Hematopoietic Stem/progenitor Cells Research Articles

Delayed macrophage targeting by clodronate liposomes worsens the progression of cytokine storm syndrome

Excessive macrophage activation and production of pro-inflammatory cytokines are hallmarks of the Cytokine Storm Syndrome (CSS), a lethal condition triggered by sepsis, autoimmune disorders, and cancer immunotherapies. While depletion of macrophages at disease onset protects from lethality in an infection-induced CSS murine model, patients are rarely diagnosed early, hence the need to characterize macrophage populations during CSS progression and assess the therapeutic implications of macrophage targeting after disease onset. In this study, we identified MHCII+F4/80+Tim4- macrophages as the primary contributors to the pro-inflammatory environment in CSS, while CD206+F4/80+Tim4+ macrophages, with an anti-inflammatory profile, become outnumbered. Additionally, we observed an expansion of Tim4- macrophages coinciding with increased hematopoietic stem progenitor cells and reduction of committed myeloid progenitors in bone marrow and spleen. Critically, macrophage targeting with clodronate liposomes at disease onset prolonged survival, while their targeting in mice with established CSS exacerbated disease severity, leading to a more dramatic loss of Tim4+ macrophages and an imbalance in pro- versus anti-inflammatory Tim4− macrophage ratio. Our findings highlight the significance of timing in macrophage-targeted interventions for effective management of CSS and suggest potential therapeutic strategies for diseases characterized by uncontrolled inflammation, such as sepsis.

Development of a quantitative prediction algorithm for human cord blood-derived CD34+ hematopoietic stem-progenitor cells using parametric and non-parametric machine learning models

The transplantation of CD34+ hematopoietic stem-progenitor cells (HSPCs) derived from cord blood serves as the standard treatment for selected hematological, oncological, metabolic, and immunodeficiency disorders, of which the dose is pivotal to the clinical outcome. Based on numerous maternal and neonatal parameters, we evaluated the predictive power of mathematical pipelines to the proportion of CD34+ cells in the final cryopreserved cord blood product adopting both parametric and non-parametric algorithms. Twenty-four predictor variables associated with the cord blood processing of 802 processed cord blood units randomly sampled in 2020–2022 were retrieved and analyzed. Prediction models were developed by adopting the parametric (multivariate linear regression) and non-parametric (random forest and back propagation neural network) statistical models to investigate the data patterns for determining the single outcome (i.e., the proportion of CD34+ cells). The multivariate linear regression model produced the lowest root-mean-square deviation (0.0982). However, the model created by the back propagation neural network produced the highest median absolute deviation (0.0689) and predictive power (56.99%) in comparison to the random forest and multivariate linear regression. The predictive model depending on a combination of continuous and discrete maternal with neonatal parameters associated with cord blood processing can predict the CD34+ dose in the final product for clinical utilization. The back propagation neural network algorithm produces a model with the highest predictive power which can be widely applied to assisting cell banks for optimal cord blood unit selection to ensure the highest chance of transplantation success.

Modification of Lhx2 activity for ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells.

Hematopoietic stem cells (HSCs) obtained from patient-derived human induced pluripotent stem cells (iPSCs) are a promising tool for curing various hematological disorders. We previously demonstrated that enforced expression of the LIM-homeobox transcription factor Lhx2, which is essential for mouse embryonic hematopoiesis, leads to generation of engraftable and expandable hematopoietic stem cells (HSCs) from mouse iPSCs. However, it remained unknown whether Lhx2 can induce HSCs from human iPSCs. Here, we investigated the effect of Lhx2 overexpression on hematopoietic differentiation of human iPSCs. Unexpectedly, Lhx2 severely inhibited proliferation of human iPSC-derived hematopoietic cells. Thus, Lhx2 exhibited differential effects on mouse and human hematopoietic cells. Further studies implied that the inhibitory effect of Lhx2 on human iPSC-derived hematopoietic cells was due to insufficient transcriptional activation ability. Therefore, we modified Lhx2 to strengthen its activity as a transcriptional activator. This modified Lhx2 could induce ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells (HSPCs). We believe that these findings will facilitate the development of a method to efficiently produce HSCs from human iPSCs.

From banked human cord blood to induced pluripotent stem cells: New opportunities and promise in induced pluripotent stem cell banking (Review).

