates that MPL p.Y591D, p.Y591H, and p.R592Q variants do not act as primary oncogenic drivers but may function only as disease modifiers.
Despite therapeutic advancements and improved patient outcomes, relapsed and refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) after anti-CD19 chimeric antigen receptor T-cell (CART19) therapy due to antigen loss remains a critical unmet clinical need. In this study, we identify that integrin α4 is consistently expressed on B-ALL cells before and after CART19. CRISPR/Cas9-mediated CD19 knockout in primary B-ALL cells did not alter integrin α4 expression, further suggesting stable integrin α4 expression independent of CD19 a stable target. Using the United States Food and Drug Administration (FDA)-approved anti-integrin α4 antibody natalizumab (NZM), we demonstrated effective disruption of leukemia cell adhesion to both VCAM-1, the primary integrin α4 ligand, and to stromal OP9 cells, thereby critically reducing interactions with the leukemia-supportive microenvironment. Most importantly, NZM treatment markedly extended survival in NSG mice engrafted with post-CART19-relapsed B-ALL compared with controls. Our work establishes integrin α4 as an ideal marker for identifying leukemia cells in patients receiving CART19.
