Ionizing radiation and chemotherapy significantly impair hematopoietic stem and progenitor cell (HSPC) function, increasing the risk of bone marrow failure and secondary malignancies. Mesenchymal stromal cells (MSCs), critical regulators within the hematopoietic niche, maintain HSPC quiescence, self-renewal, survival, and differentiation. However, the specific pro-regenerative signaling pathways activated by MSCs in human HSPCs remain incompletely defined. Here, we show that bone marrow-derived MSCs effectively suppress irradiation-induced apoptosis and preserve the in vivo repopulation capacity of human HSPCs. Transcriptomic analysis of HSPCs revealed a pronounced upregulation of CREB target genes following MSC co-culture, consistent with increased activation of the cAMP/CREB signaling pathway. Mechanistically, MSC-secreted prostaglandin E2 (PGE2) emerged as a key mediator of cAMP induced response in HSPCs. Notably, the protective effect of Forskolin/IBMX persisted for up to 72 hours post-irradiation and significantly enhanced HSPC self-renewal. At the molecular level, we revealed reduced pro-apoptotic ASPP1 and PUMA expression, elevated p21 and stabilized anti-apoptotic MCL1 and BCL-XL proteins in human HSPCs treated with cAMP pathway agonists. Overall, our findings highlight the pivotal role of PGE2/cAMP/CREB signaling axis as a central mediator of MSC-mediated protection of human HSPCs under genotoxic stress and identify pharmacological cAMP activation as a promising strategy to protect human HSPCs against DNA damage-induced hematotoxicity.

Background. Imetelstat is a first-in-class human telomerase (hTERT) inhibitor, decreasing the upregulated telomerase activity of neoplastic hematopoietic stem and progenitor cells (HSPCs). By suppressing hTERT, Imetelstat selectively eliminates dysplastic clones, enabling restoration of normal hematopoiesis. Recently approved by the FDA for the treatment of red blood cell transfusion–dependent (RBC-TD) lower-risk myelodysplastic syndromes (LR-MDS) patients after erythropoiesis-stimulating agent (ESA) failure, its precise mechanism of action remains unclear. We provide the first in vivo evidence that Imetelstat enhances erythroid maturation by alleviating ineffective erythropoiesis and reducing bone marrow (BM) clonal burden in responding LR-MDS patients.Methods. Comprehensive erythroid flow cytometry (FC) and targeted next-generation sequencing (t-NGS) analyses were performed on BM samples from LR-MDS patients enrolled in the phase III IMerge trial (. NCT02598661). FC analysis included total CD71⁺ erythroid cells (Erytot), early CD34+/CD117⁺, and late CD117⁻ erythroid precursors. The erythroid ratio (ER) was defined as the ratio of late to early precursors. BM erythroid indices and variant allele frequency (VAF) of driver somatic mutations were longitudinally assessed at baseline, +6 and +12 months after Imetelstat initiation; extended FC data were available at +24 months for one ongoing responder.Results. Four representative cases were analyzed: (a) a long responder (>1 year RBC transfusion independence), (b) a transient responder (<1 year RBC-TI), (c) a non-responder, and (d) a placebo-treated patient. The sustained responder displayed marked and persistent decreases in Erytot population (29%→11% at 24 months) accompanied by increased ER (2.36→7.33), consistent with enhanced erythroid maturation (Figure 1a). The transient responder exhibited a similar pattern during response (Erytot 17%→2%, ER 3→6.14 at 6 months) but relapsed by 12 months, with Erytot expansion (30%) and ER reduction (2.22). No significant changes were observed in non-responder or placebo cases. Clonal dynamics mirrored erythroid trends: BM VAFs of driver mutations decreased during hematologic response (22%→4.7% in the ongoing responder at 12 months, 32.9% →4.3% in the short responder at 6 months) and re-expanded at relapse (4.3%→22.6% in the transient responder at 12 months, Figure 1b). As for erythroid populations, BM VAF did not change significantly over time in both the non-responder and the placebo-treated patient. Conclusions. Imetelstat treatment in LR-MDS promotes erythroid maturation and reduces BM clonal burden during clinical response, supporting a dual mechanism of action that combines telomerase-mediated clonal suppression with restoration of effective erythropoiesis. These findings provide in vivo mechanistic insight into how telomerase inhibition reverses ineffective erythropoiesis in MDS and underpin the durable clinical responses observed in the IMerge trial.

