Intra-procedure white blood cell monitoring as a predictor of collection efficiency in mononuclear cell apheresis.

Development and characterization of anti-CXCR4 chimeric antigen receptor T cells.

Anti-thymocyte globulin (ATG Thymoglobulin®) and anti-T-lymphocyte globulin (ATLG Grafalon®) are commonly used in hematopoietic stem cell transplantation (HSCT) for in vivo T-cell depletion, aiming to reduce graft failure and graft-versus-host disease (GvHD). Despite their widespread use, the precise mechanisms by which ATG/ATLG affect immune recovery post-transplantation remain partially understood. In this study, in 289 pediatric HSCT patients treated with ATG (n=213) or ATLG (n=76), the relationship between longitudinal serum levels of the active T-lymphocyte-binding component of ATG/ATLG and immune reconstitution was evaluated. High levels of active ATG/ATLG did not prevent the recovery of neutrophils or NK-cells; however, T-cell recovery was delayed until active ATG/ATLG concentrations fell below 1AU/mL. This is in apparent contradiction with the fact that ATG/ATLG are known not only to contain antibodies against antigens present on T-cells but also to antigens present on other immune and non-immune cells, including B-, NK-cells, granulocytes, monocytes and hematopoietic stem and progenitor cells (HSPC). To address this, we assessed the binding capacities of ATG and ATLG to immune cell subsets and HSPC both in vitro directly from the vial and in vivo from patients serum samples taken at multiple time points pre- and post-HSCT. Shortly after infusion, we observed a rapid reduction of ATG and ATLG binding to all cell types except for T-cells. This real-life specificity of ATG and ATLG explains their selective impact on T-cell reconstitution. These findings enhance our understanding of the mechanisms of action of ATG and ATLG and may contribute to optimization of these therapies.

Hematopoietic acute radiation syndrome (H-ARS) is a serious clinical condition caused by exposure to high-dose ionizing radiation that leads to the depletion of hematopoietic stem and progenitor cells with the collapse of the function of bone marrow. Despite the clinical significance of H-ARS, there have been few advances in H-ARS research owing to the dearth of physiologically relevant human models that mimic the complex bone marrow microenvironment. In this study, we used a stage-specific mesodermal differentiation protocol to establish human bone marrow organoids (hBMOs) derived from hiPSCs. The resulting organoids exhibited stromal-vascular architecture, supported multilineage hematopoiesis, and contained CD34+ hematopoietic populations, as confirmed by scRNA-seq and flow cytometry. To investigate the impact on the hematopoietic cell population, hBMOs were exposed to γ-irradiation at doses of 3, 6, and 9Gy. The organoids exhibited a marked depletion of hematopoietic cell populations and disruption of niche architecture, which are hallmarks of radiation-induced hematopoietic cell damage. To evaluate the therapeutic potential of hBMOs in H-ARS, hBMOs transplanted into lethally irradiated NSG mice significantly improved survival with successful engraftment of human hematopoietic cells within the host. These findings establish hBMOs as a robust and translational human model of radiation-induced hematopoietic cell damage.

Bone marrow hematopoietic stem and progenitor cells (HSPCs) sense immune activation and instruct systemic immunity. However, the alterations of HSPCs in autoimmune diseases, which are driven by an active immune response, and their impact on disease activity and progression are not clear. Neuromyelitis optica spectrum disorder (NMOSD) is a B cell-mediated autoimmune neurological disease characterized by pathogenic autoantibodies against aquaporin-4 (AQP4-IgG). We observed aberrant bone marrow granulopoiesis in samples from individuals with NMOSD, which was accompanied by B cell clonal expansion. Aberrant granulopoiesis was mediated by hyperactivated JAK-STAT signaling, leading to an increase in ISG15+ neutrophils that produced B cell-activating factor (BAFF). These BAFF-producing neutrophils were sufficient to drive maturation of antibody-secreting cells and autoantibody production in vitro. Aberrant granulopoiesis was also observed in individuals with NMOSD receiving B cell depletion therapy who experienced relapse; in contrast, belimumab, a monoclonal antibody against BAFF, reduced autoantibody titers and number of relapses. Thus, targeting the bone marrow niche may present a treatment strategy for NMOSD and perhaps other B cell-mediated autoimmune diseases.

The bone marrow microenvironment comprises a complex network of hematopoietic stem cells, immune cells, stromal cells, and non-cellular components such as the extracellular matrix and soluble factors, collectively maintaining the homeostasis of hematopoietic stem and progenitor cells. It is highly vulnerable to pathological perturbations induced by hematologic malignancies, solid tumors, inflammatory stress, and therapeutic exposure, which collectively destabilize microenvironmental homeostasis and promote hematopoietic failure, malignant progression, and immune dysregulation. Natural products exhibit unique advantages in modulating the blood microenvironment due to their structural diversity, multitarget effects, and low toxicity. Their biological activities span multiple mechanistic dimensions, including redox regulation, metal ion homeostasis, signaling inhibition, and microenvironment remodeling. However, the intrinsic relationships between their chemical structures and biological functions have not yet been systematically elucidated. Therefore, from a translational medicine perspective, this review focuses on elucidating the pharmacological mechanisms by which natural products regulate the hematopoietic microenvironment. We systematically summarize their chemical basis and structure–activity relationships, together with recent advances in extraction techniques, chemical modification, and targeted delivery strategies. The aim is to bridge the gap between chemical research on natural products and their clinical therapeutic applications, providing a framework and innovative directions for drug development targeting hematological diseases.