Umbilical cord blood (CB) is a valuable source of haematopoietic stem/progenitor cells (HSCs) and is known for the therapeutic use of these cells in treating blood disorders. However, challenges such as a high running cost and the increasing availability of treatment alternatives have made the effort to sustain CB banks difficult. This prompts the need to revisit the current CB banking initiatives to retain the relevance in this ever‑changing era parallel to the fast‑pacing development of cell‑based therapeutic technology. Cellular reprogramming has shown to have successfully converted adult somatic cells into human induced pluripotent stem cells (hiPSCs), which promise wider applications in regenerative medicine, personalized treatment and tissue engineering. CB is the youngest, primitive adult cell source that has not been affected by any prior, acquired disorders. Hence, using CB as a source of candidate cells for generating hiPSCs may be a new opportunity for banking, albeit with challenges. The present review summarizes the rise and fall of CB usage and banking for clinical therapy, the considerations in reprogramming CB into hiPSCs, the safety concerns regarding the use of hiPSC‑derived cells in clinical transplantation and the prospect of using CB‑derived hiPSCs.

Cellular taxonomy of the preleukemic bone marrow niche of acute myeloid leukemia.

Leukemias arise from recurrent clonal mutations in hematopoietic stem/progenitor cells (HSPCs) that cause profound changes in the bone marrow microenvironment (BMM) favoring leukemic stem cell (LSC) growth over normal HSPCs. Understanding the cross talk between preleukemic mutated HSPCs and the BMM is critical to develop novel therapeutic strategies to prevent leukemogenesis. We hypothesize that preleukemic-LSCs (pLSCs) induce BMM changes critical for leukemogenesis. Using our AML-murine model, we performed single-cell RNA-sequencing of preleukemic BMM (pBMM) cells. We found normal HSC (nHSC)-regulating LepR+ mesenchymal stem cells, and endothelial cells were decreased, along with increases in CD55+ fibroblasts and pericytes. Preleukemic CD55+ fibroblasts had higher proliferation rates and decreased collagen expression, suggesting extracellular matrix remodeling during leukemogenesis. Importantly, co-culture assays found preleukemic CD55+ fibroblasts expanded pLSCs significantly over nHSCs. In conclusion, we have identified a distinct pBMM and a novel CD55+ fibroblast population that is expanded in pBMM that promote fitness of pLSCs over nHSCs.

New insights into the endothelial origin of hematopoietic system inspired by "TIF" approaches.

Hematopoietic stem progenitor cells (HSPCs) are derived from a specialized subset of endothelial cells named hemogenic endothelial cells (HECs) via a process of endothelial-to-hematopoietic transition during embryogenesis. Recently, with the usage of multiple single-cell technologies and advanced genetic lineage tracing techniques, namely, "TIF" approaches that combining transcriptome, immunophenotype and function/fate analyses, massive new insights have been achieved regarding the cellular and molecular evolution underlying the emergence of HSPCs from embryonic vascular beds. In this review, we focus on the most recent advances in the enrichment markers, functional characteristics, developmental paths, molecular controls, and the embryonic site-relevance of the key intermediate cell populations bridging embryonic vascular and hematopoietic systems, namely HECs and pre-hematopoietic stem cells, the immediate progenies of some HECs, in mouse and human embryos. Specifically, using expression analyses at both transcriptional and protein levels and especially efficient functional assays, we propose that the onset of Kit expression is at the HEC stage, which has previously been controversial.

The Different Responsiveness of C3- and C5-deficient Murine BM Cells to Oxidative Stress Explains Why C3 Deficiency, in Contrast to C5 Deficiency, Correlates with Better Pharmacological Mobilization and Engraftment of Hematopoietic Cells.

