RESUMO
BACKGROUND: The use of mobilizing agents for hematopoietic stem cell (HSC) transplantation is insufficient for an increasing number of patients. We previously reported lipid made endocannabinoid (eCB) ligands act on the human bone marrow (hBM) HSC migration in vitro, lacking long term stability to be therapeutic candidate. In this study, we hypothesized if a novel 2-AG-loaded polycaprolactone (PCL)-based nanoparticle delivery system that actively targets BM via phosphatidylserine (Ps) can be generated and validated. METHODS: PCL nanoparticles were prepared by using the emulsion evaporation method and characterized by Zetasizer and scanning electron microscopy (SEM). The encapsulation efficiency and release profile of 2-AG were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The presence of cannabinoid receptors (CBRs) in HSCs and monocytes was detected by flow cytometry. Cell morphology and viability were assessed using transmission electron microscopy (TEM), SEM, and the WST-1 viability assay. The migration efficacy of the 2-AG and 2-AG-loaded nanoparticle delivery system on HSCs and HPSCs (TF-1a and TF-1) and monocytes (THP-1) was evaluated using a transwell migration assay. RESULTS: The 140-225 nm PCL nanoparticles exhibited an increasing polydispersity index (PDI) after the addition of Ps and 2-AG, with a surface charge ranging from - 25 to -50 mV. The nanoparticles released up to 36% of 2-AG within the first 8 h. The 2-AG-Ps-PCL did not affect cellular viability compared to control on days 5 and 10. The HSCs and monocytes expressed CB1R and CB2R and revealed increased migration to media containing 1 µM 2-AG-Ps-PCL compared to control. The migration rate of the HSCs toward monocytes incubated with 1 µM 2-AG-Ps-PCL was higher than that of the monocytes of control. The 2-AG-Ps-PCL formulation provided a real time mobilization efficacy at 1 µM dose and 8 h time window via a specific CBR agonism. CONCLUSION: The newly generated and validated 2-AG-loaded PCL nanoparticle delivery system can serve as a stable, long lasting, targeted mobilization agent for HSCs and as a candidate therapeutic to be included in HSC transplantation (HSCT) protocols following scale-up in vivo preclinical and subsequent clinical trials.
Assuntos
Células-Tronco Hematopoéticas , Nanopartículas , Poliésteres , Humanos , Poliésteres/química , Nanopartículas/química , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Movimento Celular/efeitos dos fármacos , Endocanabinoides/farmacologia , Medula Óssea/metabolismo , Medula Óssea/efeitos dos fármacosRESUMO
Sustaining lifelong hematopoiesis requires maintenance, proliferation, and differentiation of hematopoietic stem cells. Thrombopoietin is a cytokine essential for regulation of hematopoietic stem cells as well as differentiation and maturation of megakaryocytes required for platelet production. Due to these properties, thrombopoietin agonists have been used to treat bone marrow failure syndromes such as aplastic anemia. Through analysis of thrombopoietin gene-deficient mice, my colleagues and I have demonstrated the mechanism of action of thrombopoietin receptor agonists in hematopoietic stem cell maintenance and differentiation. This review focuses on governance of homeostasis in the hematopoietic system by thrombopoietin signaling.
Assuntos
Células-Tronco Hematopoéticas , Receptores de Trombopoetina , Transdução de Sinais , Trombopoetina , Trombopoetina/metabolismo , Trombopoetina/farmacologia , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Receptores de Trombopoetina/metabolismo , Receptores de Trombopoetina/agonistas , Diferenciação CelularRESUMO
Age is a risk factor for hematologic malignancies. Attributes of the aging hematopoietic system include increased myelopoiesis, impaired adaptive immunity, and a functional decline of the hematopoietic stem cells (HSCs) that maintain hematopoiesis. Changes in the composition of diverse HSC subsets have been suggested to be responsible for age-related alterations, however, the underlying regulatory mechanisms are incompletely understood in the context of HSC heterogeneity. In this study, we investigated how distinct HSC subsets, separated by CD49b, functionally and molecularly change their behavior with age. We demonstrate that the lineage differentiation of both lymphoid-biased and myeloid-biased HSC subsets progressively shifts to a higher myeloid cellular output during aging. In parallel, we show that HSCs selectively undergo age-dependent gene expression and gene regulatory changes in a progressive manner, which is initiated already in the juvenile stage. Overall, our studies suggest that aging intrinsically alters both cellular and molecular properties of HSCs.
