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1.
Methods Mol Biol ; 2585: 71-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36331766

RESUMO

Flow cytometry is a technology that rapidly detects and measures physical and chemical characteristics of single cells or particles. It is a powerful tool for many areas of research, in particular, immunology, which allows for simultaneous analysis of different immune cell populations in a tissue. Here we describe the procedures to quantify and/or purify various B fractions in mouse bone marrows by flow cytometry using their signature surface markers. This method is useful to study B-cell development during steady-state or emergency hematopoiesis such as viral infections.


Assuntos
Linfócitos B , Medula Óssea , Camundongos , Animais , Citometria de Fluxo/métodos , Hematopoese , Células da Medula Óssea
2.
J Cardiol ; 81(1): 3-9, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35165011

RESUMO

Bone marrow-derived hematopoietic and immune cells play important roles in the onset and progression of cardiovascular diseases. Recent genetic analyses have discovered that clonal expansion of bone marrow hematopoietic stem/progenitor cells carrying somatic gene mutations is common and is increasing with age in healthy individuals who do not show any hematologic disorders, termed as clonal hematopoiesis. It is emergingly recognized that clonal hematopoiesis is a significant risk factor for cardiovascular diseases rather than a cumulative incidence risk of blood cancers. JAK2V617F, a gain-of-function mutation, has been identified as one of the most important mutations in clonal hematopoiesis as well as the most frequent driver mutation in myeloproliferative neoplasms. Hematopoietic cell clones harboring JAK2V617F are causally associated with the pathogenesis of cardiovascular diseases. Here, we will review the key of JAK2V617F-mediated clonal hematopoiesis including identification, prevalence, and biological impacts, linking to cardiovascular diseases and the related mechanisms. Clonal hematopoiesis with JAK2V617F may be a novel therapeutic target for cardiovascular diseases, connected to precision medicines by detecting its presence.


Assuntos
Doenças Cardiovasculares , Hematopoese , Humanos , Hematopoese/genética , Hematopoiese Clonal/genética , Células-Tronco Hematopoéticas/patologia , Doenças Cardiovasculares/genética , Fatores de Risco , Mutação
3.
Ann Lab Med ; 43(2): 145-152, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36281508

RESUMO

Background: Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of blood cells originating from somatically mutated hematopoietic stem cells, is common among the elderly and is associated with an increased risk of hematologic malignancies. We investigated the clinical, mutational, and transcriptomic characteristics in elderly Korean individuals with CHIP mutations. Methods: We investigated CHIP in 90 elderly individuals aged ≥60 years with normal complete blood counts at a tertiary-care hospital in Korea between June 2021 and February 2022. Clinical and laboratory data were prospectively obtained. Targeted next-generation sequencing of 49 myeloid malignancy driver genes and massively parallel RNA sequencing were performed to explore the molecular spectrum and transcriptomic characteristics of CHIP mutations. Results: We detected 51 mutations in 10 genes in 37 (41%) of the study individuals. CHIP prevalence increased with age. CHIP mutations were observed with high prevalence in DNMT3A (26 individuals) and TET2 (eight individuals) and were also found in various other genes, including KDM6A, SMC3, TP53, BRAF, PPM1D, SRSF2, STAG1, and ZRSR2. Baseline characteristics, including age, confounding diseases, and blood cell parameters, showed no significant differences. Using mRNA sequencing, we characterized the altered gene expression profile, implicating neutrophil degranulation and innate immune system dysregulation. Conclusions: Somatic CHIP driver mutations are common among the elderly in Korea and are detected in various genes, including DNMT3A and TET2. Our study highlights that chronic dysregulation of innate immune signaling is associated with the pathogenesis of various diseases, including hematologic malignancies.