The liver-derived circulating in peripheral blood and intrinsic cell-expressed complement known as complosome orchestrate the trafficking of hematopoietic stem/progenitor cells (HSPCs) both during pharmacological mobilization and homing/engraftment after transplantation. Our previous research demonstrated that C3 deficient mice are easy mobilizers, and their HSPCs engraft properly in normal mice. In contrast, C5 deficiency correlates with poor mobilization and defects in HSPCs' homing and engraftment. The trafficking of HSPCs during mobilization and homing/engraftment follows the sterile inflammation cues in the BM microenvironment caused by stress induced by pro-mobilizing drugs or myeloablative conditioning for transplantation. Therefore, to explain deficiencies in HSPC trafficking between C3-KO and C5-KO mice, we evaluated the responsiveness of C3 and C5 deficient cells to low oxidative stress. As reported, oxidative stress in BM is mediated by the activation of purinergic signaling, which is triggered by the elevated level of extracellular adenosine triphosphate (eATP) and by the activation of the complement cascade (ComC). In the current work, we noticed that BM lineage negative cells (lin-) isolated from C3-KO mice display several mitochondrial defects reflected by an impaired ability to adapt to oxidative stress. In contrast, C5-KO-derived BM cells show a high level of adaptation to this challenge. To support this data, C3-KO BM lin- cells were highly responsive to eATP stimulation, which correlates with enhanced levels of reactive oxygen species (ROS) generation and more efficient activation of intracellular Nlrp3 inflammasome. We conclude that the enhanced sensitivity of C3-KO mice cells to oxidative stress and better activation of the Nox2-ROS-Nlrp3 inflammasome signaling axis explains the molecular level differences in trafficking between C3- and C5-deficient HSPCs.

Molecular profiling of pre- and post- 5-azacytidine myelodysplastic syndrome samples identifies predictors of response.

Treatment with the hypomethylating agent 5-azacytidine (AZA) increases survival in high-risk (HR) myelodysplastic syndrome (MDS) patients, but predicting patient response and overall survival remains challenging. To address these issues, we analyzed mutational and transcriptional profiles in CD34+ hematopoietic stem/progenitor cells (HSPCs) before and following AZA therapy in MDS patients. AZA treatment led to a greater reduction in the mutational burden in both blast and hematological responders than non-responders. Blast and hematological responders showed transcriptional evidence of pre-treatment enrichment for pathways such as oxidative phosphorylation, MYC targets, and mTORC1 signaling. While blast non-response was associated with TNFa signaling and leukemia stem cell signature, hematological non-response was associated with cell-cycle related pathways. AZA induced similar transcriptional responses in MDS patients regardless of response type. Comparison of blast responders and non-responders to normal controls, allowed us to generate a transcriptional classifier that could predict AZA response and survival. This classifier outperformed a previously developed gene signature in a second MDS patient cohort, but signatures of hematological responses were unable to predict survival. Overall, these studies characterize the molecular consequences of AZA treatment in MDS HSPCs and identify a potential tool for predicting AZA therapy responses and overall survival prior to initiation of therapy.

Extensive immunophenotypic sub-population analysis of StemRegenin1 expanded haematopoietic stem/progenitor cells

BackgroundEx vivo haematopoietic stem/progenitor cell (HSPCs) expansion constitutes an important area of research, and has the potential to improve access to umbilical cord blood (UCB) as a source of stem cells for haematopoietic stem cell transplantation (HSCT). The ability to improve stem cell dose and thereby reduce delayed engraftment times, which has plagued the use of UCB as a stem cell source since inception, is a recognised advantage. The extent to which cluster of differentiation (CD)34 sub-populations are affected by expansion with StemRegenin1 (SR1), and whether a particular subtype may account for better engraftment than others, is currently unknown. The purpose of this study was to determine the impact of SR1-induced HSPC expansion on CD34+ immunophenotypic subsets and gene expression profiles.MethodsUCB-derived CD34+ HSPCs were characterised before (D0) and after expansion (D7) with SR1 using an extensive immunophenotypic panel. In addition, gene expression was assessed and differentially expressed genes were categorised into biological processes.ResultsA dose-dependent increase in the number of CD34+ HSPCs was observed with SR1 treatment, and unbiased and extensive HSPC immunophenotyping proved to be a powerful tool in identifying unique sub-populations within the HSPC repertoire. In this regard, we found that SR1 promotes the emergence of HSPC subsets which may aid engraftment post expansion. In addition, we observed that SR1 has a minimal effect on the transcriptome of 7-day expanded CD34+ HSPCs when compared to cells expanded without SR1, with only two genes being downregulated in the former.ConclusionThis study revealed that SR1 selects for potentially novel immunophenotypic HSPC subsets post expansion and has a minimal effect on the transcriptome of 7-day expanded HSPCs when compared to vehicle controls. Whether these distinct immunophenotypic sub-populations possess greater engraftment capacity remains to be tested in animal models.

Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

BackgroundSeveral fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis.MethodsWe created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules.ResultsWe showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo.ConclusionsOur study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Proteomics reveals dynamic metabolic changes in human hematopoietic stem progenitor cells from fetal to adulthood

BackgroundHematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly, the relationships between dynamic metabolic alterations and their functions remain poorly characterized.MethodsIn this study, we comprehensively described the proteomics of HSPCs in the human fetal liver (FL), umbilical cord blood (UCB), and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay, RT‒PCR, and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production, the metabolic state, and the expansion of human HSPCs, HSPCs were treated with buthionine sulfoximine (BSO), an inhibitor of glutathione synthetase, and N-acetyl-L-cysteine (NAC).ResultsWe investigated the metabolomic landscape of human HSPCs from the fetal, perinatal, and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays, mitochondrial activity, ROS level, glucose uptake, and protein synthesis rate analysis supported our findings. In addition, immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs, indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore, the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time.ConclusionThe metabolic landscape of human HSPCs at three developmental stages (FL, UCB, and aBM), combined with proteomics and functional validations, substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life.

Therapeutic efficacy of intracerebral hematopoietic stem cell gene therapy in an Alzheimer’s disease mouse model

The conditions supporting the generation of microglia-like cells in the central nervous system (CNS) after transplantation of hematopoietic stem/progenitor cells (HSPC) have been studied to advance the treatment of neurodegenerative disorders. Here, we explored the transplantation efficacy of different cell subsets and delivery routes with the goal of favoring the establishment of a stable and exclusive engraftment of HSPCs and their progeny in the CNS of female mice. In this setting, we show that the CNS environment drives the expansion, distribution and myeloid differentiation of the locally transplanted cells towards a microglia-like phenotype. Intra-CNS transplantation of HSPCs engineered to overexpress TREM2 decreased neuroinflammation, Aβ aggregation and improved memory in 5xFAD female mice. Our proof of concept study demonstrates the therapeutic potential of HSPC gene therapy for Alzheimer’s disease.

Sheng Xue Ning as a Novel Agent that Promotes SCF-Driven Hematopoietic Stem/Progenitor Cell Proliferation to Promote Erythropoiesis.

Stimulating erythropoiesis is essential in the treatment of various types of anemia. Sheng Xue Ning (SXN) is commonly used in China as an iron supplement to treat iron deficiency anemia, renal anemia, and anemia in pregnancy. This research reports a novel effect of SXN in enhancing the proliferation of hematopoietic stem/progenitor cell (HSPC) to promote erythropoiesis in the bone marrow, which is distinct from conventional iron supplements that primarily aid in the maturation of red blood cells. Employing a model of hematopoietic dysfunction induced by X-ray exposure, we evaluated the efficacy of SXN in restoring hematopoietic function. SXN significantly promoted the recovery of peripheral erythroid cells and enhanced the proliferation and differentiation of Lin-/c-KIT+/Sca-1+ HSPC in mice exposed to X-ray irradiation. Our results showed that SXN elevated the expression of stem cell factor (SCF) and activated the SCF/c-KIT/PI3K/AKT signaling pathway, facilitating the proliferation and differentiation of HSPC. In vitro, SXN markedly enhanced the proliferation of bone marrow nucleated cell (BMNC) and the colony-forming capacity of BFU-E, CFU-E, and CFU-GM, while also elevating the expression of proteins involved in the SCF/c-KIT/PI3K/AKT pathway in BMNC. Additionally, SXN enhanced the proliferation and differentiation of mesenchymal stem cell (MSC) and increased SCF secretion. In conclusion, SXN demonstrates the capacity to enhance erythropoiesis by upregulating SCF expression, thereby promoting HSPC proliferation and differentiation via the SCF/c-KIT/PI3K/AKT pathway. SXN may offer a new strategy for improving the activity of HSPC and promoting erythropoiesis in the treatment of hematopoiesis disorders.