Assuntos
Envelhecimento , Células-Tronco Hematopoéticas , Camundongos Endogâmicos C57BL , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Animais , Envelhecimento/genética , Envelhecimento/fisiologia , Camundongos , Diferenciação Celular , Linhagem da Célula/genética , Hematopoese/genética , Células Mieloides/metabolismo , Células Mieloides/citologia , Masculino , Regulação da Expressão Gênica , FemininoRESUMO
Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole-genome CRISPR-Cas9 screening showed that Toll-like receptor 3 (Tlr3) signaling regulated b2m expression. Targeting b2m or tlr3 reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive element (RE) transcripts. Overall, our data suggest that RE-associated double-stranded RNA could interact with TLR3 to stimulate surface expression of B2m on hematopoietic stem and progenitor cells. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality.
Assuntos
Calreticulina , Células-Tronco Hematopoéticas , Macrófagos , Fagocitose , Receptor 3 Toll-Like , Microglobulina beta-2 , Animais , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo , Calreticulina/metabolismo , Calreticulina/genética , Proliferação de Células , Sistemas CRISPR-Cas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Several 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: Our 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.
Assuntos
Citocinas , Proteínas de Fusão Oncogênica , Transdução de Sinais , Humanos , Animais , Citocinas/metabolismo , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Regulação Leucêmica da Expressão Gênica , Criança , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
BACKGROUND: Ex 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. METHODS: UCB-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. RESULTS: A 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. CONCLUSION: This 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.
Assuntos
Antígenos CD34 , Sangue Fetal , Células-Tronco Hematopoéticas , Imunofenotipagem , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Diferenciação Celular , Transplante de Células-Tronco Hematopoéticas/métodos , PurinasRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is linked to diverse aging-related diseases, including hematologic malignancy and atherosclerotic cardiovascular disease (ASCVD). While CHIP is common among older adults, the underlying factors driving its development are largely unknown. To address this, we performed whole-exome sequencing on 8,374 blood DNA samples collected from 4,187 Atherosclerosis Risk in Communities Study (ARIC) participants over a median follow-up of 21 years. During this period, 735 participants developed incident CHIP. Splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) and TET2 CHIP grow significantly faster than DNMT3A non-R882 clones. We find that age at baseline and sex significantly influence the incidence of CHIP, while ASCVD and other traditional ASCVD risk factors do not exhibit such associations. Additionally, baseline synonymous passenger mutations are strongly associated with CHIP status and are predictive of new CHIP clone acquisition and clonal growth over extended follow-up, providing valuable insights into clonal dynamics of aging hematopoietic stem and progenitor cells. This study also reveals associations between germline genetic variants and incident CHIP. Our comprehensive longitudinal assessment yields insights into cell-intrinsic and -extrinsic factors contributing to the development and progression of CHIP clones in older adults.