Assuntos
Hematopoiese Clonal , Neoplasias Hematológicas , Idoso , Humanos , Hematopoese/genética , Transcriptoma , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Histona Desmetilases/genética , RNA Mensageiro
4.
Methods Mol Biol ; 2567: 181-189, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255702

RESUMO

Mesenchymal stromal cells (MSCs) are the crucial component of the hematopoietic stem and progenitor cell (HSPC) niche in the bone marrow. Therefore, an ex vivo culture system that recapitulates the marrow microenvironment is important to understanding the niche's regulatory role on HSPC function and improving ex vivo HSPC expansion for clinical transplantation. Herein, a procedure for ex vivo expansion of MSCs from human bone marrow cells and their identification and characterization is described. In addition, a protocol for MSC and HSPC coculture assay is presented. This MSC-HSPC coculture assay can be used for ex vivo expansion of HSPC. Furthermore, this assay is also useful for qualitative analysis of MSCs capable of supporting hematopoiesis.


Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Humanos , Técnicas de Cocultura , Células-Tronco Hematopoéticas , Hematopoese , Células da Medula Óssea , Células Cultivadas , Diferenciação Celular
5.
Methods Mol Biol ; 2567: 233-249, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255705

RESUMO

The zebrafish as a model organism is well known for its versatile genetics, rapid development, and straightforward live imaging. It is an excellent model to study hematopoiesis because of its highly conserved ontogeny and gene regulatory networks. Recently developed highly specific transgenic reporter lines have allowed direct imaging and tracking of hematopoietic stem and progenitor cells (HSPCs) in live zebrafish. These reporter lines can also be used for fluorescence-activated cell sorting (FACS) of HSPCs. Similar to mammalian models, HSPCs can be transplanted to reconstitute the entire hematopoietic system of zebrafish recipients. However, the zebrafish provides unique advantages to study HSPC biology, such as transplants into embryos and high-throughput chemical screening. This chapter will outline the methods needed to identify, isolate, and transplant HSPCs in zebrafish.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais Geneticamente Modificados , Mamíferos/metabolismo
6.
Methods Mol Biol ; 2567: 113-126, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255698

RESUMO

Regulation of hematopoiesis is dependent upon interactions between hematopoietic stem/progenitor cells and niche components, requiring a highly diverse array of different cell-cell interactions and cell signaling events. The overwhelming diversity of the components that can regulate hematopoiesis, especially when factoring in how the cell surface and intracellular protein expression profiles of hematopoietic stem/progenitor cells and niche components differ between homeostatic conditions and stressed conditions such as aging and irradiation, can make utilizing techniques like flow cytometry daunting, particularly while examining small cell populations such as hematopoietic stem cells (HSCs). Due to the complexity of the hematopoietic system, high-dimensional single-cell genomics and proteomics are constantly performed to understand the heterogeneity and expression profiles within this system. This chapter describes one such single-cell assay, which utilizes mass cytometry Time of Flight (CyTOF) technology to determine differences in expression profile within HSC, using changes in HSC populations due to gender and aging.


Assuntos
Hematopoese , Nicho de Células-Tronco , Nicho de Células-Tronco/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas , Comunicação Celular , Fenótipo
7.
Methods Mol Biol ; 2567: 99-112, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255697

RESUMO

Hematopoietic stem cells (HSCs) are responsible for the generation and maintenance of pools of multipotent precursors that ultimately give rise to all fully differentiated blood and immune cells. Proper identification and isolation of HSCs for functional analysis has greatly facilitated our understanding of both normal and abnormal adult hematopoiesis. Whereas adult hematopoiesis in mice and humans is driven by quiescent HSCs that reside almost exclusively within the bone marrow (BM), developmental hematopoiesis is characterized by a series of transient progenitors driving waves of increasingly mature hematopoietic cell production that occur across multiple anatomical sites. These waves of hematopoietic cell production are also responsible for the generation of distinct immune cell populations during development that persist into adulthood and contribute uniquely to adult immunity. Therefore, methods to properly isolate and characterize fetal progenitors with high purity across development become increasingly important not only for defining developmental hematopoietic pathways, but also for understanding the contribution of developmental hematopoiesis to the immune system. Here, we describe and discuss methods and considerations for the isolation and characterization of HSCs from the fetal liver, the primary hematopoietic organ during fetal development.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Humanos , Adulto , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea , Diferenciação Celular , Fígado/metabolismo
8.
Methods Mol Biol ; 2567: 127-140, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255699

