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1.
Elife ; 122024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38573813

RESUMO

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.


Assuntos
Glicólise , Fosfofrutoquinase-2 , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Anaerobiose , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fosforilação Oxidativa , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
2.
BMC Complement Med Ther ; 24(1): 158, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38610025

RESUMO

BACKGROUND: A triplet chemotherapy regimen of docetaxel, cisplatin, and 5-fluorouracil (TPF) is used to treat head and neck squamous cell carcinoma; however, it is toxic to bone marrow mesenchymal stem cells (BMSCs). We previously demonstrated that Ganoderma spore lipid (GSL) protect BMSCs against cyclophosphamide toxicity. In this study, we investigated the protective effects of GSL against TPF-induced BMSCs and hematopoietic damage. METHODS: BMSCs and C57BL/6 mice were divided into control, TPF, co-treatment (simultaneously treated with GSL and TPF for 2 days), and pre-treatment (treated with GSL for 7 days before 2 days of TPF treatment) groups. In vitro, morphology, phenotype, proliferation, senescence, apoptosis, reactive oxygen species (ROS), and differentiation of BMSCs were evaluated. In vivo, peripheral platelets (PLTs) and white blood cells (WBCs) from mouse venous blood were quantified. Bone marrow cells were isolated for hematopoietic colony-forming examination. RESULTS: In vitro, GSL significantly alleviated TPF-induced damage to BMSCs compared with the TPF group, recovering their morphology, phenotype, proliferation, and differentiation capacity (p < 0.05). Annexin V/PI and senescence-associated ß-galactosidase staining showed that GSL inhibited apoptosis and delayed senescence in TPF-treated BMSCs (p < 0.05). GSL downregulated the expression of caspase-3 and reduced ROS formation (p < 0.05). In vivo, GSL restored the number of peripheral PLTs and WBCs and protected the colony-forming capacity of bone marrow cells (p < 0.05). CONCLUSIONS: GSL efficiently protected BMSCs from damage caused by TPF and recovered hematopoiesis.


Assuntos
Antineoplásicos , Ganoderma , Células-Tronco Mesenquimais , Animais , Camundongos , Camundongos Endogâmicos C57BL , Docetaxel , Cisplatino , Espécies Reativas de Oxigênio , Esporos Fúngicos , Hematopoese , Fluoruracila , Lipídeos
3.
Proc Natl Acad Sci U S A ; 121(16): e2318155121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38602917

RESUMO

Tissue development occurs through a complex interplay between many individual cells. Yet, the fundamental question of how collective tissue behavior emerges from heterogeneous and noisy information processing and transfer at the single-cell level remains unknown. Here, we reveal that tissue scale signaling regulation can arise from local gap-junction mediated cell-cell signaling through the spatiotemporal establishment of an intermediate-scale of transient multicellular communication communities over the course of tissue development. We demonstrated this intermediate scale of emergent signaling using Ca2+ signaling in the intact, ex vivo cultured, live developing Drosophila hematopoietic organ, the lymph gland. Recurrent activation of these transient signaling communities defined self-organized signaling "hotspots" that gradually formed over the course of larva development. These hotspots receive and transmit information to facilitate repetitive interactions with nonhotspot neighbors. Overall, this work bridges the scales between single-cell and emergent group behavior providing key mechanistic insight into how cells establish tissue-scale communication networks.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Hematopoese , Transdução de Sinais , Comunicação Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo
4.
PLoS One ; 19(4): e0300623, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38564577

RESUMO

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.


