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 knock-in in primate hematopoietic stem and progenitor cells.

Aging is a dominant risk factor for chronic diseases characterized by the functional decline of tissues and organs. During aging, the hematopoietic system declines in regenerative capacity-seemingly attributable to increases in DNA damage, replicative stress, and autophagic flux-resulting in skewing towards a myeloid lineage and away from a lymphoid lineage. Here, we characterized the transcriptomic and cellular landscape of the aged C57Bl/6J mouse hematopoietic system using a combination of bulk RNAseq and single cell RNAseq (scRNAseq). We show that aging leads to global transcriptional alterations in bulk peripheral blood mononuclear cells (PBMCs), lineage marker-depleted bone marrow cells (Lin-BM), and in hematopoietic stem and progenitor cells (HSPCs), immunophenotypically lineage marker negative (Lin-) Sca1+ cKit+ (LSK+). These changes indicate widespread activation of inflammatory processes, namely in PBMCs and Lin-BM cells. Interestingly, there is also a downregulation of cell cycle genes in HSPCs during aging. ScRNAseq across 39 hematopoietic cell types revealed age-related skewing in cell composition. Aged PBMCs showed significant decreases in CD4 and CD8 naïve cells concomitant with increases in CD4/8 memory and CD8 exhausted T cell populations. Lin-BM cells showed significant myeloid skewing in common myeloid progenitor (CMP) cells, as well as in the HSC population. We also identified a unique HSC population marked by increased Vwf, Wwtr1, and Clca3a1 expression that does not exist in young HSCs, thus likely marking true aged HSCs. Collectively, this work should serve as a useful resource for understanding and therapeutically targeting the aged hematopoietic system.

Very cold but still too warm; functional characterization of transient warming events in stem cell grafts.

Hematopoietic progenitor cell collection (HPC-C) by apheresis after granulocyte-colony stimulating factor (G-CSF) stimulation is a routine worldwide procedure. The first apheresis procedure is typically performed on Day 5 of G-CSF administration. We aimed to evaluate whether initiating the apheresis procedure on Day 4, with 1 day shorter G-CSF exposure for the donor, affects collection outcomes. This study included healthy, unrelated donors who underwent HPC-C by apheresis after G-CSF administration between April 2022 and February 2024 at our center. Of 182 unrelated donors, 34.6% underwent apheresis on Day 4 of G-CSF treatment and 65.4% on Day 5. There was no significant difference between the two groups in terms of collected CD34+ cell yield (p = 0.369) or the need for a second apheresis procedure (p = 0.74). Successful mobilization was achieved in 93.7% of donors who underwent a single apheresis procedure on Day 4 and in 95% of those who underwent a single apheresis procedure on Day 5 (p = 0.477). In donors requiring two consecutive apheresis procedures, the collected CD34+ cell yield was higher in those who initiated apheresis on Day 4, although this difference was not statistically significant (p = 0.067). This is the first study comparing HPC-C outcomes on Days 4 and 5 of G-CSF administration exclusively in unrelated donors. Our data indicate no difference in the collected CD34+ cell yield or the need for a second apheresis procedure between the two groups. Initiating apheresis on Day 4 may allow for 1 day less G-CSF exposure without compromising collection outcomes.

Unveiling the power of quinazoline derivatives: a new frontier in targeted cancer therapy.

Vitamin C Deficiency and Hemorrhagic Complications in Post-Allogeneic Hematopoietic Progenitor Cell Transplant Patients: A Case Series

Acute myocardial infarction (AMI) impacts the regenerative capacity of hematopoietic stem and progenitor cells (HSPCs) following injury, but it remains unclear if these functional alterations persist beyond the initial ischemic event.A minimally invasive mouse model of recurrent myocardial infarction was established using echocardiography-guided coronary interventions. Bone marrow HSPCs were quantified and analyzed for proliferation by flow cytometry and BrdU incorporation. Peripheral blood leukocytes and inflammatory cytokines (IL-6, G-CSF) were measured by flow cytometry and ELISA. Bone marrow extracellular TGF-β1 was assessed by ELISA, and its functional role was evaluated through antibody-mediated inhibition.The minimally invasive infarction model was validated through electrocardiography, cardiac biomarkers, echocardiography, and cardiac pathology staining. Fourteen days post-I/R or sham treatment, bone marrow hematopoiesis returned to a steady state with no significant differences in Lin-Sca-1+c-Kit+ (LSK), hematopoietic stem cell (HSC), multipotent progenitor (MPP), and granulocyte/macrophage progenitor (GMP) cell numbers. However, after a secondary ischemic challenge, there was a dampened reactive hematopoiesis indicated by reduced HSPCs, compared to the first ischemic event. BrdU incorporation analysis showed decreased HSPC proliferation activity during the reparative phase after initial ischemic challenge, linking dampened hematopoiesis to decreased HSPCs proliferation. Additionally, early recurrent infarct mice had fewer neutrophils released in peripheral blood and lower serum IL-6 and G-CSF levels. Elevated TGF-β1 levels were detected in bone marrow extracellular fluid during the reparative phase of cardiac-ischemic injury, and inhibiting TGF-β1 reversed the dampened reactive hematopoiesis of HSPCs in an early recurrent MI setting.Our data suggest that initial myocardial ischemia challenges blunt bone marrow reactive hematopoiesis to subsequent ischemic stress, with decreased HSPC proliferation contributing to diminished regenerative capacity. TGF-β1 in bone marrow extracellular fluid may mediate this decreased HSPC proliferation.