RESUMO

Radiation exposure is particularly damaging to cells of the hematopoietic system, inducing pancytopenia and bone marrow failure. The study of these processes, as well as the development of treatments to prevent hematopoietic damage or enhance recovery after radiation exposure, often require analysis of bone marrow cells early after irradiation. While flow cytometry methods are well characterized for identification and analysis of bone marrow populations in the nonirradiated setting, multiple complications arise when dealing with irradiated tissues. Among these complications is a radiation-induced loss of c-Kit, a central marker for conventional gating of primitive hematopoietic populations in mice. These include hematopoietic stem cells (HSCs), which are central to blood reconstitution and life-long bone marrow function, and are important targets of analysis in these studies. This chapter outlines techniques for HSC identification and analysis from mouse bone marrow postirradiation.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Animais , Medula Óssea , Células da Medula Óssea , Transplante de Medula Óssea , Camundongos Endogâmicos C57BL
9.
Methods Mol Biol ; 2580: 355-377, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36374469

RESUMO

While the zebrafish has for some time been regarded as a powerful model organism with which to study early events in hematopoiesis, recent evidence suggests that it also ideal for unraveling the molecular requirements for T cell development in the thymus. Like mammals, zebrafish possess an adaptive immune system, comprising B lymphocytes as well as both the γδ and αß lineages of T cells, which develop in the thymus. Moreover, the molecular processes underlying T cell development in zebrafish appear to be remarkably conserved. Thus, findings in the zebrafish model will be of high relevance to the equivalent processes in mammals. Finally, molecular processes can be interrogated in zebrafish far more rapidly than is possible in mammals because the zebrafish possesses many unique advantages. Here, we describe these unique attributes and the methods by which they can be exploited to investigate the role of novel genes in T cell development.


Assuntos
Hematopoese , Peixe-Zebra , Animais , Peixe-Zebra/genética , Diferenciação Celular , Hematopoese/genética , Linfócitos T , Mamíferos
10.
Physiol Rev ; 103(1): 649-716, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36049115

RESUMO

Somatic mosaicism, the occurrence of multiple genetically distinct cell clones within the same tissue, is an evitable consequence of human aging. The hematopoietic system is no exception to this, where studies have revealed the presence of expanded blood cell clones carrying mutations in preleukemic driver genes and/or genetic alterations in chromosomes. This phenomenon is referred to as clonal hematopoiesis and is remarkably prevalent in elderly individuals. While clonal hematopoiesis represents an early step toward a hematological malignancy, most individuals will never develop blood cancer. Somewhat unexpectedly, epidemiological studies have found that clonal hematopoiesis is associated with an increase in the risk of all-cause mortality and age-related disease, particularly in the cardiovascular system. Studies using murine models of clonal hematopoiesis have begun to shed light on this relationship, suggesting that driver mutations in mature blood cells can causally contribute to aging and disease by augmenting inflammatory processes. Here we provide an up-to-date review of clonal hematopoiesis within the context of somatic mosaicism and aging and describe recent epidemiological studies that have reported associations with age-related disease. We will also discuss the experimental studies that have provided important mechanistic insight into how driver mutations promote age-related disease and how this knowledge could be leveraged to treat individuals with clonal hematopoiesis.


Assuntos
Doenças Cardiovasculares , Hematopoese , Humanos , Camundongos , Animais , Idoso , Hematopoese/genética , Hematopoiese Clonal/genética , Células-Tronco Hematopoéticas , Mosaicismo , Doenças Cardiovasculares/genética , Mutação
11.
Biomolecules ; 12(11)2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36421719

RESUMO

E-cadherin is a key regulator of epithelial cell-cell adhesion, the loss of which accelerates tumor growth and invasion. E-cadherin is also expressed in hematopoietic cells as well as epithelia. The function of hematopoietic E-cadherin is, however, mostly elusive. In this study, we explored the validity of mouse models to functionally investigate the role of hematopoietic E-cadherin in human hematopoiesis. We generated a hematopoietic-specific E-cadherin knockout mouse model. In mice, hematopoietic E-cadherin is predominantly expressed within the basophil lineage, the expression of which is dispensable for the generation of basophils. However, neither E-cadherin mRNA nor protein were detected in human basophils. In contrast, human hematopoietic E-cadherin marks the erythroid lineage. E-cadherin expression in hematopoiesis thereby revealed striking evolutionary differences between the basophil and erythroid cell lineage in humans and mice. This is remarkable as E-cadherin expression in epithelia is highly conserved among vertebrates including humans and mice. Our study therefore revealed that the mouse does not represent a suitable model to study the function of E-cadherin in human hematopoiesis and an alternative means to study the role of E-cadherin in human erythropoiesis needs to be developed.