Assuntos
Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Animais , Humanos , Camundongos , Transplante de Medula Óssea , Diferenciação Celular/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Camundongos Knockout
5.
Exp Mol Med ; 56(3): 549-558, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38443597

RESUMO

Hematopoiesis can occur outside of the bone marrow during inflammatory stress to increase the production of primarily myeloid cells at extramedullary sites; this process is known as extramedullary hematopoiesis (EMH). As observed in a broad range of hematologic and nonhematologic diseases, EMH is now recognized for its important contributions to solid tumor pathology and prognosis. To initiate EMH, hematopoietic stem cells (HSCs) are mobilized from the bone marrow into the circulation and to extramedullary sites such as the spleen and liver. At these sites, HSCs primarily produce a pathological subset of myeloid cells that contributes to tumor pathology. The EMH HSC niche, which is distinct from the bone marrow HSC niche, is beginning to be characterized. The important cytokines that likely contribute to initiating and maintaining the EMH niche are KIT ligands, CXCL12, G-CSF, IL-1 family members, LIF, TNFα, and CXCR2. Further study of the role of EMH may offer valuable insights into emergency hematopoiesis and therapeutic approaches against cancer. Exciting future directions for the study of EMH include identifying common and distinct EMH mechanisms in cancer, infectious diseases, and chronic autoimmune diseases to control these conditions.


Assuntos
Hematopoese Extramedular , Neoplasias , Humanos , Hematopoese , Células-Tronco Hematopoéticas , Medula Óssea , Doença Crônica
6.
Cancer Med ; 13(5): e7093, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38497538

RESUMO

BACKGROUND: The occurrence of somatic mutations in patients with no evidence of hematological disorders is called clonal hematopoiesis (CH). CH, whose subtypes include CH of indeterminate potential and clonal cytopenia of undetermined significance, has been associated with both hematologic cancers and systemic comorbidities. However, CH's effect on patients, especially those with concomitant malignancies, is not fully understood. METHODS: We performed a retrospective evaluation of all patients with CH at a tertiary cancer center. Patient characteristics, mutational data, and outcomes were collected and analyzed. RESULTS: Of 78 individuals included, 59 (76%) had a history of cancer and 60 (77%) had moderate to severe comorbidity burdens. DNMT3A, TET2, TP53, and ASXL1 were the most common mutations. For the entire cohort, the 2-year overall survival rate was 79% (95% CI: 70, 90), while the median survival was not reached. Of 20 observed deaths, most were related to primary malignancies (n = 7, 35%), comorbidities (n = 4, 20%), or myeloid neoplasms (n = 4, 20%). Twelve patients (15%) experienced transformation to a myeloid neoplasm. According to the clonal hematopoiesis risk score, the 3-year transformation rate was 0% in low-risk, 15% in intermediate-risk (p = 0.098), and 28% in high-risk (p = 0.05) patients. By multivariate analysis, transformation was associated with variant allele frequency ≥0.2 and hemoglobin <10 g/dL. CONCLUSIONS: In a population including mostly cancer patients, CH was associated with comorbidities and myeloid transformation in patients with higher mutational burdens and anemia. Nevertheless, such patients were less likely to die of their myeloid neoplasm than of primary malignancy or comorbidities.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Hematopoiese Clonal , Estudos Retrospectivos , Hematopoese/genética , Neoplasias/epidemiologia , Neoplasias/genética , Transtornos Mieloproliferativos/epidemiologia , Transtornos Mieloproliferativos/genética , Comorbidade
7.
Elife ; 132024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38497789

RESUMO

The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of the kidney in fish immune systems remains to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cells types from zebrafish kidneys via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in the kidney, suggesting the kidney acts as a secondary lymphoid organ. These results indicated that the fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune systems, and highlighted the multifaced biology of kidneys in ancient vertebrates.


Assuntos
Perciformes , Viroses , Animais , Peixe-Zebra , Hematopoese/genética , Rim , Imunidade Adaptativa , Análise de Sequência de RNA , Mamíferos
8.
Nat Commun ; 15(1): 2255, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38490977

RESUMO

An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta-gonad-mesonephros region is the site for HSC production from hemogenic endothelial cells (HEC). While several distinct regulators are involved in this process, it is not yet known whether macroautophagy (autophagy) plays a role in hematopoiesis in the pre-liver stage. Here, we show that different states of autophagy exist in hematopoietic precursors and correlate with hematopoietic potential based on the LC3-RFP-EGFP mouse model. Deficiency of autophagy-related gene 5 (Atg5) specifically in endothelial cells disrupts endothelial to hematopoietic transition (EHT), by blocking the autophagic process. Using combined approaches, including single-cell RNA-sequencing (scRNA-seq), we have confirmed that Atg5 deletion interrupts developmental temporal order of EHT to further affect the pre-HSC I maturation, and that autophagy influences hemogenic potential of HEC and the formation of pre-HSC I likely via the nucleolin pathway. These findings demonstrate a role for autophagy in the formation/maturation of hematopoietic precursors.