Dynamically adjusted cell fate decisions and resilience to mutant invasion during steady-state hematopoiesis revealed by an experimentally parameterized mathematical model

A major next step in hematopoietic stem cell (HSC) biology is to enhance our quantitative understanding of cellular and evolutionary dynamics involved in undisturbed hematopoiesis. Mathematical models have been and continue to be key in this respect, and are most powerful when parameterized experimentally and containing sufficient biological complexity. In this paper, we use data from label propagation experiments in mice to parameterize a mathematical model of hematopoiesis that includes homeostatic control mechanisms as well as clonal evolution. We find that nonlinear feedback control can drastically change the interpretation of kinetic estimates at homeostasis. This suggests that short-term HSC and multipotent progenitors can dynamically adjust to sustain themselves temporarily in the absence of long-term HSCs, even if they differentiate more often than they self-renew in undisturbed homeostasis. Additionally, the presence of feedback control in the model renders the system resilient against mutant invasion. Invasion barriers, however, can be overcome by a combination of age-related changes in stem cell differentiation and evolutionary niche construction dynamics based on a mutant-associated inflammatory environment. This helps us understand the evolution of e.g., TET2 or DNMT3A mutants, and how to potentially reduce mutant burden.

Amphiphilic cytokine traps remodel marrow adipose tissue for hematopoietic microenvironment amelioration

Hematopoietic stem cell transplantation (HSCT) is extensively employed in the treatment of hematological malignancies but is markedly constrained by the paucity of hematopoietic stem/progenitor cells (HSPCs). Recent studies have found that marrow adipose tissue (MAT) acts on hematopoiesis through complicated mechanisms. Therefore, the osteo-organoids fabricated in vivo using biomaterials loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) have been used as models of MAT for our research. To obtain sufficient amounts of therapeutic HSPCs and healthy MAT, we have developed amphiphilic chitosan (AC)-gelatin as carriers of rhBMP-2 to the regulate type conversion of adipose tissue and trap hematopoietic growth factors. Unlike medicine interventions or cell therapies, the traps based on AC not only attenuate the occupancy of adipocytes within the hematopoietic microenvironment while preserving stem cell factor concentrations, but also improve marrow metabolism by promoting MAT browning. In conclusion, this approach increases the proportion of HSPCs in osteo-organoids, and optimizes the composition and metabolic status of MAT. These findings furnish an experimental basis for regulating hematopoiesis in vivo through materials that promote the development of autologous HSPCs. Additionally, this approach presents a theoretical model of rapid adipogenesis for the study of adipose-related pathologies and potential pharmacological targets.

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities

Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematological abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting rheumatologic/immunologic symptoms or severe hematological manifestations. Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of the mesenchymal stromal cells (MSCs). Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and non-severe hematological manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematological symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM MSCs in DADA2 patients exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. Our exploration into the BM landscape of DADA2 patients sheds light on the critical hematological dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

Transcriptomic analysis of BM-MSCs identified EGR1 as a transcription factor to fully exploit their therapeutic potential

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45−, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

PIWI pathway: bridging acute myeloid leukemia stemness and cellular differentiation.

PIWI proteins are stem cell-associated RNA-binding proteins crucial for survival of germ stem cells. In cancer, PIWI proteins are overexpressed. Specifically, PIWIL4 is highly expressed in multiple cancers with the highest levels found in acute myeloid leukemia (AML), an aggressive malignancy propagated by a population of leukemia stem cells (LSCs). Bamezai et al. (Blood Journal, blood, 2023, 142, 90-105) demonstrated that PIWIL4 supports AML blasts and LSCs but is not necessary for healthy human hematopoietic progenitor stem cells (HSPCs) function in vivo. PIWIL4 in AML acts by preventing the accumulation of R-loops in key genes for LSCs persistence implicated in: DNA damage, replicative stress, and transcription arrest. We report that PIWIL4 expression significantly decreases in THP-1 monocytes exposed to a differentiating agent, suggesting a potential role for PIWIL4 in maintaining the undifferentiated state of myeloid cells. PIWIL4 overexpression could lead to the emergence of LSCs, driving leukemia propagation and maintenance. Our findings correlate with the persistent overexpression of PIWIL4 in myeloid cancers as reported by Bamezai et al., and suggest that PIWIL4 may be involved in myeloid cell differentiation. In this perspective, we highlight recent findings on the implication of PIWI pathway in maintaining AML stemness. Additionally, we propose further investigation on the role of PIWI pathway in oncogenesis and cellular differentiation as a strategy to identify biomarkers and therapeutic targets for AML.

HES1 is required for mouse fetal hematopoiesis

BackgroundHematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling.MethodsIn this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay.ResultsWe found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo.ConclusionTogether, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.