Ex vivo culture of hematopoietic stem and progenitor cells with platelet lysate: Investigating proliferation and erythroid-megakaryocytic lineage effects.

Emerging Considerations in Transfusion Medicine: Hematopoietic Stem and Progenitor Cell Collection for Gene Therapy for Sickle Cell Disease and Transfusion-Dependent Thalassemia.

Clonal hematopoiesis (CH) refers to the presence of somatic variants in hematopoietic stem and progenitor cells (HSPC) that result in expansion over time. CH of indeterminate potential (CHIP) is operationally defined as pathogenic variants in oncogenic driver genes occurring in HSPCs at variant allele frequencies ≥ 2%. CH is associated with increased risk for progressive cytopenias (also called clonal cytopenia of undetermined significance), hematological (predominantly myeloid but also lymphoid) neoplasms, cytosis (including monocytosis), and nonhematological conditions such as atherosclerotic cardiovascular and cerebrovascular disease. CH is linked to numerous other diseases including venous thromboembolism, type 2 diabetes mellitus, chronic obstructive pulmonary disease, osteoporosis, and gout, with a potential protective impact in Alzheimer's disease (AD). CH detection is becoming increasingly common due to the ubiquitous use of somatic and germline sequencing in clinical practice, particularly, in oncology. The clinical implications of CH are most relevant in therapy-related myeloid neoplasms (t-MN), with antecedent CH clones in genes such as TP53, PPM1D, and/or CHEK2 having a clear selection advantage. Furthermore, genetic predisposition to CH has provided some clarity on the origin and evolution of CH. We are currently defining the role for CH assessment in individuals with persistent (≥ 4 months) unexplained cytopenias, in patients with malignancies prior to adjuvant cytotoxic chemotherapy and/or radiation or radionuclide therapy, screening prior to autologous hematopoietic stem cell transplantation or chimeric antigen receptor T cell (CAR-T) therapy, and to work-up potentially germline mosaic variants.

Identification of Siglec-15 as a novel target for CAR-T cell therapy in acute myeloid leukemia.

MDM4 HAPLOINSUFFICIENCY LEADS TO P53-MEDIATED BONE MARROW FAILURE.

Aging-related blood disorders are linked to defects in the regenerative and multilineage differentiation ability of hematopoietic stem and progenitor cells (HSPCs). While remodeling of the bone marrow (BM) microenvironment where HSPCs reside is known to contribute to these age-associated defects, the underlying factors and mechanisms remain poorly defined. Here, we discovered that the age-related decline of the neurotransmitter neuropeptide Y (NPY) in the BM is a critical driver of HSPC dysfunction. Using mouse models, we demonstrated that NPY levels decrease in the BM with age, and that genetic NPY overexpression or exogenous NPY administration in old mice substantially reverses aging-associated phenotypic and functional defects in HSPCs. Transcriptome analysis revealed that NPY supplementation in old mice restores aging-disrupted molecular pathways in their HSCs, including oxidative stress responses, myeloid differentiation, stemness, mitochondrial activity, and RhoA signaling. However, NPY genetic loss in young mice led to a decline in HSCs regenerative capacity and increased oxidative stress. Importantly, NPY levels also decline in elderly humans, and ex vivo treatment of human BM-derived HSPCs with NPY enhances their in vivo repopulating capacity. These results suggest that NPY supplementation or preservation of NPY-producing nerve fibers could be a therapeutic strategy to rejuvenate aged HSC function.