Assuntos
Hematopoiese Clonal , Dioxigenases , Humanos , Hematopoiese Clonal/genética , Masculino , Feminino , Idoso , Estudos Longitudinais , Pessoa de Meia-Idade , Dioxigenases/genética , DNA Metiltransferase 3A , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Aterosclerose/genética , Fatores de Risco , Sequenciamento do Exoma , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Envelhecimento/genética , Incidência , MutaçãoRESUMO
Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and has been found to have anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through two receptor complexes, one of which includes OSMRß. Here, we investigated OSM-OSMRß signaling in adult mouse hematopoietic stem cells (HSCs) using the conditional Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J. We crossed Osmrfl/fl mice to interferon-inducible Mx1-Cre, which is robustly induced in adult HSCs. From these mice, we isolated HSCs by flow cytometry, stimulated with recombinant OSM or vehicle for 1 hour, and assessed gene expression changes in control versus Osmr knockout HSCs by RNA-seq. This data may be utilized to investigate OSMRß -dependent and -independent OSM signaling as well as the transcriptional effects of an IL-6 family cytokine on mouse HSCs to further define its anti-inflammatory versus pro-inflammatory properties.
Assuntos
Células-Tronco Hematopoéticas , Oncostatina M , Transdução de Sinais , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/genética , Análise de Sequência de RNA , Receptores de Oncostatina M/genética , RNA-SeqRESUMO
From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 µM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.
Assuntos
Diferenciação Celular , Canais Iônicos , Mecanotransdução Celular , Megacariócitos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Humanos , Megacariócitos/metabolismo , Megacariócitos/citologia , Diferenciação Celular/genética , Trombopoese/genética , Cálcio/metabolismo , Antígenos CD34/metabolismo , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Anemia Hemolítica Congênita/patologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Hidropisia Fetal/genética , Hidropisia Fetal/metabolismo , Hidropisia Fetal/patologia , Plaquetas/metabolismo , Pirazinas , TiadiazóisRESUMO
Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled "MYCT1 controls environmental sensing in human haematopoietic stem cells", published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine.
Assuntos
Endocitose , Terapia Genética , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Diferenciação Celular , AnimaisRESUMO
BACKGROUND: Chimeric antigen receptor (CAR)-T therapy has emerged as a promising treatment for hematologic malignancies. However, cytopenia remains one of the most frequent and challenging adverse effects of this therapy. METHODS: We conducted a retrospective analysis of 26 patients with relapsed/refractory aggressive B-cell lymphoma who received CAR-T therapy at our center. Subsequently, to investigate measures to address cytopenias following CAR-T therapy, we isolated and generated murine CAR-T cells and bone marrow-derived mesenchymal stem cells (MSCs), establishing a murine syngeneic CAR-T therapy model. We assessed the impact of MSC infusion on hematopoietic recovery post-CAR-T therapy by evaluating complete blood count, bone marrow hematopoietic stem cells and their subpopulations, bone marrow histomorphology, and hematopoiesis-related genes. RESULTS: All patients experienced cytopenias to varying degrees, with complete lineage involvement in half of the patients. Grade ≥ 3 cytopenias were observed in 88.46% of the patients. CAR-T therapy was associated with a higher incidence of biphasic, late-onset, or prolonged cytopenias. Survival analysis indicated that neutropenia and lymphopenia tended to be associated with better prognosis, whereas thrombocytopenia tended to be related to poorer outcomes. Through animal experiments, we discovered that MSCs infusion boosted HSCs and their long-term subpopulations, enhancing hematopoietic recovery, particularly in the megakaryocyte lineage, and mitigating bone marrow damage. Importantly, both in vitro and in vivo experiments demonstrated that MSCs did not compromise the activity or antitumor efficacy of CAR-T cells. CONCLUSIONS: Our findings propose MSCs infusion as a promising strategy to address cytopenias, particularly thrombocytopenia, after CAR-T therapy. This approach could help overcome certain limitations of cellular immunotherapy by enhancing hematopoietic recovery without compromising the efficacy of CAR-T cells. HIGHLIGHTS: 1 Cytopenia is a frequently observed adverse effect following CAR-T therapy, and it is often characterized by biphasic and prolonged patterns. 2 MSCs play a critical role in promoting hematopoietic recovery and mitigating bone marrow damage in a murine model of CAR-T therapy 3 The activity and antitumor efficacy of CAR-T cells were not impaired by MSCs.