Assuntos
Hemangioblastos , Células-Tronco Hematopoéticas , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Embrião de Mamíferos , Hematopoese/genética , Fatores de Transcrição/metabolismo , Autofagia/genética , Mesonefro
9.
Cancer Res Commun ; 4(3): 849-860, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38466568

RESUMO

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is a therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however, the plasticity and instability of leukemia cells counteract the establishment of treatments aimed at irreversibly inducing and maintaining their differentiation states. On the basis of our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells, we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment succeeded in inducing irreversible differentiation in AML cell lines. Mechanistically, cytosolic DNA was sensed by absent in melanoma 2 (AIM2), a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation, thereby facilitating the myeloid differentiation. Importantly, the combined therapy dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively, these findings indicate that the autophagy inhibition-cytosolic DNA-sensor signaling axis can potentiate AML differentiation therapy. SIGNIFICANCE: Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling, thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines.


Assuntos
Hematopoese , Leucemia Mieloide Aguda , Animais , Camundongos , Diferenciação Celular , Leucemia Mieloide Aguda/tratamento farmacológico , DNA/farmacologia , Autofagia/genética
10.
Stem Cell Res Ther ; 15(1): 74, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38475857

RESUMO

BACKGROUND: Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) play a pivotal role in maintaining lifelong hematopoiesis. The distinction between stem cells and other progenitors, as well as the assessment of their functions, has long been a central focus in stem cell research. In recent years, deep learning has emerged as a powerful tool for cell image analysis and classification/prediction. METHODS: In this study, we explored the feasibility of employing deep learning techniques to differentiate murine HSCs and MPPs based solely on their morphology, as observed through light microscopy (DIC) images. RESULTS: After rigorous training and validation using extensive image datasets, we successfully developed a three-class classifier, referred to as the LSM model, capable of reliably distinguishing long-term HSCs, short-term HSCs, and MPPs. The LSM model extracts intrinsic morphological features unique to different cell types, irrespective of the methods used for cell identification and isolation, such as surface markers or intracellular GFP markers. Furthermore, employing the same deep learning framework, we created a two-class classifier that effectively discriminates between aged HSCs and young HSCs. This discovery is particularly significant as both cell types share identical surface markers yet serve distinct functions. This classifier holds the potential to offer a novel, rapid, and efficient means of assessing the functional states of HSCs, thus obviating the need for time-consuming transplantation experiments. CONCLUSION: Our study represents the pioneering use of deep learning to differentiate HSCs and MPPs under steady-state conditions. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into the molecular mechanisms underlying stem cell self-renewal.


Assuntos
Aprendizado Profundo , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Hematopoese , Células-Tronco Multipotentes , Diferenciação Celular
11.
Cell ; 187(6): 1402-1421.e21, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38428422

RESUMO

Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.


Assuntos
Inflamação , Interleucina-10 , Mielopoese , Animais , Camundongos , Feto , Hematopoese , Inflamação/imunologia , Interleucina-10/imunologia , Gravidez/imunologia , Células-Tronco Hematopoéticas/citologia
12.
Int Immunopharmacol ; 130: 111760, 2024 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-38428148