Cold storage, warm outcomes: Viability of Haematopoietic stem cells after extended cryopreservation.

Clonal hematopoiesis (CH) is driven by the age-associated expansion of hematopoietic stem and progenitor cells (HSPCs) that harbor somatic driver mutations; however, the mechanisms underlying their long-term persistence remain incompletely understood. This review frames CH through the lens of inclusive fitness, proposing that mutant pre-leukemic HSPCs enhance their evolutionary success not only through intrinsic self-renewal advantages, but also via indirect effects mediated by their differentiated progeny. We synthesize evidence showing that mutant immune cells promote inflammatory microenvironments that selectively impair wild-type HSCs while reinforcing mutant self-renewal, establishing self-sustaining feedback loops that shape clonal dynamics and systemic disease risk.

Sirt7 is a member of the sirtuin family of proteins, which are NAD+-dependent deacetylases and ADP-ribosyltransferases. It is involved in a wide range of cellular processes. To study the specific role of Sirt7 in haematopoiesis during aging, the gene was specifically inactivated in hematopoietic stem cells (HSC). Vav1 promoter mediated expression of CRE recombinase in floxed Sirt7 mice resulted in specific inactivation of Sirt7 in the haematopoietic stem and progenitor cells. Young mice exhibited a normal peripheral blood count and no detectable haematological aberrancies. Peripheral blood of 19-month-old Sirt7 knockout mice revealed a diminished abundance of lymphocytes, but elevated count of monocytes compared to control mice. The number of erythrocytes, platelets and haemoglobin concentration remained unchanged. In the bone marrow of aged mice, a reduced abundance of myeloid undifferentiated cells could be observed. The development of hepatomegaly due to Sirt7 gene inactivation could indicate a myeloproliferative influence. Taken together, our data demonstrate that Sirt7 functions as a critical suppressor on haematopoietic stem cells differentiation in aged mice.

Although the safety of retroviral vector (RV) gene therapy has been improved over the last years, insertional mutagenesis is still a risk factor, as seen in some of the clinical trials targeting hematopoietic stem cells. This highlights the necessity of appropriate preclinical genotoxicity assays. Our group previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) to evaluate the risk of side effects by integrating vectors. In this study, murine hematopoietic stem and progenitor cells are transduced with RVs, and genotoxicity can be detected by a proliferation advantage under limiting dilution conditions (IVIM) or the activation of genes associated with oncogenesis and stem cell-like properties (SAGA). A limitation of SAGA is the costly microarray technology. In this study, we present the digital droplet-based SAGA-Quantification (SAGA-Q) as a cost-efficient and faster alternative. Murine samples transduced with known mutagenic vector designs consistently showed upregulation of genotoxicity predictor genes. Based on a training set of 140 IVIM samples (including untransduced controls and samples transduced with long terminal repeat-driven γRV, SIN-LV.SF, SIN-LV.EFS, SIN-LV.PGK.RAG2, SIN-LV.MND.RAG1, and SIN-LV.MND.RAG2), we used random forest prediction for reliable and fast identification of genotoxic vector designs. The relevance of the predictor genes for the immortalization process was further highlighted by an elevated expression in immortalized clones. By simplifying SAGA to SAGA-Q, we aim to increase the accessibility of genotoxicity assessment and, thus, support the safer translation of gene therapy products to clinical trials.