Durable control of HIV infection is challenged by persistent latent reservoirs, including hematopoietic stem and progenitor cells (HSPCs), which provide a unique niche for proviral silencing. Mechanisms of HIV latency in HSPCs remain poorly studied. Here, we utilized a dual-reporter HIV (89.6 VT1) and single-cell RNA sequencing to identify host factors governing latency in HSPCs. Transcriptomic profiling revealed elevated expression of several genes in latently infected cells, among them the transcriptional repressor GFI1. Functional studies showed that GFI1 suppresses HIV gene expression by binding a conserved sequence near the primer binding site within the long terminal repeat (LTR). Disruption of GFI1 DNA-binding or corepressor recruitment domains diminished its silencing effect. GFI1 also antagonized Tat and NF-κB-mediated activation, and reversal of GFI1-mediated suppression was most robust with combined HDAC inhibition and NF-κB activation. These findings position GFI1 as an important regulator of HIV latency in HSPCs.

Clec16a maintains definitive hematopoietic stem and progenitor cells via mitophagy.

Childhood is a critical period for hematopoietic development and susceptibility to hematologic disease. Here we generated a multimodal single-cell atlas of healthy human bone marrow, capturing mRNA and surface protein expression in 90,710 cells, including over 20,000 hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) from nine donors ranging from infancy to young adulthood (2-32 years). Young pediatric (YP) bone marrow (<10 years) was compositionally and molecularly distinct from adolescent and young adult (AYA) bone marrow (≥13 years), with hematopoietic output shifting from B cell dominance in YP bone marrow to myeloid and T cell bias in AYA bone marrow. Spatial transcriptomics of six bone marrow biopsies (0-23 years) confirmed these age-dependent changes. Two lymphoid progenitor (LyP) subsets regulated this lineage shift: CD127+ LyP cells with B cell-biased output were enriched before age 10, whereas CD127- LyP cells with combined lymphoid and myeloid features predominated thereafter. Stromal signaling showed corresponding age-dependent changes, with increased interleukin-7 production by bone marrow MSC in YP compared to AYA, indicating niche-mediated regulation of HSPC lineage potential during ontogeny. This single-cell atlas provides a comprehensive resource for understanding hematopoietic development and early-life origins of hematologic disease.

Hematopoietic stem and progenitor cells (HSPCs) are crucial for blood production and regeneration. While their function is known to be regulated by diverse physical cues, the impact of pervasive radiofrequency electromagnetic fields (RF-EMF), particularly through non-thermal radiofrequency radiation (RFR) mechanisms, remains poorly understood. We conducted colony-forming unit (CFU) assay in vitro and competitive transplantation assay in vivo to evaluate whether RFR influences hematopoiesis reconstitution capacity. Subsequently, the effects of RFR preconditioning on hematopoietic injury induced by ionizing radiation in mice were assessed by continuously monitoring the peripheral blood, HSPCs number, and colony-forming units. The influence of RFR on radioprotection unit frequency was evaluated using multiple gradients, non-competitive mouse transplantation models. Seahorse XF assays were employed to characterize cellular energy metabolic status, while specific fluorescent probes were utilized to detect calcium ion (Ca2+) levels in distinct cellular compartments using flow cytometry. Transcriptomic profiling was used to uncover the underlying mechanisms. HSPCs were pretreated with plasma membrane Ca2+-ATPase (PMCA) inhibitor prior to RFR exposure, and Seahorse assays along with CFU assay and competitive transplantation assay were performed to compare whether PMCA inhibition could abrogate RFR-induced HSPCs function change. To investigate the mechanism by which RFR enhanced PMCA activity inducing Ca2+ efflux, we performed fluorescence recovery after photobleaching (FRAP) assays to detect membrane fluidity. Non-thermal 2856MHz RFR enhanced HSPCs colony formation activity and reconstitution capacity, without compromising the multilineage differentiation homeostasis. RFR preconditioning accelerated hematopoietic recovery following ionizing radiation and increased radioprotection unit frequency. Mechanistically, RFR increased plasma membrane fluidity which potentiates PMCA activity, resulting in elevated Ca2+ efflux and reduced intracellular Ca2+ levels. These cellular alterations ultimately contributed to maintaining HSPCs in a low metabolic state, and consequently improving their functional capacity. Pharmacological inhibition of PMCA abolished both the functional enhancement and metabolic suppression. Our results provided the first evidence that non-thermal RFR can improve HSPCs function. The central mechanism involved RFR-induced plasma membrane fluidity, activation of PMCA, thus accelerating Ca2+ efflux and maintaining HSPCs in a metabolically quiescent state. This work provided transformative insights into electromagnetic field biology and potential transplantation strategies for radiation-induced hematopoietic injury.