Assuntos
Imunoterapia Adotiva , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , Masculino , Feminino , Pessoa de Meia-Idade , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adulto , Transplante de Células-Tronco Mesenquimais/métodos , Receptores de Antígenos Quiméricos/metabolismo , Idoso , Estudos Retrospectivos , Trombocitopenia/terapia , Hematopoese , Linfoma de Células B/terapia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Neutropenia/terapia , CitopeniaRESUMO
Down syndrome predisposes individuals to haematological abnormalities, such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood1-3. Here, to understand dysregulated haematopoiesis in Down syndrome, we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human fetal liver and bone marrow samples from 3 fetuses with disomy and 15 fetuses with trisomy. We found that differences in gene expression in Down syndrome were dependent on both cell type and environment. Furthermore, we found multiple lines of evidence that haematopoietic stem cells (HSCs) in Down syndrome are 'primed' to differentiate. We subsequently established a Down syndrome-specific map linking non-coding elements to genes in disomic and trisomic HSCs using 10X multiome data. By integrating this map with genetic variants associated with blood cell counts, we discovered that trisomy restructured regulatory interactions to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Furthermore, as mutations in Down syndrome display a signature of oxidative stress4,5, we validated both increased mitochondrial mass and oxidative stress in Down syndrome, and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Together, our single-cell, multi-omic resource provides a high-resolution molecular map of fetal haematopoiesis in Down syndrome and indicates significant regulatory restructuring giving rise to co-occurring haematological conditions.
Assuntos
Síndrome de Down , Sangue Fetal , Hematopoese , Células-Tronco Hematopoéticas , Análise de Célula Única , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Hematopoese/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Estresse Oxidativo/genética , Transcriptoma/genética , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/genética , Trissomia/genética , Fígado/metabolismo , Fígado/embriologia , Diferenciação Celular/genética , Cromatina/metabolismo , Cromatina/genética , Linhagem da Célula/genética , Medula Óssea/metabolismo , Feto/metabolismo , Feto/citologia , MultiômicaRESUMO
BACKGROUND: Obesity is accompanied by inflammation, which significantly affects the homeostasis of the immune microenvironment. Hematopoietic stem cells (HSCs), residing primarily in the bone marrow, play a vital role in maintaining and producing diverse mature blood cell lineages for the adult hematopoietic and immune systems. However, how HSCs development is affected by obese-promoting inflammation, and the mechanism by which HSC hematopoietic potency is affected by inflammatory signals originating from the obese-promoting changes on bone marrow niche remain unclear. This study elucidates the relationship between obesity-promoting inflammation and HSC fate determination. METHODS: The obesity mice model was established by feeding C57BL/6J mice a high-fat diet (HFD) containing 60% kcal fat. After 6 weeks, HSCs were analyzed using flow cytometry and identified key inflammation cytokine. Transcriptome sequencing techniques were used to discern the distinct pathways in HSCs. Ultimately, confirming the biological mechanism of obesity-induced HSC fate changes via Anakinra blocking specific inflammatory signals. RESULTS: Obesity caused by HFD changed the physical and biochemical properties of the bone marrow niche. In the HFD mice, the population of long-term HSCs in the bone marrow was decreased and facilitated HSCs differentiation towards the myeloid lineage. In addition, HFD increased expression of the inflammatory factor IL-1ß in the bone marrow, and a significantly increased expression of IL-1r1 and active p38/MAPK signaling pathway were detected in the HSCs. Inhibition of IL-1ß further normalized the expression of genes in p38/MAPK pathway and reversed HSC fate. CONCLUSIONS: These findings have been demonstrated that the p38/MAPK signaling pathway in HSCs is activated by elevated levels of IL-1ß within the HSC niche in obese models, thereby regulating HSC differentiation. It suggested a direct link between obesity-promoting inflammation and myeloid differentiation bias of HSCs in the HFD mice.