Upon aging, hematopoietic stem cells show accumulation of DNA damage that has been causally linked with their functional decline, with debatable role of proliferative events. In this study, we sought to enquire the effect of increased proliferation rate in hematopoietic stem and progenitor cells (HSPCs) on hematopoietic aging. Multiple rounds of blood withdrawals were performed during adult life to maintain a higher proliferation rate in HSPC population in mice. Our experiments showed little effect of increased proliferation on age-associated functional decline in the hematopoietic system. However, we noted a decrease in the double-strand breaks accumulated with age after the serial bleeding regimen. Analysis of scRNA-Seq data from mouse and human HSPCs showed enrichment of DNA damage response pathways. Importantly, we demonstrate that the induction of HSPC proliferation in aged mice was sufficient to activate the DNA damage response in vivo and decrease the load of double-strand breaks. Hence, these results show that repeated blood withdrawals equivalent to clinical blood donation clear DNA damages without impacting the functioning of HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and sustain long-term self-renewal. Deriving patient-specific HSPCs has immense therapeutic potential to overcome the shortage of compatible donors for transplantation. In zebrafish, hemogenic endothelium (HE) is a specialized collection of dorsal aortic endothelial cells (ECs) that give rise to HSPCs. Our data reveal cysteine rich intestinal protein 2 (crip2) has a previously unrecognized function in establishing the proper EC environment for HSPC specification. To investigate the requirement of crip2, we generated loss-of-function alleles in crip2 and crip3, a gene family member with cardiovascular expression. crip2-/-;crip3-/- (cripDM) embryos exhibit decreased HSPC emergence with impaired lineage derivative production. Single cell RNA-sequencing of kdrl:mCherry+ ECs reveals upregulation of vascular development signature and failure to repress Notch signals during the vital transition of HE specification to HSPC emergence. Moreover, our data underscore that inhibition of Notch promotes HSPC generation in cripDM embryos and Crip genes operate through NF-κB to limit Notch. Identification of Crip2 as a novel regulator of Notch repression in HE will enhance our understanding of cues necessary to improve human HSPC production in vitro.

Background Acute myeloid leukemia (AML) is a malignant disorder originating from myeloid hematopoietic stem and progenitor cells. Despite the availability of current treatment options, a significant number of patients fail to achieve complete remission after initial chemotherapy. CD33, a transmembrane protein highly expressed on AML cells, serves as a promising therapeutic target. This study aimed to develop and evaluate chimeric antigen receptor T cells (CAR-T) and antibody-drug conjugates (ADC) based on humanized antibodies, specifically targeting CD33, to assess their potential efficacy against AML. Methods Monoclonal antibodies specific to human CD33 were generated by immunizing mice and then humanized. These humanized antibodies were then used to construct CAR-T cells and ADCs, and their cytotoxic properties were evaluated both in vitro and in vivo . In the in vivo experiments, mice bearing Molm13-Luciferase tumor cells were assigned to different treatment groups and were administered with saline, Gemtuzumab-MMAE, or Clone3HM-MMAE. Results The in vitro experiments revealed that several antibody clones, including Clone2HM, Clone3HM, Clone5HM, Clone6HM, and Clone7HM, displayed strong cytotoxic effects against Molm13-Luciferase tumor cells when conjugated with MMAE, outperforming the positive control antibody Gemtuzumab-MMAE. In the in vivo studies, mice treated with Clone3HM-MMAE showed a significant reduction in tumor signals, which nearly disappeared in the latter stages of the experiment. This led to a substantially longer survival time compared to other groups. Additionally, the body weight of mice in all treatment groups remained stable throughout the treatment period, indicating a favorable safety profile. Conclusion The CAR-T cells and ADCs developed in this study, based on humanized antibodies, showed significant anti-tumor efficacy in the AML model. Clone3HM-MMAE, in particular, demonstrated excellent anti-tumor activity along with a strong safety profile. These results strongly support the further development of targeted therapeutic strategies for AML.

Hematopoietic stem and progenitor cells (HSPC) produce all cells of the blood and immune system in a process known as hematopoiesis. During infection, there is an increased demand for immune cells which causes HSPC to rapidly and transiently modify cellular output, a response described as emergency hematopoiesis. Small molecules from the host environment may contribute to signals that regulate emergency hematopoiesis, providing a means to influence important processes during infection. Environmental exposures have long been associated with altered immune responses in human population and experimental studies. Specifically, chemicals that bind the aryl hydrocarbon receptor (AHR) modulate immune responses in a broad range of contexts, including during viral infection. Separate studies have shown that AHR signaling also influences steady-state hematopoiesis. Using two different AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2-(1H-indol-3-ylcarbonyl)-4-thiazole-carboxylic acid methyl ester (ITE), we characterized the impact of AHR activation on the proportion of HSPC and lineage-committed progenitor cells over the course of an influenza A virus infection in mice. AHR activation via these two ligands had a distinct impact on HSPC yet affected monocytes in the blood and lung similarly. For example, AHR activation with TCDD, but not ITE, increased myeloid-biasing among HSPC. However, the frequency of monocytes in the lung was reduced by either TCDD or ITE treatment. Using Vav1CreAhrfxfx mice, we showed that these effects depend on AHR expression in hematopoietic cells. Collectively, these findings highlight the differential effects of AHR ligands and their role in regulating emergency hematopoiesis in response to a common respiratory pathogen.