Radiation adaptive response (RAR) significantly improves resistance to subsequent high-dose radiation exposure. While hematopoietic stem and progenitor cells (HSPCs) are known to be crucial for radiation protection, their specific roles and response mechanisms in RAR remain unclear. Systematic investigation of these processes could reveal valuable therapeutic targets for developing HSPCs-focused radiation protection strategies. We establish a radiation-adaptive mouse model by administering a priming dose of 0.5 Gy γ rays after a 14-day interval and then investigate the protective effects and underlying mechanisms of RAR on HSPCs. Our results demonstrate that 0.5 Gy pre-irradiation significantly enhances the radioresistance of HSPCs against subsequent 6.5 Gy irradiation, attenuates the radiation-induced suppression of both HSPCs quantity and repopulation capacity, and consequently facilitating the restoration of hematopoiesis. Additionally, 0.5 Gy irradiation alone increases the DNA damage and apoptosis of HPCs and LSKs, which in turn activates dormant HSCs but attenuates their long-term repopulating capability. Notably, 0.5 Gy irradiation activates the IL-17 signaling pathway in LSKs and upregulates IL-17A expression in both plasma and jejunum. Neutralization of IL-17A not only abrogates the RAR induced by 0.5 Gy pre-irradiation but also attenuates the protective effect of this pre-irradiation on HSPCs. This study systematically elucidates the radioprotective effects of RAR on mouse bone marrow cell subsets, unveils the important potential role of the IL-17 signaling pathway in RAR, and provides new insights into the underlying mechanisms of radiation-adaptive protection. It also provides theoretical support for the development of targeted intervention strategies in radiation protection for HSPCs.

Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA). The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings. APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34+ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34+. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity. Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.

Despite intriguing roles for the Succinate receptor (Sucnr1) in inflammation, few studies have explored its role in hematopoiesis. Here, we show that low SUCNR1 represents a marker for reduced overall and progression-free survival in acute myeloid leukemia (AML) patients. Succinic acid, which displays Sucnr1-dependent and independent effects, promotes disease in mouse models of pre-leukemic myelopoiesis, AML and AML xenografts, expressing low SUCNR1. In vivo global or hematopoietic deletion of Sucnr1 induces expansion of hematopoietic stem and progenitor cells (HSPC) and hematopoiesis, whilst Sucnr1-tomato+ HSPC display restricted engraftment potential. Mechanistically, activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC and preserves HSPC transcriptional programs via control of S100a8/S100a9. Blocking S100a9 with tasquinimod rescues the defects of Sucnr1 knock-out mice, and combined with a potent Sucnr1 agonist shows therapeutic value in AML mice. In AML xenografts, single-cell RNA-sequencing reanalyses confirm SUCNR1 as a therapeutic vulnerability in patients. Together, Sucnr1 signaling restricts hematopoiesis at least partially through HSPC and via control of S100a8/S100a9. Its dysregulation emerges as contributor to malignancy that opens therapeutic avenues for AML patients.

A 3D "nichoid" boost for gene-engineered blood stem cells.