Assuntos
Dieta Hiperlipídica , Células-Tronco Hematopoéticas , Interleucina-1beta , Camundongos Endogâmicos C57BL , Obesidade , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Células-Tronco Hematopoéticas/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Obesidade/metabolismo , Obesidade/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Dieta Hiperlipídica/efeitos adversos , Masculino , Sistema de Sinalização das MAP Quinases , Inflamação/metabolismo , Inflamação/patologia , Transdução de Sinais , Diferenciação CelularRESUMO
Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. VldlrhighHSCs show higher erythroid potential, which is enhanced after acute anemia induction. VldlrhighHSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions.
Assuntos
Anemia , Apolipoproteínas E , Diferenciação Celular , Células-Tronco Hematopoéticas , Lipoproteínas , Animais , Anemia/metabolismo , Anemia/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Lipoproteínas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismo , Receptores de LDL/genética , Masculino , Cromatina/metabolismo , Eritropoese/genética , Células Eritroides/metabolismoRESUMO
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, Aß 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.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Camundongos Transgênicos , Microglia , Animais , Doença de Alzheimer/terapia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Terapia Genética/métodos , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Camundongos , Microglia/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Peptídeos beta-Amiloides/metabolismo , Diferenciação Celular , Humanos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Diferenciação Celular , Proliferação de Células , Eritropoese , Células-Tronco Hematopoéticas , Transdução de Sinais , Fator de Células-Tronco , Animais , Eritropoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Camundongos , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
In the realm of hematopoiesis, hematopoietic stem cells (HSCs) serve as pivotal entities responsible for generating various blood cell types, initiating both the myeloid and lymphoid branches within the hematopoietic lineage. This intricate process is marked by genetic variations that underscore the crucial role of genes in regulating cellular functions and interactions. Recognizing the significance of genetic factors in this context, this article delves into a genetic perspective, aiming to unravel the biological factors that govern the transition from one cell's fate to another within the hematopoietic system. To gain deeper insights into the genetic traits of three distinct blood cell types-HSCs, erythroblasts (EBs), and megakaryocytes (MKs)-we conducted a comprehensive transcriptomic analysis. Leveraging diverse hematopoietic cell datasets from healthy individuals, sourced from The BLUEPRINT consortium, our investigation targeted the identification of genetic variants responsible for changes in gene expression levels and epigenetic modifications across the entire human genome in each of these cell types. The total number of normalized expressed transcripts includes 14,233 novel trinity lncRNAs, 13,749 mRNAs, and 3092 lncRNAs. This scrutiny revealed a total of 31,074 transcripts, with a notable revelation that 14,233 of them were previously unidentified or novel lncRNAs, highlighting a substantial reservoir of genetic information yet to be explored. Examining their expression across distinct lineages further unveiled 2845 differentially expressed (DE) mRNAs and 354 DE long noncoding RNAs (lncRNAs) notably enriched among the three distinct blood cell types: HSCs, EBs, and MKs. Our investigation extended beyond mRNA to focus on the dynamic expression of lncRNAs, revealing a well-defined pattern that played a significant role in regulating differentiation and cell-fate specification. This coordination of lncRNA dynamics extended to aberrations in both mRNA and lncRNA transcriptomes within HSCs, EBs, and MKs. We specifically characterized lncRNAs with preferential expression in HSCs, as well as in various downstream differentiated lineage progenitors of EBs and MKs, providing a comprehensive perspective on lncRNAs in human hematopoietic cells. Notably, the expression of lncRNAs exhibited substantial cell-to-cell variation, a phenomenon discernible only through single-cell analysis. The comparative analysis undertaken in this study provides valuable insights into the distinctive genetic signatures guiding the differentiation of these crucial hematopoietic cell types.
Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas , Megacariócitos , RNA Longo não Codificante , Transcriptoma , Humanos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Linhagem da Célula/genética , Megacariócitos/metabolismo , Megacariócitos/citologia , RNA Longo não Codificante/genética , Hematopoese/genética , Eritroblastos/metabolismo , Eritroblastos/citologia , Perfilação da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Diferenciação Celular/genéticaRESUMO
Microglia play fundamental roles in multiple pathological primary and secondary processes affecting the central nervous system that ultimately result in neurodegeneration and for this reason they are considered as a key therapeutic target in several neurodegenerative diseases. Microglia-targeted therapies are directed at either restoring or modulating microglia function, to redirect their functional features toward neuroprotection. Among these strategies, hematopoietic stem cell gene therapy have proven to be endowed with a unique potential for replacing diseased microglia with engineered, transplant progeny cells that can integrate and exert relevant beneficial effects in the central nervous system of patients affected by inherited and acquired neurodegenerative conditions.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Doenças Neurodegenerativas , Humanos , Terapia Genética/métodos , Terapia Genética/tendências , Doenças Neurodegenerativas/terapia , Doenças Neurodegenerativas/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Microglia/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologiaRESUMO
Acute myeloid leukemia (AML) is a devastating disease initiated and maintained by a rare subset of cells called leukemia stem cells (LSCs). LSCs are responsible for driving disease relapse, making the development of new therapeutic strategies to target LSCs urgently needed. The use of mass spectrometry-based metabolomics profiling has enabled the discovery of unique and targetable metabolic properties in LSCs. However, we do not have a comprehensive understanding of metabolite differences between LSCs and their normal counterparts, hematopoietic stem and progenitor cells (HSPCs). In this study, we used an unbiased mass spectrometry-based metabolomics analysis to define differences in metabolites between primary human LSCs and HSPCs, which revealed that LSCs have a distinct metabolome. Spermidine was the most enriched metabolite in LSCs compared with HSPCs. Pharmacological reduction of spermidine concentrations decreased LSC function but spared normal HSPCs. Polyamine depletion also decreased leukemic burden in patient-derived xenografts. Mechanistically, spermidine depletion induced LSC myeloid differentiation by decreasing eIF5A-dependent protein synthesis, resulting in reduced expression of a select subset of proteins. KAT7, a histone acetyltransferase, was one of the top candidates identified to be down-regulated by spermidine depletion. Overexpression of KAT7 partially rescued polyamine depletion-induced decreased colony-forming ability, demonstrating that loss of KAT7 is an essential part of the mechanism by which spermidine depletion targets AML clonogenic potential. Together, we identified and mechanistically dissected a metabolic vulnerability of LSCs that has the potential to be rapidly translated into clinical trials to improve outcomes for patients with AML.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Espermidina , Animais , Humanos , Camundongos , Acetiltransferases , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Metaboloma , Metabolômica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Espermidina/metabolismoRESUMO
Most gene functions have been discovered through phenotypic observations under loss of function experiments that lack temporal control. However, cell signaling relies on limited transcriptional effectors, having to be re-used temporally and spatially within the organism. Despite that, the dynamic nature of signaling pathways have been overlooked due to the difficulty on their assessment, resulting in important bottlenecks. Here, we have utilized the rapid and synchronized developmental transitions occurring within the zebrafish embryo, in conjunction with custom NF-kB reporter embryos driving destabilized fluorophores that report signaling dynamics in real time. We reveal that NF-kB signaling works as a clock that controls the developmental progression of hematopoietic stem and progenitor cells (HSPCs) by two p65 activity waves that inhibit cell cycle. Temporal disruption of each wave results in contrasting phenotypic outcomes: loss of HSPCs due to impaired specification versus proliferative expansion and failure to delaminate from their niche. We also show functional conservation during human hematopoietic development using iPSC models. Our work identifies p65 as a previously unrecognized contributor to cell cycle regulation, revealing why and when pro-inflammatory signaling is required during HSPC development. It highlights the importance of considering and leveraging cell signaling as a temporally dynamic entity.