Assuntos
Basófilos , Hematopoese , Humanos , Camundongos , Animais , Basófilos/metabolismo , Linhagem da Célula/genética , Hematopoese/genética , Caderinas/genética , Caderinas/metabolismo , Camundongos Knockout
12.
Cell Rep ; 41(6): 111592, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36351383

RESUMO

Steady-state extramedullary hematopoiesis during adulthood is an emerging field of great interest. The meninges contain both innate and adaptive immune cells, which provide immunosurveillance of the central nervous system (CNS). Hematopoietic progenitors that give rise to meningeal immune cells remain elusive. Here, we report that steady-state meninges of adult mice host hematopoietic stem cells (HSCs), as defined by long-term, efficient, multi-lineage reconstitution and self-renewal capacity in the meninges, blood, spleen, and bone marrow of sublethally irradiated adult recipients. HSCs lodge in the meninges after birth with local expression of pro-hematopoietic niche factors. Meningeal HSCs are locally maintained in homeostasis and get replenished from the blood only when the resident pool is reduced. With a tissue-specific expression profile, meningeal HSCs can provide the CNS with a constant supply of leukocytes more adapted to local microenvironment.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/fisiologia , Medula Óssea , Baço , Meninges , Camundongos Endogâmicos C57BL
13.
Stem Cells Transl Med ; 11(11): 1123-1134, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36398586

RESUMO

Several decades have passed since the generation of the first embryonic stem cell (ESC) lines both in mice and in humans. Since then, stem cell biologists have tried to understand their potential biological and clinical uses for their implementation in regenerative medicine. The hematopoietic field was a pioneer in establishing the potential use for the development of blood cell products and clinical applications; however, early expectations have been truncated by the difficulty in generating bonafide hematopoietic stem cells (HSCs). Despite some progress in understanding the origin of HSCs during embryonic development, the reproduction of this process in vitro is still not possible, but the knowledge acquired in the embryo is slowly being implemented for mouse and human pluripotent stem cells (PSCs). In contrast, ESC-derived hematopoietic cells may recapitulate some leukemic transformation processes when exposed to oncogenic drivers. This would be especially useful to model prenatal leukemia development or other leukemia-predisposing syndromes, which are difficult to study. In this review, we will review the state of the art of the use of PSCs as a model for hematopoietic and leukemia development.


Assuntos
Leucemia , Células-Tronco Pluripotentes , Humanos , Camundongos , Animais , Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo
14.
Nat Commun ; 13(1): 7064, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400777

RESUMO

The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.


Assuntos
Basófilos , Hematopoese , Camundongos , Animais , Contagem de Leucócitos , Diferenciação Celular/genética , Células-Tronco/metabolismo
15.
Yi Chuan ; 44(9): 756-771, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36384953

RESUMO

Hematopoiesis plays an important role(s) in maintenance and physiology of life. Hematopoiesis in vertebrates mainly includes self-renewal of the hematopoietic stem cells, proliferation and differentiation of the hematopoietic progenitor cells, and maturation of the blood cells. The regulation of hematogenesis involves a variety of transcription factors, membrane receptors, hematopoietic growth factors, and microRNAs, which interact with each other and form a variety of signaling pathways and signal networks. MicroRNAs are a class of non-coding RNAs widely distributed in eukaryotic cells and play important roles in the hematopoietic process. The expression of microRNAs is regulated by transcription factors involved in hematopoietic signaling pathways. In turn, their expression can inhibit or down-regulate those of transcription factors involved in hematopoietic related signaling pathways and other related regulatory factors, thereby affecting the signaling pathways related to hematopoiesis and ultimately the hematopoietic process. In this review, we introduce the hematopoiesis processes and related signal pathways in vertebrates, focusing on the relationships between microRNAs and hematopoietic transcription factors and signal pathways, and summarizing the recent research progress of microRNAs in hematopoiesis.