BackgroundMaternal obesity is a global health challenge with profound consequences for offspring health. While its impact on metabolic programming has been widely studied, far less is known about how maternal obesity shapes the fetal immune system. The fetal bone marrow (FBM) is the central site of hematopoietic stem and progenitor cell (HSPC) development, and disruptions in this niche can have lifelong effects on immunity, infection susceptibility, and inflammatory disease risk. In this study, we examined FBM hematopoiesis in a nonhuman primate model of spontaneous maternal obesity.MethodsUsing spectral flow cytometry, single-cell RNA sequencing, and functional differentiation assays, we mapped progenitor composition, lineage trajectories, and immune function in offspring exposed to maternal obesity compared with lean controls. These complementary approaches allowed us to capture cellular frequencies and transcriptional programs, while trajectory and signaling analyses provided insight into how progenitor maturation and intercellular communication are disrupted by maternal obesity.ResultsOur findings reveal that maternal obesity decreases CD34+ HSPCs and common lymphoid progenitor populations, while expanding megakaryocyte-erythroid and granulocyte-monocyte progenitors. Pseudotime analysis demonstrated altered maturation, with cells accumulating at early differentiation states. Transcriptional profiling uncovered a strong inflammatory bias, with myeloid progenitors upregulating alarmins, interferon-stimulated genes, and proinflammatory mediators. Functionally, monocytes derived from obese FBM showed impaired migratory and colony-stimulating capacity, coupled with exaggerated TNFα responses to LPS stimulation.ConclusionTogether, these results demonstrate that maternal obesity, even in the absence of obesogenic diet, disrupts fetal bone marrow hematopoiesis by altered HSPC maturation, reprogramming lineage trajectories, and inducing inflammatory bias.

Recent research has challenged a long-held view of the brain as an immune-privileged organ, revealing active immunosurveillance with therapeutic relevance. Using a new genetically engineered mouse model of ZFTA-RELA ependymoma, a childhood brain tumor, we characterized an immune circuit between the tumor and antigen-presenting hematopoietic stem and progenitor cells (HSPCs) in the skull bone marrow. The presentation of antigens by HSPCs to CD4+ T cells biased HSPC lineages toward myelopoiesis and polarized CD4+ T cells to regulatory T cells, culminating in tumor immunotolerance. Remarkably, normalizing hematopoiesis with a single infusion of antibodies directed against cytokines enriched in the cerebrospinal fluid of mice bearing ZFTA-RELA ependymomas, choroid plexus carcinomas or group 3 medulloblastoma-all aggressive childhood brain tumors-disrupted this process and caused profound tumor regression. These findings demonstrate the existence of a skull bone marrow-tumor immunological interface and suggest that modulating the local supply of myeloid cells could represent a less toxic therapeutic strategy for aggressive childhood brain tumors.

In vivo production of an anti-HIV antibody in mice by non-viral gene knockin in primate hematopoietic stem and progenitor cells.

Effective T cell reconstitution in people living with HIV is central to durable immune control and cure strategies. Sustained thymic output underpins T cell recovery and requires continuous seeding by T cell-committed progenitors originating in the bone marrow (BM). Using the SIV/rhesus macaque model, we identified a thymus-seeding progenitor (TSP; CD4⁻CD8⁻CD34⁺CD38⁻CD7⁺) in BM declining rapidly following SIV infection. This loss closely associated with reduction in T cell lineage committed differentiation of BM-derived hematopoietic stem and progenitor cells (HSPCs). Importantly, both the decline in TSPs and the impairment of pre-thymic T cell potential were strongly associated with early loss of viral control, independent of peripheral T cell dynamics. Plasma interleukin-6 (IL-6) levels robustly predicted the magnitude of TSP loss and the restriction of T cell-biased HSPC differentiation. Integrated transcriptomic and proteomic analyses revealed inflammatory imprinting of HSPCs characterized by activation of the IL-6-JAK-STAT axis, inflammasome engagement, and coordinated suppression of key T cell specification factors, including RUNX1, FYN, and ZAP70. In a nonanimal model of thymopoiesis, IL-6 exposure of rhesus macaque and human HSPCs inhibited their transition from DN1 (CD38⁻) to DN2 (CD38⁺) TSP states, indicating an early block in T cell lineage commitment. Conversely, ex vivo IL-6 receptor blockade restored thymocyte differentiation to levels comparable to untreated controls. Collectively, these findings demonstrate that pathogenic inflammation restricts pre-thymic T cell development early after infection, directly contributing to loss of viral control. These findings have important implications for understanding the mediators of anti-viral T cell immunity and HIV cure.