RESUMO

Bone marrow macrophages (Mφ) are essential components of the bone marrow niche that regulate the function of hematopoietic stem cells. Poor graft function and inhibition of hematopoietic production can result from abnormal macrophage function; however, the underlying mechanism is unclear. Clodronate liposomes (Clo-Lip) have been used widely to deplete macrophages and study their functions. Our previous results showed that Clod-Lip-mediated clearance of macrophages plays a vital role in regulating hematopoietic reconstruction after allogeneic hematopoietic cell transplantation (HCT). In this study, using an isogenic hematopoietic stem cell transplantation model, we found that Clod-Lip-mediated clearance of macrophages suppressed hematopoietic reconstruction by inhibiting the homing process of hematopoietic cells. We also demonstrated that macrophage depletion inhibited the direct supportive effect of macrophages on hematopoietic stem and progenitor cells and erythroid differentiation but promoted the production of megakaryocytic progenitors ex vivo. We showed that macrophages increase CD49e expression on hematopoietic stem and progenitor cells (HSPCs). However, CD49e inhibitors did not support the proliferative effect of macrophages on hematopoietic cells. In contrast, macrophage E-selectin/ intercellular cell adhesion molecule-1 (ICAM-1) may be involved in directly regulating HSPCs. In conclusion, macrophage depletion with Clo-Lip partially disrupts bone marrow hematopoiesis after HCT by impeding donor cell homing and macrophage-HSPCs interactions.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Integrina alfa5 , Integrina alfa5/metabolismo , Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas/métodos , Hematopoese , Macrófagos/metabolismo
13.
Nat Immunol ; 25(4): 590-591, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38514889

Assuntos
Hematopoese , Humanos
14.
Nature ; 627(8005): 839-846, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38509363

RESUMO

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Estresse Fisiológico , Animais , Feminino , Masculino , Camundongos , Envelhecimento/fisiologia , Infecções Bacterianas/patologia , Infecções Bacterianas/fisiopatologia , Vasos Sanguíneos/citologia , Linhagem da Célula , Eritropoese , Fator Estimulador de Colônias de Granulócitos/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hemorragia/patologia , Hemorragia/fisiopatologia , Linfopoese , Megacariócitos/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Mielopoese , Crânio/irrigação sanguínea , Crânio/patologia , Crânio/fisiopatologia , Esterno/irrigação sanguínea , Esterno/citologia , Esterno/metabolismo , Estresse Fisiológico/fisiologia , Tíbia/irrigação sanguínea , Tíbia/citologia , Tíbia/metabolismo
15.
Stem Cell Reports ; 19(4): 469-485, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38518784

RESUMO

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.


Assuntos
Neoplasias Hematológicas , Hematopoese , Camundongos , Animais , Diferenciação Celular/genética , Células-Tronco Hematopoéticas , Histona Acetiltransferases/genética
16.
Elife ; 132024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38526524

RESUMO

During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.


Assuntos
Hematopoese , Macrófagos , Animais , Camundongos , Hematopoese/genética , Células-Tronco Hematopoéticas , Diferenciação Celular , Eritropoese , Fígado , Nicho de Células-Tronco/genética
17.
Bioinformatics ; 40(4)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38485690

RESUMO

MOTIVATION: The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. RESULTS: Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. AVAILABILITY AND IMPLEMENTATION: The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.


Assuntos
Hematopoiese Clonal , Neoplasias Hematológicas , Adulto , Humanos , Sequenciamento de Nucleotídeos em Larga Escala , Software , Reprodutibilidade dos Testes , Mutação , Hematopoese/genética
18.
Stem Cell Reports ; 19(4): 486-500, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38458190

RESUMO

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM) are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single-cell mRNA-seq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM confirmed that the loss of these molecules significantly reduced the ability of OM to augment the osteoblast-mediated hematopoietic-enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells.


Assuntos
Células-Tronco Hematopoéticas , Megacariócitos , Animais , Humanos , Camundongos , Megacariócitos/metabolismo , Regulação para Cima , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/fisiologia , Osteoblastos/metabolismo , Nicho de Células-Tronco/fisiologia
19.
Development ; 151(7)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38451068

RESUMO

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.


Assuntos
Células-Tronco Mesenquimais , Peixe-Zebra , Camundongos , Animais , Peixe-Zebra/genética , Células-Tronco Hematopoéticas/metabolismo , Hematopoese , Embrião de Mamíferos , Mesonefro , Gônadas
20.
Commun Biol ; 7(1): 374, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38548886

RESUMO

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.


Assuntos
Hematopoese , Megacariócitos , Humanos , Megacariócitos/metabolismo , Diferenciação Celular/genética , Hematopoese/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo
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