Assuntos
MicroRNAs , Animais , MicroRNAs/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Fatores de Transcrição/metabolismo
16.
Rinsho Ketsueki ; 63(10): 1422-1429, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-36351651

RESUMO

Hematopoietic stem cells (HSC) have self-renewal as well as multilineage differentiation capacity and maintain hematopoiesis throughout life. HSC transplantation (HSCT) is performed as a curative therapy for hematopoietic malignancies and nonmalignant hematopoietic disorders. Furthermore, bone marrow, mobilized peripheral blood, and cord blood are available sources for HSCT. HLA compatibility is the most critical factor for a successful HSCT. The HSC number in a graft is also invaluable for engraftment. Moreover, it is challenging to obtain an abundant number of HSC for patients with obesity, particularly, in cord blood. HSC ex vivo expansion is an appropriate solution for this problem. Extrinsic factors to expand and maintain HSCs, such as cytokines are identified from analysis of HSCs and their niche. Thus, HSC ex vivo expansion is improved by adding them in culture medium; however, it is still difficult for therapeutic applications. Recently, several small molecular compounds have been reported to facilitate ex vivo expansion of HSC. Clinical trials that transplant ex vivo expanded cord blood have been already expanded, and some trials demonstrate reduction of time to hematopoietic recovery. Thus, we anticipate that ex vivo expanded cord blood transplantation will be applied widely in the future.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Hematopoese , Proliferação de Células , Diferenciação Celular , Sangue Fetal
17.
Commun Biol ; 5(1): 1268, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400843

RESUMO

Tissue stem cells maintain themselves through self-renewal while constantly supplying differentiating cells. Two distinct models have been proposed as mechanisms of stem cell homeostasis. According to the classical model, there is hierarchy among stem cells, and master stem cells produce stem cells by asymmetric division; whereas, according to the recent model, stem cells are equipotent and neutrally compete. However, the mechanism remains controversial in several tissues and species. Here, we developed a mathematical model linking the two models, named the hierarchical neutral competition (hNC) model. Our theoretical analysis showed that the combination of the hierarchy and neutral competition exhibited bursts in clonal expansion, which was consistent with experimental data of rhesus macaque hematopoiesis. Furthermore, the scaling law in clone size distribution, considered a unique characteristic of the recent model, was satisfied even in the hNC model. Based on the findings above, we proposed the criterion for distinguishing the three models based on experiments.


Assuntos
Hematopoese , Células-Tronco , Animais , Macaca mulatta , Homeostase , Células Cultivadas
18.
Cell Stem Cell ; 29(11): 1512-1514, 2022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36332568

RESUMO

A recent study1 demonstrates how hematopoietic stem cells (HSCs) contribute minimally to blood and immune cell production during development and only become active postnatally. The work also reveals how Mecom expression can be used to distinguish rare HSCs from the more abundant progenitors that arise to maintain embryonic hematopoiesis.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Desenvolvimento Embrionário , Fatores de Transcrição/metabolismo , Diferenciação Celular
19.
Cell Stem Cell ; 29(11): 1562-1579.e7, 2022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36332570

RESUMO

During fetal development, human hematopoietic stem cells (HSCs) colonize the bone marrow (BM), where they self-renew and sustain hematopoiesis throughout life; however, the precise timepoint at which HSCs seed the BM is unclear. We used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells (HSPCs) and their microenvironment from 10 to 14 post-conception weeks (PCWs). We further demonstrated that functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs. In contrast, functional HSCs were not detected in the spleen by 14 PCWs. By comparing the niche-HSPC interactions between BM and spleen, we identified ligand-receptor pairs likely to be involved in fetal HSC migration and maintenance. Our work paves the way for research into the mechanisms underlying HSC colonization in human fetal BM and provides invaluable resources for future studies on HSC development.


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Adulto , Humanos , Camundongos , Animais , Hematopoese/genética , Células da Medula Óssea , Análise de Sequência de RNA
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