The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for cell intrinsic regulators of hematopoiesis, we perform a genome-wide in vivo hematopoietic stem and progenitor cell (HSPC)-based CRISPR knockout screen. We discover SAGA complex members, including Tada2b and Taf5l, as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibits hematopoiesis in vivo, causing a buildup of immature hematopoietic cells in the bone marrow. The SAGA complex deposits histone H3 lysine 9 acetylation (H3K9ac) and removes histone H2B ubiquitination (H2Bub). Loss of Tada2b leads to a reduction in H3K9ac levels and altered H2Bub enrichment in HSPCs, implicating disruption of SAGA complex activity. This is associated with upregulation of interferon pathway genes, reduced mitochondrial activity, and increased megakaryocyte progenitor cell commitment. Loss of these factors also enhances the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome cell line model. In summary, this study identifies the SAGA complex as an important regulator of hematopoiesis.

Characterized by somatic mutations (e.g., DNMT3A) in blood cells, clonal hematopoiesis (CH) is an age-related process wherein mutated hematopoietic stem and progenitor cells (HSPCs) expand. This expansion thereby increases the risk of all-cause mortality, myeloid hematologic malignancies and other nonmalignant disorders, yet the risk factors that contribute to CH are still largely unknown. Periodontitis induces low-grade systemic inflammation and affects an estimated 62% of dentate adults globally, which may influence CH-associated pathologies. Periodontitis was modeled by bilateral maxillary second molar ligation in mice; CH was established using hematopoietic-specific Dnmt3a R878H mutant mice. Periodontal bone destruction was assessed via micro-computed tomography and H/E-staining. Changes in bone marrow HSPCs, peripheral blood cells, and gingival immune cells were analyzed by flow cytometry. Key molecular mediators were identified through transcriptomic sequencing of sorted gingival myeloid cells and serum cytokine arrays. Results showed that ligature-induced periodontitis (LIP) promoted Dnmt3a R878H-driven clonal hematopoiesis, manifested as a myeloid-biased phenotype characterized by increased myeloid cells in the gingiva and peripheral blood. The selective enrichment of the Dnmt3a R878H clones during LIP is primarily because Dnmt3a R878H HSCs exhibit enhanced resistance and maintain competitive advantages within inflammatory microenvironments. Transcriptomic analysis revealed upregulation of Ccl17 in gingival R878H myeloid cells, which was corroborated by elevated serum and bone marrow levels of CCL17. The CCL17 upregulation drove myeloid cells recruitment to the gingiva, exacerbating periodontitis while simultaneously reinforcing Dnmt3a R878H HSC expansion. This study highlights the necessity of controlling local chronic inflammation, such as periodontitis, in the clinical management of CH.

Despite major advances in acute reperfusion therapies, patients surviving ischemic stroke or myocardial infarction remain at high risk for long-term cardiovascular and metabolic comorbidities. Emerging evidence identifies trained immunity, the long-lasting reprogramming of innate immune progenitors, as a central driver of this interorgan communication. Sterile insults such as stroke or myocardial infarction imprint persistent inflammatory memory via long-lasting reprogramming of bone marrow hematopoietic progenitors, biasing myelopoiesis and generating proinflammatory monocytes that target distant organs. This central trained immunity explains how a single ischemic event can precipitate cardiac dysfunction, accelerate atherosclerosis, or exacerbate metabolic disease, thereby contributing to multimorbidity in vascular patients. Understanding these systemic immune circuits provides a conceptual framework for developing interventions that interrupt maladaptive inflammatory memory. Finally, we discuss emerging therapeutic strategies to prevent maladaptive innate immune memory and mitigate chronic vascular inflammation and multimorbidity.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in heterogeneous systems. However, this technique often results in the reanalysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we develop PIP-seq for Rare-cell Enrichment and Sequencing (PURE-seq). PURE-seq allows direct FACS sorting of cells into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably sequences ultrarare cells, with 1 hour of sorting capturing tens of target cells at a rarity of 1 in 1,000,000. Leveraging this extreme sensitivity, we use PURE-seq to isolate and single-cell sequence circulating tumor cells from metastatic melanoma patient blood, obtaining detailed single cancer cell gene expression profiles. Additionally, we use PURE-seq to examine hematopoietic stem and progenitor cells from young, old and middle-aged mice. Transcriptomic analysis identifies Egr1 as a putative master regulator of murine hematopoietic stem and progenitor cell aging, demonstrating PURE-seq's utility as a discovery platform for basic science applications. PURE-seq offers a simple and highly sensitive method for single-cell sequencing ultra-rare cells.