Articular cartilage, a highly specialised and avascular tissue, exhibits limited regenerative potential following trauma or degenerative conditions such as osteoarthritis (OA). Conventional surgical interventions, including microfracture and autologous chondrocyte implantation (ACI), have shown limited long-term efficacy due to donor site morbidity and restricted cell proliferation. In this context, mesenchymal stromal cells (MSCs) have emerged as a promising alternative owing to their multipotency, self-renewal capacity, and low immunogenicity. While bone marrow (BM) remains the traditional source of MSCs, recent studies have reported that peripheral blood-derived mesenchymal stromal cells (PB-MSCs) may possess chondrogenic, osteogenic, and adipogenic potential comparable to that of BM-derived MSCs. PB-MSCs can be harvested through minimally invasive methods, thereby avoiding the complications associated with BM aspiration. Experimental evidence indicates that PB-MSCs exhibit strong cell viability, proliferative potential, and the ability to synthesise cartilage-specific extracellular matrix proteins, such as type II collagen and sulphated glycosaminoglycans, within three-dimensional scaffolds. Immunophenotypically, PB-MSCs express mesenchymal markers including CD29, CD44, CD90, and CD105 while lacking hematopoietic markers CD34 and CD45. Flow cytometry analyses reveal that CD105+ populations increase following cryopreservation, highlighting their clinical utility. In contrast to these experimentally defined PB-MSCs, the term peripheral blood stem cells (PBSCs) is used in clinical studies to describe heterogeneous, non-cultured peripheral blood-derived cell preparations, typically enriched in hematopoietic stem and progenitor cells following granulocyte colony-stimulating factor (G-CSF) mobilisation, without full mesenchymal characterisation. In vitro studies confirm successful tri-lineage differentiation, whereas in vivo investigations have demonstrated effective cartilage regeneration using PB-based clinical approaches, including postoperative intra-articular administration of hyaluronic acid (HA) combined with PBSCs, as well as implantation of PBSCs covered with a collagen membrane. Furthermore, advancements in biomaterial engineering, such as poly(ethylene glycol)-cysteine-arginine-glycine-aspartic acid (PEG-CRGD) hydrogels, have enhanced PB-MSC adhesion, proliferation, and chondrogenic differentiation while promoting immunomodulation through M2 macrophage polarisation. Despite these promising outcomes, the available evidence remains limited and heterogeneous, with substantial variability in cell definitions, experimental models, and clinical study designs, which currently constrains definitive conclusions regarding clinical efficacy. Future research should focus on optimising isolation protocols, understanding molecular pathways governing PB-MSC chondrogenesis, and standardising clinical applications. Overall, PB-MSCs represent a viable, less invasive, and translationally relevant cell source for cartilage regeneration and regenerative orthopaedic therapies.

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.

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.

Introduction: Myelofibrosis (MF) is a clonal hematological disease originating by the sequential acquisition of somatic mutations in hematopoietic stem and progenitor cells (HSPC). Alongside so-called “driver mutations” triggering the constitutive activation of JAK-STAT pathway and inducing myeloproliferation, MF may present with additional mutations often affecting epigenetic regulators. The JAK-inhibitor Ruxolitinib (Ruxo) effectively relieves MF symptoms but rarely eradicates the malignant clone, with highly variable responses among patients. Understanding the molecular mechanisms underlying Ruxo heterogeneous efficacy is essential to improve therapeutic strategies.Methods: To elucidate the clonal dynamics associated with Ruxo response, we conducted a longitudinal single-cell (SC) proteogenomic study on 12 MF patients (6 responders and 6 non-responders, as defined by clinical features) at diagnosis and after at least 6 months of Ruxo therapy. Cryopreserved peripheral blood mononuclear cells and CD34+ HSPC from each time point were analyzed through Tapestri platform. Next, longitudinal samples from a Responder and a Non-responder patient form the same cohort were subjected to SC-RNA+protein analysis by means of 10X genomics platform.Results: Ruxo responders showed a marked reduction in circulating CD34+ cells and a decrease in JAK2V617F allele frequency in granulocytes. SC-genomic analysis revealed that the mutation acquisition order determines Ruxo sensitivity: patients in whom the driver mutation either occurred alone or preceded mutations in TET2 or ASXL1 generally responded to therapy, while those in whom epigenetic mutations arose first exhibited limited therapeutic benefit. Using SC-proteomics we identified 14 HSPC and differentiated cell clusters and differences in the clonal dynamics of CD34+ and CD34- cells were observed. Non-responders displayed post-treatment expansion of highly mutated monocytes, suggesting myeloid lineage–driven resistance, whereas responders exhibited an increase in wild-type monocytes. Conversely, in CD34+ cells from responders, we observed both a reduction in driver-homozygous cells and an expansion of heterozygous co-mutated clones despite Ruxo treatment. Notably, despite MF being a primarily myeloid disorder, a fraction of mutated T and B cells was also detected, particularly in non-responders. SC-RNA+protein profiling recapitulated the cellular heterogeneity observed in the SC-proteogenomic dataset, revealing persistent activation of JAK-mediated interferon-response signaling in non-responder monocytes upon treatment, while this pathway was suppressed in responders.Conclusions: Our data demonstrate that the order of mutation acquisition impacts Ruxo response in MF patients. Ruxo primarily affects JAK2-only mutated clones. As a result, co-mutated clones may evade this treatment and outcompete other neoplastic cell populations, thus contributing to disease persistence.

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

Emerging evidence suggests the development of malaria parasites in the human bone marrow. Whether glucose-6-phosphate dehydrogenase (G6PD) deficiency affects parasite growth in erythroblast has yet to be determined. Here, we examine the invasion and development of Plasmodium falciparum in human erythroblasts of subjects carrying the most common variant of G6PD Viangchan (871G>A) in Southeast Asia. Erythroblasts were generated by differentiation of erythroids of human CD34-positive hematopoietic stem and progenitor cells isolated from peripheral blood. The results showed that P. falciparum parasites invade polychromatic erythroblasts and develop into mature trophozoites. The percentages and stages of parasitized erythroblasts were not different between the subjects with G6PD normal, heterozygous, or hemizygous G6PD Viangchan. While the sample size (n = 6) limited statistical analysis, these preliminary findings suggest that protection against malaria is not observed in this in vitro erythroblast model, supporting further validation in expanded cohorts.

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

Transient mRNA-based CD117 CAR T cells effectively target acute myeloid leukemia in vitro for potential use as a preconditioning strategy.

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.

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

Hematopoietic stem and progenitor cells (HSPCs) arise from hemogenic endothelium via the endothelial-to-hematopoietic transition (EHT), a process requiring precise mitochondrial quality control. Here, we identify Clec16a, an E3 ubiquitin ligase, as a conserved regulator of embryonic HSPC emergence. In zebrafish and HEK293T models, Clec16a is enriched in hemogenic endothelium, and its loss disrupts arterial identity, impairs EHT, and reduces lymphoid, erythroid, and myeloid lineages. Transcriptomic and proteomic analyses show that Clec16a deficiency compromises mitophagy by promoting aberrant K48-linked ubiquitination and proteasomal degradation of ATG5, leading to mitochondrial dysfunction and elevated reactive oxygen species. These findings establish Clec16a as an essential regulator linking ubiquitin signaling, mitophagy, and hematopoietic fate specification. Our study defines a mitophagy-dependent checkpoint that safeguards mitochondrial homeostasis during developmental hematopoiesis and provides insight into the metabolic control of hematopoietic disorders.

Targeting the NLRP3 inflammasome signalling in acute myeloid leukemia: Mechanisms, therapeutics, and future directions.

Human immunodeficiency virus (HIV) remains a persistent global health burden, as combination antiretroviral therapy (ART) achieves sustained viral suppression but fails to eliminate long-lived latent reservoirs. Stem cell-based therapeutic strategies have emerged as transformative approaches with the potential to induce durable remission and, ultimately, a functional cure. Clinical proof-of-concept has been established through allogeneic hematopoietic stem cell transplantation (HSCT) using CCR5Δ32/Δ32 donor cells, demonstrating that durable resistance to viral entry can result in prolonged HIV remission. Building on these landmark observations, recent advances in autologous gene-edited hematopoietic stem and progenitor cells and induced pluripotent stem cell (iPSC)-derived immune effectors have accelerated the development of scalable, patient-specific interventions. The convergence of stem cell biology with precision genome-editing platforms, including CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), has enabled targeted disruption of viral entry pathways and host dependency factors, while offering new strategies to address viral latency and immune reconstitution. Despite significant challenges related to treatment-associated toxicity, manufacturing complexity, long-term safety, and ethical considerations, rapid progress in cellular engineering and translational immunology continues to advance the field toward curative outcomes. This review critically synthesizes recent progress in stem cell-based HIV therapeutics, elucidates the underlying mechanistic frameworks, evaluates emerging clinical and preclinical evidence, and outlines future directions required to achieve a durable functional cure.

MDM4 HAPLOINSUFFICIENCY LEADS TO P53-MEDIATED BONE MARROW FAILURE.